TJVM Vol. 48 No. 4 December 2018

THE THAI JOURNAL OF VETERINARY MEDICINE Office: Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330 Thailand Tel. 66(2) - 218 9556 Fax. 66(2) - 255 3910 Advisory Committee: Prof. Dr. Roongroje Thanawongnuwech Asst. Prof. Dr. Thawatchai Sakphuaram Dean President of the Veterinary Council of Thailand Prof. Dr. Alongkorn Amonsin Dr. Somchuan Ratanamungklanon Associate Dean of Academic International President of the Thai Veterinary Medical Association and Student Affairs under the Royal Patronage Prof. Dr. Kaywalee Chatdarong Dr. Boonlert Preechatangkij Associate Dean of Research and Innovation President of Chulalongkorn University Veterinary Alumni Association Editorial Board: Annop Kunavongkrit (Thailand) Andrew Ponter (France) Atichat Bramasa (Thailand) Andrej Madej (Sweden) Chollada Buranakarl (Thailand) Eileen L. Thacker (USA) Jiroj Sasipreeyajan (Thailand) Elisabeth Persson (Sweden) Han-Soo Joo (USA) Marissak Kalpravidh (Thailand) Karen L. Keller (USA) Mongkol Techakumphu (Thailand) Oliver Sparagano (UK) Narongsak Chaiyabutr (Thailand) Stanley H. Done (UK) Peerasak Chanprateep (Thailand) Stig Einarsson (Sweden) Pranee Tuntivanich (Thailand) Takashi Aoki (Japan) Roongroje Thanawongnuwech (Thailand) Teresa Y. Morishita (USA) Somchai Chanpongsang (Thailand) Masashi Maita (Japan) Padet Tummaruk (Thailand) Eric Lombardini (USA) - Anusak Kijtawornrat (Thailand, Editor-in-Chief) Anudep Rangsipipat (Thailand) Boonrit Thongsong (Thailand) Theerayuth Kaewamatawong (Thailand) Piyarat Chansiripornchai (Thailand) Chenphop Sawangmake (Thailand) Journal Management and Membership: Mrs.Karoon Sanchu, Mrs.Kourwan Promngram, Mr.Kritsana Phanchinda and Mrs.Junya Petchkongthong The Veterinary Library and Information Center Faculty of Veterinary Science, Chulalongkorn University, Bangkok, 10330 Thailand Tel. 66(2)- 218 9556-7 Email: [email protected] https://www.tci-thaijo.org/index.php/tjvm This publication will be indexed and abstracted in Science Citation Index Expanded (SciSearch®), SCOPUS, CABI, ProQuest and EBSCO Publisher: Chulalongkorn University Printing House (5305-128/600) Phayathai Road, Pathumwan, Bangkok, 10330 Thailand Tel. 66(2) - 218 3557-63, Fax. 66(2) - 218 3551



The Thai Journal of Veterinary Medicine Vol. 48 No. 4 December 2018 Contents Review Article 515 Review of the cardiovascular toxicity of amitriptyline treatment for canine neuropathic pain Saikaew Sutayatram, Kumpanart Soontornvipart, Piyasiri Glangosol Original Article 529 Comparison of isolation techniques for bone marrow derived canine mesenchymal stem cells (MSCs) 541 and the compatibility of MSCs loaded onto polycarpolactone hydroxyapatite 551 Chalika Wangdee, Sasithorn Panasophonkul, Napaphat Thadavirul, Kumpanart Soontornvipart, Prasit Pavasant, Pitt Supaphol, Mongkol Techakumphu, Theerawat Tharasanit 559 Anti-proliferative and total ERK1/2 inhibitory effects of plant flavonols on Human cervical cancer 567 (HeLa) cells 573 583 Sookruetai Boonmasawai, Ladawan Sariya, Arpron Leesombun, Kridsada Chaichoun, Jarupha Taowan, 595 Orathai Thongjuy 603 High infection rate of Zika virus in mosquitoes collected from an area of active Zika virus transmission 613 in eastern Thailand 623 Apiwat Tawatsin, Atchara Phumee, Usavadee Thavara, Patcharawan Sirisopa, Wanapa Ritthison, 631 Kitsanaphong Thammakosol, Proawpilart Intayot, Yutthana Joyjinda, Supaporn Wacharapluesadee, 639 Thiravat Hemachudha, Padet Siriyasatien 645 Hemostatic efficacy of sheep-derived fibrin glue for liver biopsy in swine 655 663 Siriluck Luckanahasaporn, Theerawat Tharasanit, Passakorn Briksawan, Sumit Durongphongthron Small dosages of alpha1 adrenoceptor antagonist on goat semen quality and seminal fluid volume Sakdichod Kimsakulvech, Sookruetai Boonmasawai, Dulyatud Gronsang, Chowalit Nakthong, Yupaporn Lanamtiang Computed tomographic contrast enhancement and tumor angiogenesis in canine oral tumors Urapa Klansnoh, Wijit Banlunara, Nan Choisunirachon The potential use of phytoestrogen containing the herb, Pueraria mirifica, for bone healing in osteoporotic monkeys Donlaporn Kittivanichkul, Nan Choisunirachon, Kumpanart Soontornvipart, Suchinda Malaivijitnond Comparison of sperm freezability and fertility among Hmong Black-Bone, Barred Plymouth Rock and Thai Native (Pradu Hang Dam) chickens Saennamphung Somtaw, Pornjit Sonseeda, Nalinee Tubtimtong, Yupin Phasuk, Thevin Vongpralub Efficacy of different vaccination programs of recombinant HVT-NDV vaccine against genotype VII NDV Challenge in broiler chickens Nataya Charoenvisal, Bubpa Supannamoke, Rik Koopman, Jiroj Sasipreeyajan A preliminary study on diversity of midgut microbiota in Aedes aegypti (Linnaeus) and Culex quinquefasciatus (Say) collected from Bangkok, Thailand Sonthaya Tiawsirisup, Jirinnut Sirijutalak, Mananya Sondang, Manunya Supol, Nut Ansusinha, Ranida Tuanudom, Channarong Rodkhum Plasmid profile of Enterococcus faecium and Enterococcus faecalis isolated from pigs, pork and humans in Thai-Laos border provinces Wink Phyo Thu, Nuananong Sinwat, Rungtip Chuanchuen Oxidative stress and antioxidant in canine cutaneous mast cell tumors Arayaporn Macotpet, Ekkachai Pattarapanwichien, Fanan Suksawat, Patcharee Boonsiri Impact of feed characteristics for monitoring AFB1 contamination in commercial concentrate feeds used in small holder dairy farms, Chiangmai, Thailand Wasana Chaisri, Wantanwa Mongkon, Yoshigo Sugita-Konishi, Witaya Suriyasathaporn Comparative effect of DL-methionine and DL-methionine hydroxy analogue supplemented diet on productive performance, fat accumulation and lipid profile in blood of meat-type ducks Chanwit Kaewtapee, Chaiyapoom Bunchasak Survival time and prognosis factors in hypertrophic cardiomyopathy cats with congestive heart failure Lalida Tantisuwat, Panduangjai Puangampai, Pornchanok Panpakdee, Thitinan Tangarsasilp, Sirilak Disatian Surachetpong Emergence of a classical variant of porcine epidemic diarrhea virus novel to Thailand responsible for the milder clinical disease in a herd previously infected with a pandemic variant Gun Temeeyasen, Anchalee Srijangwad, Thitima Tripipat, Angkana Tantituvanont, Dachrit Nilubol

Effectiveness of sentinel rodents for surveillance of exposure to undocumented bacterial pathogens in 671 animal research facility 681 Shih-Keng Loong, Haryanti-Azura Mohammad-Wali, Nurul-Asma-Anati Che-Mat-Seri, 689 Nur-Hidayana Mahfodz, Dzuzaini Mohd-Ghazali, Pooi-Fong Wong, Sazaly AbuBakar 699 Short Communication Effects of supplementing varying levels of 1,3-diacylglycerol (DAG) on growth performance, apparent nutrient digestibility and selected fecal microbial populations in growing pigs Wen Chao Liu, Kwan Sik Yun, In Ho Kim Comparative ultrasonographic and computed tomographic images of the adrenal glands of healthy cats Panrawee Phoomvuthisarn, Kiatpichet Komin, Nan Choisunirachon Case Report Surgical treatment for tubular colonic duplication communicating to retroperitoneal space in an adult dog Nithida Boonwittaya, Piyathip Choochalermporn, Sunee Kunakornsawat, Teerapol Sirinarumitr Diagnostic Forum 707 709 ECG Quiz 711 Chollada Buranakarl, Saikaew Sutayatram Ophthalmology Snapshot Nalinee Tuntivanich What is Your Diagnosis? Ninlawan Thammasiri, Nan Choisunirachon

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Review Article Review of the cardiovascular toxicity of amitriptyline treatment for canine neuropathic pain Saikaew Sutayatram1* Kumpanart Soontornvipart2 Piyasiri Glangosol3 Abstract Nowadays, the lifespan of pets has been increased by improvements in veterinary practice but age-related disorders are also increasing. Several nervous system attenuations are well-known to be the result of aging and/or pathological lesions. In many cases, neuropathic pain (NP) can develop and cause a significant impact on the clinical outcome and quality of life. With the difficulty in identification of the specific cause of NP, most NP management is planned according to clinical manifestations and recommended trial therapy. The drug of choice for trial therapy seems to be amitriptyline, a tricyclic antidepressant with multiple effects on neurotransmitter receptors and ion channels. However, the pharmacological effects of amitriptyline depend on several pathophysiological factors, including the level of drug exposure, drug metabolism capacity, concurrent disease and cellular responsiveness. In addition to its therapeutic effects, amitriptyline also possesses several adverse effects, especially cardiovascular toxicity. However, the information on its toxicity in veterinary clinical practice is very limited. In this article, we review the use of amitriptyline in NP management and its cardiovascular attenuation using both human medicine and veterinary publications. The aim of this review is to highlight the importance of routine cardiovascular monitoring and to provide a list of parameters that are associated with amitriptyline cardiovascular toxicity. Keywords: amitriptyline, canine, cardiovascular toxicity, neuropathic pain 1Department of Veterinary Physiology, Faculty of Veterinary Science, Chulalongkorn University, Henri Dunant Road, Pathumwan, Bangkok 10330, Thailand 2Department of Veterinary Surgery, Faculty of Veterinary Science, Chulalongkorn University, Henri Dunant Road, Pathumwan, Bangkok 10330, Thailand 3The Small Animal Teaching Hospital, Faculty of Veterinary Science, Chulalongkorn University, Henri Dunant Road, Pathumwan, Bangkok 10330, Thailand *Correspondence: [email protected] Thai J Vet Med. 2018. 48(4): 515-528.

516 Sutayatram S. et al. / Thai J Vet Med. 2018. 48(4): 515-528. Introduction sensations are commonly reported as squeezing, pressure, burning, electric shock-like and stabbing Neuropathic pain (NP), a pain sensation due sensations (Truini et al., 2013). Also, NP is often to a somatosensory abnormality, requires correction of exhibited as a chronic pain that can be classified as both the primary cause and deviated nervous activity. hyperesthesia (pain from subthreshold noxious However, evaluation of the nervous activity is not a stimulus), allodynia (pain from non-noxious stimulus), simple procedure, especially at the primary level of a and dysaesthesia (intermittent spontaneous pain) health care unit. Also, the NP pathological processes (Woolf and Mannion, 1999; Cashmore et al., 2009). The usually take time for development and progression. etiology and the pathogenesis of NP has been Therefore, most NP cases manifest as chronic pain and extensively elaborated upon in several studies in both their treatment plans are mainly based upon trial human and veterinary medicine (Mathews, 2008; treatment constructed from medical publications, Garcia-Larrea, 2014; Jiménez-Yedra et al., 2014; Moore, including clinical studies, case reports and guidelines. 2016). However, in veterinary practice, a In human medicine, NP is a frequent clinical comprehensive neurological examination can only be sign in patients with peripheral and central nervous performed in a small fraction of the referral animal system diseases and causes significant distress (Truini hospitals and control clinical studies are also very et al., 2013). The incident rates of NP in human studies limited. Hence, veterinary NP management is largely vary from 1-3% of patients in the acute pain service based upon human medical knowledge. With the (Hayes et al., 2002) to approximately 12% of the growing number of animal patients with NP, trial patients with chronic pain sensation who seek medical therapy plans using several neuromodulating consultation (Pérez et al., 2009). However, in another medications and physical treatments have been study using the Leeds Assessment of Neuropathic increasing. Among those medications, amitriptyline, a Symptoms and Signs questionnaire, NP was detected classic member of the tricyclic antidepressants (TCAs), in 47% of all participants and the NP showed a is usually prescribed as one of the first-line drugs in significant impact on their sleep and daily life (Hans et many somatosensory system disorders (Mathews, al., 2007). As a result, NP has been studied and 2008; Grubb, 2010). Although it is an antidepressant, it reported extensively, especially in patients with stroke, affects and alters several central and peripheral spinal cord disease, multiple sclerosis, peripheral nervous functions, as well as ion channel activities, that neuropathy from diabetic mellitus and chemotherapy are involved in the NP pathophysiology (Song et al., and chronic osteoarthritis (Ohtori et al., 2012). This has 2000; de Leon-Casasola, 2007). Nevertheless, its led to the establishment of several clinical practice versatile actions are also capable of producing several guidelines for NP (Cruccu et al., 2004; Moulin et al., adverse effects, especially in patients at risk of 2014; National Institute for Health and Care Excellence, increased pharmacological responsiveness and active 2017; Western Australian Therapeutic Advisory drug retention. In human medicine and animal toxicity Group, 2017). models, amitriptyline is known to have cardiovascular toxicity in terms of both electrical and mechanical In veterinary practice, a study of pain functions but there is no clinical report on this topic in prevalence and type at a veterinary teaching hospital canine patient. Therefore, we aimed to review the indicated that approximately 8% of dogs and 7% of cats rationale of using amitriptyline for NP management suffered from NP (Muir et al., 2004). Furthermore, in and the cardiovascular toxicity manifestation in both those animals with pain from all types, aging was humans and dogs. We hope that all of this associated with a higher rate of pain. In addition, NP amitriptyline information may be of benefit for was reported to emerge from various etiologies (e.g., veterinary practice. entrapment, trauma, inflammation and degeneration of nervous tissue), problems (e.g., pelvic fracture, Neuropathic pain spinal cord injury, intervertebral disc herniation, discospondylitis, vertebral osteomyelitis, diabetic The somatosensory system conveys neural neuropathy, tumors of the nervous system, information, including proprioception, and the inflammatory bowel disease and pancreatitis), and sensations of touch, heat, pressure and pain from both surgical procedures (e.g., amputation and inguinal internal and external stimuli. Therefore, it plays a hernia repair) (Mathews, 2008). Among these, major role in the body’s perception and response to all intervertebral disc disease (IVDD) is the most common stimuli. However, impairment and abnormal function cause of spinal disease in dogs (Rusbridge, 2015) and at any path of the nervous system can lead to the pathogenesis related with spinal degeneration is somatosensory system disorders, in which perception progressive with aging (Adams and Dolan, 2012). and response to the somatic stimuli are inappropriate. Nowadays, with the higher number of pets and their The symptoms of somatosensory impairment are longer life expectancy due to advanced medical related to body sensations and manifest as management, the chronic pain cases that fit with the neuromuscular signs such as sensory deficits (Kessner NP criteria have been increasing in veterinary clinics. et al., 2016) and NP (Moore, 2016). However, only a few clinical NP cases, for instance, canine peripheral nerve lesion and discospondylitis, According to the International Association for and feline postsurgical pain (O'Hagan, 2006; Cashmore the Study of Pain, NP is “pain initiated or caused by a et al., 2009; Grubb, 2010) have been published in the primary lesion or dysfunction of the nervous system”. veterinary field. This under-recognition in veterinary In other words, NP is an unpleasant sensory sensation studies might be due to the difficulties in the induced by various pain stimuli from a lesion or identification and evaluation of NP (Moore, 2016). disease of the somatosensory nerve ending. These pain

Sutayatram S. et al. / Thai J Vet Med. 2018. 48(4): 515-528. 517 The NP diagnosis and severity grading in gabapentin, amitriptyline and amantadine, an N- humans require an integration of the information on methyl-D-aspartate (NMDA) receptor antagonist the history of nervous system injury, an absence of (KuKanich, 2013; Jiménez-Yedra et al., 2014; Mathews evidence of ongoing nervous system injury, type of et al., 2014; Moore, 2016). The same medications are pain (e.g., allodynia, hyperalgesia or dysesthesias) and, used in cats except for pregabalin where there is no most importantly, self-reporting by the patients of their pharmacokinetic study or clinical research in cats pain characteristics (burning, stabbing or electric (Baltzer, 2010; KuKanich, 2013; Epstein et al., 2015; shock-like). Unfortunately, pets cannot provide this Clark et al., 2017). For non-pharmacological most crucial part of NP diagnosis, the self-reporting of management, acupuncture is the most common the pain sensation. Therefore, NP assessment in pets modality in pets with chronic pain, while medical employs gathering of information from the history of massage, thermal modification, nutritional the nervous system injury or diseases, history taking supplementation and environmental modification can from the owners about animal behavior patterns and be used as adjunctive pain management (Mathews, pain expressions, especially any chronic pain that 2008; Corti, 2014; Epstein et al., 2015; Moore, 2016). could not be improved by conventional analgesics, such as nonsteroidal anti-inflammatory drugs and In Thailand, a multimodal approach is opioids, and any chronic pain that impaired the pet’s usually recommended for NP management in animals. quality of life, together with the findings obtained from For instance, for dogs with spinal cord injury induced a through clinical examination and specific sensory by either IVDD or motor vehicle accidents that showed testing for allodynia and hyperalgesia by experienced no pain improvement with conventional analgesics, veterinarians. Common behavioral patterns in dogs veterinarians may also prescribe gabapentin and/or and cats with NP reported by the owners are repeated amitriptyline with a course of acupuncture (if scratching, biting and chewing at the specific part of applicable) as a clinical trial for NP management. the body and spontaneous pain expression with gentle Although gabapentin is considered the first choice to petting (Grubb, 2010; Mathews et al., 2014). With try in dogs with NP, if the NP shows no improvement advanced diagnostic tools and improved veterinary with 2-4 weeks of gabapentin treatment, amitriptyline neuromuscular and orthopedic examination protocols, may be added to the NP management. The reasons for such as using electrodiagnostic testing in feline choosing these two drugs are mostly because of their diabetic neuropathy evaluation (Mizisin et al., 2002), availability for veterinary use, low cost and fewer drug NP detection and management are becoming more regulations, compared with other drugs. Moreover, informative. several clinical trials of gabapentin have been done in dogs (Cashmore et al., 2009; Wagner et al., 2010; Management of Neuropathic pain Crociolli et al., 2015). However, with the short half-life (approximately 3-4 hours) of gabapentin in dogs The management of NP aims to both treat the (Vollmer et al., 1986), the need for frequent use (every primary cause of the somatosensory system disorders 8 hours) may limit the application and might be the (if applicable) and to alleviate NP with a specific cause of mixed outcomes in clinical trials that prescribe treatment depending on the suspected mechanism. gabapentin only twice a day. Even though Hence, NP management, especially in the early state, is amitriptyline has no experimental studies or controlled a multimodal approach that combines several methods clinical trials on its analgesic ability in dogs, it is and medications to achieve the maximum pain control. recommended by several veterinary publications Later on, when the severity of the NP is reduced, NP (Mathews, 2008; KuKanich, 2013; Moore, 2016). management can be tapered down and fewer Furthermore, there has been a case report in two dogs combinations of medications or, rarely, a single with NP that showed clinical improvement with medication may be able to maintain the analgesic amitriptyline within the first week of treatment effect. In general, the types of NP management can be (Cashmore et al., 2009). classified into pharmacological and non- pharmacological managements. Amitriptyline Typically, pharmacological management of Amitriptyline, a classic member of the TCAs, NP in veterinary medicine applies knowledge from possesses diverse neuromodulatory actions resulting human clinical trials, meta-analysis studies and in antidepressant and analgesic effects. Its guidelines. Most of the publications recommend TCAs antidepressant action is caused by the blocking of brain (e.g., amitriptyline, nortriptyline and imipramine), serotonin and noradrenaline re-uptake (Bendtsen et al., serotonin/noradrenaline reuptake inhibitors (e.g., 1996) as well as the serotonin receptor function (Fuxe duloxetine and venlafaxine) and alpha2-delta subunit et al., 1977). Its analgesic effect results from numerous of voltage-gated calcium channel modulators (e.g., mechanisms, including reduction in the brain pregabalin and gabapentin) over other medications, serotonin and noradrenaline reuptake at the including opioids (e.g., tramadol, buprenorphine, presynaptic level, suppression of ion channel activities oxycodone, morphine and methadone) and topical (e.g., tetrodotoxin-sensitive and tetrodotoxin-resistant medications (e.g., alpha2-adrenergic agonist, sodium channels, and voltage-gated potassium and amantadine and lidocaine) (de Leon-Casasola, 2007; calcium channels) (Song et al., 2000; de Leon-Casasola, Finnerup et al., 2015). On the other hand, in animals, 2007), inhibition of the NMDA receptor function there has been no controlled study for NP treatment. (Watanabe et al., 1993), and augmentation of the However, commonly recommended medications for gamma-aminobutyric acid (GABA) receptor activity chronic pain induced by NP in dogs are pregabalin, (Dharmshaktu et al., 2012). Another analgesic

5186 Sutayatram S. et al. / Thai J Vet Med. 2018. 48(4): 515-528. mechanism of amitriptyline is related to its anti- improvement in pets varies from as fast as 48 hours to inflammatory effects (Rafiee et al., 2017). Thus, approximately 2-4 weeks after starting the medication amitriptyline and other TCAs are considered as the (Mathews, 2008). In responsive cases, amitriptyline can first-line medication for human patients with NP, gradually attenuate pain over time. For instance, a case especially in cases of stroke, spinal cord injury and report in three dogs with NP showed that two dogs multiple sclerosis (Dharmshaktu et al., 2012; Moulin et responded well with amitriptyline at 1.1-1.3 mg/kg al., 2014; Deng et al., 2016). Likewise, amitriptyline has orally twice daily within the first week, whereas the been suggested for treating canine and feline NP third dog given amitriptyline at 1.4 mg/kg orally twice associated with several causes (Mathews, 2008; daily for two weeks, had the side effect of mental KuKanich, 2013; Moore, 2016). A trial course of alteration without any pain improvement (Cashmore amitriptyline prescription is also strongly suggested et al., 2009). For discontinuation, amitriptyline should for pets suffering from NP induced by diabetic be slowly tapered down (Boothe, 2011; Plumb, 2018). neuropathy, inflammatory bowel disease and feline interstitial cystitis (Mathews, 2008). Importantly, TCAs treatment is not always safe. In addition to their therapeutic effects, numerous Pharmacokinetic studies of orally side effects of amitriptyline have been reported and administered amitriptyline in dogs reveal that range from mild reactions to a lethal problem. Mild amitriptyline exhibits a peak plasma concentration at adverse effects include developing mental alteration, 0.5-2 hours after ingestion with a short half-life of 5-6 gastrointestinal upset, anticholinergic sign, alopecia hours (Boeck and Jørgensen, 1980; Kukes et al., 2009; and allergic skin reaction (Kerr et al., 2001). The serious Norkus et al., 2015). Amitriptyline has a higher cardiovascular problems associated with amitriptyline bioavailability in fasted dogs than that in fed dogs (91% treatment in humans include myocardial infarction, and 69%, respectively) suggesting that food in the GI stroke, arrhythmias, hypotension or hypertension and tract might affect the drug’s absorption (Norkus et al., tachycardia, according to the U.S. Food and Drug 2015). In the blood circulation, the lipophilic TCAs are Administration (FDA) prescribing information. All of bound to the plasma protein and distributed into most these side effects can develop when administered body tissues and maternal milk (Wismer, 2000; Plumb, within or above the therapeutic doses, although lethal 2018). The hepatic metabolism of the drug is very reactions are more frequently found in overdose cases. important for amitriptyline elimination as 95% of this In dogs, a TCA overdose can alter mentation, reduce drug is metabolized by hepatic cytochrome P450 gastrointestinal movement and cause several other oxidative enzymes (Thorn, 2017). In dogs, the level of clinical signs such as dyspnea, vocalizing, ataxia, nortriptyline, an active metabolite of amitriptyline that seizure, hyperthermia, hypotension, arrhythmia and also produces pharmacological effects, showed a death (Wismer, 2000; Plumb, 2018). Information on the positive correlation with amitriptyline exposure adverse effects of amitriptyline at therapeutic dosages (Norkus et al., 2015). Nortriptyline showed a longer in dogs is insufficient, where only mental alteration half-life in humans, at approximately 16-38 hours and vomiting have been mentioned (Cashmore et al., (Verbree et al., 2016). This overlapped circulation of 2009; Norkus et al., 2015). However, in experimental amitriptyline and nortriptyline could aggravate their amitriptyline intoxication studies, conscious dogs had pharmacological effects. Besides hepatic function, clinical signs of sedation, vomiting, restlessness and renal clearance plays a major role in inactive- seizure (Boeck and Jørgensen, 1980). Most importantly, metabolite elimination (Rudorfer and Potter, 1999). these dogs developed cardiovascular toxicity that Therefore, hepatic and renal functions, as well as other could lead to their death without proper treatment medications that require the same groups of hepatic (Boeck and Jørgensen, 1980; Nattel et al., 1984; enzymes for their metabolism, heavily affect the Sasyniuk et al., 1986; Yokota et al., 1987; Wismer, 2000). plasma concentration of amitriptyline and its active metabolite. To prevent the side effects and toxicities of Cardiovascular toxicity associated with TCAs, proper titration is crucial. TCAs should be amitriptyline prescribed at a low dose once daily at bedtime and then the dosage can be increased every 3-7 days depending Cardiovascular toxicity is the most prominent on the patient’s tolerance to the therapeutic dose side effect of TCAs found in both therapeutic regimens (Dharmshaktu et al., 2012). and overdose cases, as it significantly impacts the clinical outcome. Most importantly, this cardiovascular Although, there have been no attenuation can cause hemodynamic disruption and pharmacological analysis on the effectiveness of TCA death in severe cases. In fact, this side effect is the major treatment of NP in dogs and cats, the recommended cause of death in TCA toxicity (Williams and Sherter, oral dose of amitriptyline in dogs in recent veterinary 1971). The toxicity can develop in both patients with pharmacology textbook is 3-4 mg/kg every 12 hours and without previous cardiovascular defects. (Plumb, 2018). This dosage is extrapolated from the However, the severity of these problems is dependent pharmacokinetic data in a canine study and other on the TCA plasma concentration and cardiovascular human clinical studies (Kukes et al., 2009), and is status (Glassman et al., 1988). In patients who higher than the previously recommended dose of 1-2 experienced cardiovascular side effects, plasma mg/kg once or twice daily (Mathews, 2008; KuKanich, concentrations of TCAs could be as low as 50 ng/ml, 2013). In cats, the recommended oral dose is 0.5-1 which is lower than the optimal concentration (150-300 mg/kg or 2.5-12.5 mg per cat, once daily at night ng/ml) for the therapeutic effect (Simpson et al., 1983). (Mathews, 2008; Baltzer, 2010; Plumb, 2018). However, Thus, the use of TCAs has raised safety concerns over amitriptyline is considered as an extra-label drug for the past few decades (Yekehtaz et al., 2013). Several both dogs and cats (Plumb, 2018). The onset of clinical

Sutayatram S. et al. / Thai J Vet Med. 2018. 48(4): 515-528. 551197 physicians and medical associations have suggested various origins. Sinus tachycardia is the most common that a more intensive monitoring should be mandatory cardiovascular side effect in TCAs toxicity cases, while in all patients, especially for geriatric, patients with ventricular dysrhythmias (e.g., ventricular premature underlying cardiovascular problems such as contraction (VPC), idioventricular rhythm, ventricular myocardial ischemia, conduction disturbance, tachycardia and ventricular fibrillation), as well as ventricular arrhythmia and atrial fibrillation, as well as other ECG abnormalities, including supraventricular patients receiving a high dose of TCAs (Burrows et al., tachycardia, bradycardia, sinus arrest and pulseless 1976; Burgess et al., 1979; Glassman et al., 1993; Kerr et electrical activity are less prevalent (Thanacoody and al., 2001). As electrocardiogram (ECG) alterations in Thomas, 2005). The combination of an anticholinergic most of the cardiotoxicity cases associated with TCAs effect and a hyper-adrenergic state induced by the could be detected before the onset of clinical signs inhibition of norepinephrine (NE) reuptake seems to be (Açikalin et al., 2010), ECG monitoring has become a the primary cause of sinus tachycardia associated with more essential tool than monitoring the serum drug amitriptyline in dogs, and this augmentation in the concentration for risk stratification of the toxicity of heart rate has been positively correlated with the dose TCAs in medical practice (Verbree et al., 2016). or plasma concentration of amitriptyline (Boeck and Jørgensen, 1980; Ikeda et al., 1996). The hyper- Likewise, association between TCAs and adrenergic state is suspected to be a major mechanism cardiovascular status has been found in animals. For for the increased sinus rate, as concomitant instance, in dog studies, the administration of a high administration of propranolol, a beta-adrenergic dose of amitriptyline has caused serious blocker, can prevent the elevation of heart rate induced cardiovascular problems that were positively by amitriptyline in anesthetized post-myocardial correlated with the amitriptyline plasma levels. infarction dogs (Ikeda et al., 1996). In humans, Moreover, amitriptyline showed a more pronounced amitriptyline also has a tendency to increase the heart effect on infarcted myocardium than normal rate, especially in patients that have a lower heart rate myocardium (Boeck and Jørgensen, 1980; Nishimoto et before receiving medication (Glassman and Bigger, al., 1990). Interestingly, a single oral amitriptyline 1981). This augmentation in the heart rate could be a administration at approximately 8 mg/kg could subclinical finding and the increased heart rate was produce a plasma concentration range from 22 to 127 approximately 16 beats per min in the chronic ng/ml (Norkus et al., 2015), while chronic moderate dosage (Ziegler and Biggs, 1977). amitriptyline administration (0.7-3.3 mg/kg twice daily) for canine behavioral problems resulted in a For ventricular dysrhythmias, the ventricular serum concentration from less than 20 to 350 ng/ml tachyarrhythmia induced by amitriptyline in dogs (Reich et al., 2000). Therefore, canine patients receiving could result from an increased triggered activity rather recommended amitriptyline dosage should be than an enhanced automaticity (Nattel, 1985). In a monitored for cardiovascular side effects as well. study on rabbits, the inhibition of presynaptic NE Cardiovascular monitoring, at least heart rate and reuptake, which could enhance the myocardial rhythm, is also recommended during amitriptyline catecholamine sensitivity, was found to play a major treatment in veterinary practice (Papich, 2011). role in the ventricular dysrhythmia induced by TCAs, including amitriptyline (Barth et al., 1975). These As mentioned above, amitriptyline as well as ectopic impulse generation abnormalities can also other TCAs have central and peripheral originate from the infarcted area. As can be seen in the neuromodulatory and multi-ion channel blocking myocardial infarction dog model, amitriptyline effects that can affect not only neuronal alteration but increased the inducible ventricular arrhythmia events also cardiovascular function. In severe cases, the in these dogs (Nishimoto et al., 1990). In addition, the cardiovascular toxicity that results from TCAs can cardiac sodium and potassium channel blockades of cause serious cardiac electrical alterations and TCAs, including amitriptyline and nortriptyline, can cardiovascular mechanical disturbances. prolong the cardiac action potential duration and refractory period resulting in an increased risk of Electrocardiogram alterations: Abnormal cardiac dysrhythmias from ectopic foci, and most importantly electrical properties induced by TCAs, especially Torsade de pointes, a lethal polymorphic ventricular during an overdose, are often present as sinus tachycardia associated with QT interval prolongation tachycardia, ventricular tachycardia, ventricular (Thanacoody and Thomas, 2005; Verbree et al., 2016). fibrillation, Torsade de pointes, bradyarrhythmia and There are several other risk-amplifying factors of conductive block (Abeyaratne et al., 2016). From all of Torsade de pointes that should be considered, such as the TCAs actions, catecholamine reabsorption age-related drug clearance attenuations, high plasma inhibition at the presynaptic adrenergic nerve ending drug concentrations, other confounding medications, of the heart, as well as the anticholinergic and sodium electrolyte disturbances, mainly hypokalemia and channel blockade effects on cardiac tissue are well- hypomagnesemia, as well as coexisting cardiac known causes of cardiotoxicity. These TCAs effects problems (Wenzel-Seifert et al., 2011). Interestingly, the alter cardiac electrical functions both the impulse medication dosage was not considered to be a sensitive generation and the conduction property leading to a predictor for cardiotoxicity, especially in geriatrics wide variety of cardiac electrical disturbances and whose plasma drug level relied greatly on other clinical hemodynamic manifestations. pharmacokinetic factors (Hefner et al., 2018). However, several researchers have suggested that TCAs induce In terms of cardiac impulse generation, TCAs an antiarrhythmic effect due to their inhibition of the can augment both enhanced automaticity and inward sodium current, the same mechanism as class I triggered activity. Accordingly, TCAs affect both pacemaker and ectopic foci leading to arrhythmia from

52106 Sutayatram S. et al. / Thai J Vet Med. 2018. 48(4): 515-528. antiarrhythmic drugs, and some TCAs, including dose. Amitriptyline also increased the dispersion of the imipramine, were found to be safe for use in patients ventricular refractoriness measured from the effective with ventricular ectopic beats (Glassman and Bigger, refractory period in post-myocardial infarction canine 1981; Glassman et al., 1993). Bradyarrhythmias have models but a combination administration with been reported rarely (Brackenridge et al., 1968; Lebre et propanol could not alter this period (Ikeda et al., 1996). al., 1995) and the slower heart rate was most likely to These findings indicated that direct cardiac result from the delayed conduction effects, especially suppression by the “quinidine-like” effect was the at the atrioventricular (AV) node (Thanacoody and principal mechanism for some types of the ventricular Thomas, 2005). arrhythmias, as the delay in the ventricular conduction and the prolongation of the refractory period can Besides dysrhythmia, the conductive block is significantly increase the risk of ventricular reentry also a hallmark of the cardiac toxicity of TCAs, arrhythmia. Nevertheless, most of the mild to especially in overdose patients. Various degrees of moderate amitriptyline cardiovascular effects were cardiac electrical conduction alterations in both the reversible within a few days in dogs (Boeck and myocardium and the cardiac conductive pathway can Jørgensen, 1980; Xin and Liu, 1986). be induced by TCAs at the level of the AV nodal and the ventricular conductions. This conductive side Although there has been no clinical report on effect, which mimics class I antiarrhythmics, is these conduction disturbances in canine patients described as a “quinidine-like” effect that can slow the receiving TCAs at a therapeutic dosage. A study in distal conduction system, such as His-Purkinje canines with behavioral problems that required conduction, rather than AV nodal conduction. In chronic treatment of TCAs revealed that only one dog patients using TCAs, a right bundle branch block from a total of 39 dogs had RBBB that might be (RBBB) pattern, as detected by ECG, and a reduced associated with medication (Reich et al., 2000). Last conduction velocity of the bundle of His, the bundle year, we also examined a dog with IVDD induced NP branches, and Purkinje fibers, as measured from His that was treated with oral amitriptyline at 0.3 mg/kg bundle electrocardiography, have been reported twice daily who exhibited RBBB (figure 1) and (Burrows et al., 1976). The prolongation of ventricular hypertension after 3 days of amitriptyline treatment. conduction in some cases can produce ECG alterations The cardiovascular disturbances in this dog were not from a normal sinus wave form to a left bundle branch seen before amitriptyline administration and the block (LBBB), RBBB and bilateral bundle branch block, cardiac structure was unremarkable after amitriptyline as well as a complete heart block, especially in administration, as evaluated by ECG and amitriptyline overdose cases (Brackenridge et al., 1968; echocardiography. Hence, the potential association Kramarz and Foryś, 2013; Yekehtaz et al., 2013; Li and between RBBB and amitriptyline could not be ignored. Lamichhane, 2017). Another possible explanation for Moreover, this geriatric dog also had an ongoing the attenuation of the cardiac distal conduction is problem of multiple hepatic nodules (approximately myocardial damage, which has also been reported in 1.5-4.1 cm in diameter) with moderate to severe bile cases of amitriptyline and nortriptyline overdose retention and increased liver enzymes that had been (Brackenridge et al., 1968). Moreover, in patients with manifested for years. Therefore, this dog was at risk of a TCA overdose, prolongation of the QRS duration and TCA-induced cardiovascular toxicity even at a low corrected QT interval (QTc) are important risk dosage of amitriptyline, compared with other younger stratifications for the prediction of complications and healthier dogs. (Verbree et al., 2016), especially for amitriptyline, where its serum level had a significant correlation with Other ECG waveform alterations associated QTc prolongation (Hefner et al., 2018). On the other with TCAs have been reported in several cases, hand, prolongation of the PR interval or QRS duration including a shortened QT interval, ST elevation or were strong indications of a supra-TCA therapeutic depression, as well as diphasic, inversed and window (Glassman and Bigger, 1981). alternated T waves (Bolognesi et al., 1997; Enslin and Nikolić, 2005; Abeyaratne et al., 2016; Lubna et al., Likewise, amitriptyline can depress the 2018). In a study in an anesthetized dog model, canine Purkinje fiber depolarization rate (Vmax) via amitriptyline at a plasma concentration of blockade of cardiac sodium channels (Nattel, 1985) and approximately 100-fold more than the therapeutic the dose-dependent conductive block associated with concentration caused a significantly lower QT interval TCAs in animals. A single oral dose of amitriptyline at and QTc than those values before the drug 70 mg/kg in dogs resulted in prolongation of the PR administration (Lubna et al., 2018), which is contrast to and QRS intervals that progressed to RBBB and LBBB the typical long QT syndrome and a torsadogenic within the first few hours after drug administration potential of amitriptyline described in other literature. (Boeck and Jørgensen, 1980). Concordantly, Moreover, the inhibition of the inward sodium current intravenous injection of amitriptyline at 0.3-1 mg/kg in and the ST elevation induced by amitriptyline in anesthetized dogs resulted in a marked prolongation several cases fit with the criteria of Bruganda of the PR and QRS intervals, as well as QTc. Moreover, syndrome. Thus, the suggestion that amitriptyline the dogs developed ventricular fibrillation or cardiac might be able to produce Bruganda syndrome is of arrest at a dose of 10 mg/kg (Yokota et al., 1987). concern. Furthermore, the combination of arrhythmias Nishimoto and colleagues (1990) also found that and conduction disturbances can also develop, as their intravenous injection of amitriptyline at 1 mg/kg mechanisms and target cells do not always overlap. significantly slowed the ventricular conduction in The ventricular conduction block occurs at the normal myocardium, and the ventricular conduction ventricular conductive tissue, while the ventricular of infarcted myocardium was delayed at the higher ectopic beat may be associated with an increased

Sutayatram S. et al. / Thai J Vet Med. 2018. 48(4): 515-528. 52117 trigger activity of the ventricular myocardium. QTc in patients with bundle branch block using a Therefore, prolongation of the QT interval or the modified JT and JTc equation, that takes the QRS bundle branch block pattern can present interval and uses a fixed adjustment value for each simultaneously with the ventricular ectopic beat or gender, rather than the normal measurement of the QT tachycardia in some cases (Brackenridge et al., 1968). interval or QTc, to quantify the risk of serious arrhythmia, especially Torsade de pointes (Yankelson For cardiac electrical monitoring, a routine et al., 2018). Unfortunately, there has not been a study ECG monitoring is not the gold standard for on the JT correction for the bundle branch block in dogs arrhythmia detection. However, sustained and QTc calculated from the ECG of dogs with RBBB abnormality in the heart rate and ECG patterns can or LBBB cannot be used directly to estimate the risk of easily be detected by routine ECG monitoring, and the developing serious arrthymias. Therefore, ECG VPC that could be detected from a 2-minute ECG screening before starting amitriptyline and regular recording showed a high correlation with high ECG monitorings during the course of treatment frequency VPC using 24-hour ECG recordings should be planned in all cases, especially in patients (Evenson et al., 2000). Thus, regular ECG monitoring is with a preexisting cardiovascular problem (Glassman still strongly recommended in all patients receiving and Bigger, 1981). The cardiac electrical toxicity TCAs but more caution should be used when induced by TCAs in dogs requires further cardiac interpreting the QTc from cases that have a bundle evaluation tools together with clinical manifestation to branch block pattern, as this ECG pattern can reduce improve the risk-stratification and to determine the the accuracy of the QTc measurement. Lately, severity of the problem. researchers have tried to evaluate the QT interval and Figure 1 Lead II ECG trace obtained from a geriatric dog after 3 days of amitriptyline treatment showing the sinus rhythm with short R wave, deep and broad S wave, and slightly prolonged QRS complex referred to right bundle branch block (RBBB). Cardiac mechanical attenuations: Myocardial application, most of the animal studies used TCAs at depressions due to the toxicity of TCAs can be very high doses and so the findings may more expressed as a conductive defect and mechanical accurately relate to the overdose cases. Whereas, TCAs dysfunction. Several mechanisms are involved in the are administrated in therapeutic regimens, the direct negative inotropic effect, such as outward myocardial dysfunction may not develop into clinical potassium current augmentation, as well as inward impairment in healthy subjects. sodium and calcium current inhibition via multichannel blockade effects of TCAs (Casis and At a therapeutic dosage, the effect of Sánchez-Chapula, 1998; Minoura et al., 2012). The amitriptyline on the left ventricular performance was reduction in myocardial contractility, evaluated from inconclusive. Most of the early phase studies evaluated the maximal rate of rise of left ventricular pressure the patients’ myocardial contractility using an indirect (LVdP/dtmax), has been shown in many animal cardiac index from the seismocardiogram and ECG. The parameters from this method are the systolic time models, including dogs, and amitriptyline affected the intervals (STI), e.g. left ventricular ejection time contraction in both healthy and infarcted myocardium (LVET), the aortic pre-ejection period (PEP), (Nishimoto et al., 1990; Lubna et al., 2018). This PEP/LVET ratio, and the electromechanical systole cardiotoxicity was exhibited in a dose-dependent (QS2). The STI measurement details have been manner. At a therapeutic plasma level, amitriptyline elaborated on thoroughly in the study by Reant and did not alter either ECG or mechanical cardiac colleagues (2010). In healthy volunteers, amitriptyline performance, while it increased the LVdP/dtmax at a could augment the ventricular contractility measured from seismocardiogram and ECG, or depress the level of 10-fold higher than the therapeutic plasma ventricular systolic function depicted by the PEP level. The ventricular mechanical function was prolongation (Kopera et al., 1980; Warrington et al., significantly depressed in the anesthetized dog model 1989). In clinical studies, amitriptyline produced some at a 100-fold higher level than the therapeutic plasma mild ventricular performance attenuations, as level (Lubna et al., 2018). However, in these dogs, the determined by the STI alterations, including the cardiac output relied more on the heart rate than the prolongation of the PEP, increased PEP index and ventricular contraction suggesting that this PEP/LVET ratio, as well as by 2D-echocardiographic contractility depression was not lethal. For clinical

522 Sutayatram S. et al. / Thai J Vet Med. 2018. 48(4): 515-528. ventricular performance attenuation, in terms of the In contrast, TCA-mediated cardiovascular reduction in the ejection fraction (Burgess et al., 1979; toxicity can also manifest as hypertension. This side Galetta et al., 1993). effect has been presented in several clinical reports, such as in patients with panic disorders (Louie et al., Besides subclinical contractile dysfunction, 1992), depression (Hessov, 1971) and amitriptyline TCAs, especially amitriptyline and clomipramine, poisoning (Brackenridge et al., 1968). This have raised concerns as drug-induced hypertensive effect could result from autonomic cardiomyopathies due to their cardiotoxicity and imbalance at the cardiac level leading to increased potential associations with clinical cardiovascular cardiac output and by increased NE function at the outcomes (Feenstra et al., 1999; Montastruc et al., 2010). alpha1-adrenergic receptor of the vessel resulting in The mild ventricular dysfunctions in those patients are augmentation of vasoconstriction and total peripheral more noticeable under cardiac stress, including the resistance (Licht et al., 2009). exercise stress test and cardiovascular comorbidity. In the case of a severe ventricular conductive block, In dogs, the hemodynamic alterations dyssynergic ventricular septal motion can be found in associated with TCAs are also inconclusive and the the echocardiogram (Li and Lamichhane, 2017). information is solely provided by acute toxicity Therefore, preexisting cardiovascular problems and studies. Oral administration of amitriptyline at toxic other coincident factors can superimpose the levels showed mixed results in terms of the blood amitriptyline outcome in terms of ventricular pressure in laboratory dogs, where stable, markedly performance. Unlike humans, there is no available reduced or increased blood pressure were presented controlled clinical study in canine patients on the by different dogs in the same study setting (Boeck and effects of amitriptyline on left ventricular performance. Jørgensen, 1980). However, none of these dogs exhibited any serious hemodynamic disturbance that Although the STI showed a high feasibility required treatment. On the other hand, in another dog and correlation with the left ventricular ejection study, the intravascular administration of fraction in clinical practice, other important factors, amitriptyline at a high dose resulted in a marked such as left ventricular preload and afterload, as well reduction in systolic blood pressure followed by a as the cardiac electrical status significantly impact slight increase towards baseline value (Yokota et al., upon the STI values (Reant et al., 2010). With the 1987). Although, the average mean arterial blood various cardiac electrical effects of amitriptyline, pressure of each dog was within the physiological including alterations in heart rate and ventricular level, half of the dogs died from cardiac arrest and conduction ability, the evaluation of the ventricular ventricular fibrillation. Moreover, administration of performance using STI should be done with more amitriptyline intravenously at doses to give a plasma caution. Thus, more comprehensive cardiovascular drug concentration of 1-, 10- and 100-fold therapeutic evaluations should be performed in all at-risk patients. doses to anesthetized dogs caused no alteration in their cardiovascular parameters at a low dose, and a Hemodynamic disturbances: Orthostatic hypotension temporary significant reduction in the total vascular is one of the most serious cardiovascular toxicity effects resistance with an elevated heart rate, cardiac output of TCAs, particularly in the elderly, as this postural and left ventricular contractility at a medium dose, hypotension can cause intense physical injuries such as without any significant change in the mean arterial hip fracture, open wounds and head concussion blood pressure (Lubna et al., 2018). However, at a high (Glassman and Bigger, 1981). Among the drugs in the dose, the mean arterial blood pressure was transiently TCA family, orthostatic hypotension is more common decreased together with total vascular resistance, while with imipramine than amitriptyline and nortriptyline. the heart rate, cardiac output and left ventricular In a control study, the administration of amitriptyline contractility were augmented. Nevertheless, no dog at a therapeutic dosage for seven consecutive days developed lethal cardiohemodynamic collapse. These produced a significant increase in the postural results indicate that the cardiac function that hypotension, as measured by the differences in both determines the cardiac output was the controlling the systolic standing blood pressure and the diastolic factor for the cardiohemodynamic outcome in these recumbent blood pressure. However, none of these studies. Hence, blood pressure monitoring alone is healthy participants exhibited signs of neither sufficient for risk stratification nor severity unconsciousness (Kopera, 1978). The exact mechanism evaluation of TCA toxicity. of TCAs-induced orthostatic hypotension has not been clearly elucidated. However, links have been made Amitriptyline cardiovascular toxicity treatment: For between the postural hypotension and the increased all cases of TCA poisoning by acute ingestion, peripheral vasodilatation due to alpha1-adrenergic elimination of further gastrointestinal absorption is the receptor blockade, the negative inotropic effect, as well first step that should be performed within the first hour as life-threatening arrthymias. These effects can also of medication exposure. In dogs, inducing emesis explain the systemic hypotension in the patients with should be performed only in the absence of clinical TCA intoxication, in which systemic hypotension is a signs, as emesis can cause aspiration in animals with a common hemodynamic alteration (Brackenridge et al., low level of consciousness. Rather, repeated oral 1968; Kerr et al., 2001; Verbree et al., 2016). This administration of activated charcoal together with a hypotensive effect can be intensified by other cathartic to reduce the gastrointestinal absorption of concurrent problems, including dehydration, systolic the TCAs and increase the drug elimination is dysfunction and other medications that suppress recommended (Wismer, 2000). Since TCAs can cardiovascular functions. suppress the gastrointestinal motility leading to

Sutayatram S. et al. / Thai J Vet Med. 2018. 48(4): 515-528. 523 increased absorption time, a magnesium salt cathartic aggravate the “quinidine-like” effect and prolong the should not be used. In humans, gastric lavage is repolarization effect of TCAs (Burrows et al., 1976; another treatment option for gastric decontamination Sasyniuk et al., 1986; Kerr et al., 2001). Instead, class II but it has shown inconclusive results in reducing the antiarrhythmics, including propranolol, would be gastrointestinal drug absorption (Kerr et al., 2001; more beneficial considering the catecholamine Body et al., 2011). However, airway intubation with reuptake inhibition effect of TCAs (Brackenridge et al., oxygenated air is highly recommended for all 1968). However, these beta-adrenergic blockers have unconscious patients to prevent iatrogenic aspiration the potential to depress myocardial contractility, and and further hypoxia. so they may further decrease the cardiac output and blood pressure (Kerr et al., 2001). Most importantly, For the initial clinical assessment at least a sufficient cardiopulmonary resuscitation time with physical examination, ECG, blood pressure and blood close monitoring are very important in TCA toxicity gas monitoring should be used. Intravenous fluid cases, as several cardiac arrest patients survived after therapy is recommended to treat TCA-induced 3-5 hours of external cardiac massage and ventilator hypotension. However, in hypotensive cases that are (Southall and Kilpatrick, 1974; Abeyaratne et al., 2016). not responsive to fluid therapy, administration of adrenergic agonists can be used for positive inotrope Other side effects of TCAs, such as metabolic and vasopressor effects. Epinephrine and NE have and electrolyte disturbances, especially magnesium been found to be more effective in treatment of TCA- and potassium, should also be corrected. Magnesium induced hypotension than dopamine, because the sulfate administration for myocardial stabilization and dopamine efficacy can be depressed by the fact that early after-depolarization suppression is suggested in TCAs can cause NE depletion at the presynaptic nerve humans (Kan et al., 2014). Correction of hypokalemia terminal (Kerr et al., 2001; Body et al., 2011). is also crucial, especially in patients with prolonged QT intervals. The central nervous system side effects of In more complicated cases including patients TCAs usually present as self-limiting seizures. with cardiovascular toxicity (i.e. QRS prolongation, However, in patients with recurrent seizures, dysrhythmias or hypotension) or acidosis, in which benzodiazepines and barbiturates are drugs of choice their blood gas can be closely monitored, intravenous (Kerr et al., 2001; Verbree et al., 2016). administration of sodium bicarbonate is a standard therapy in humans (Body et al., 2011). For canine Conclusion patients, administration of sodium bicarbonate at 2-3 mEq/kg is recommended as well (Wismer, 2000). In conclusion, amitriptyline is relatively Sodium bicarbonate treatment has shown several effective in NP management but it can produce clinical benefits in cases of TCA poisoning (Verbree et al., side effects, including mental alteration and various 2016). First of all, it can increase the sodium gradient cardiovascular disturbances, even at a low dose when across the sodium channels leading to alleviation of the prescribed to high risk patients. Therefore, using negative effects of TCAs on the sodium channel amitriptyline in at-risk cases, most importantly those function and action potential generation (Sasyniuk et that are aging or with cardiovascular comorbidity or al., 1986). In addition, sodium bicarbonate can buffer liver function deficiency, requires thorough physical the acid, and so the plasma pH is maintained at a mild examination throughout the treatment, while alkalosis range. The alkalosis serves two main cardiovascular function evaluation, including ECG purposes of (i) to facilitate the TCA dissociation from and blood pressure monitors, should also be routinely the cardiac sodium channel to reduce the performed to evaluate any acute adverse effect, and to arrhythmogenic effect and (ii) to increase the TCA predict the physiological alteration induced by chronic binding ability to plasma proteins and decrease the TCAs treatment. availability of the pharmacologically active form. The preferred plasma pH in the treatment of TCA toxicity Acknowledgements is 7.45-7.55 in humans and at least 7.50 in dogs (Nattel et al., 1984, Body et al., 2011). Therefore, the We would like to thank the Small Animal alkalinisation effect of sodium bicarbonate Teaching Hospital, Faculty of Veterinary Science, administration is more effective than only increasing Chulalongkorn University, for providing clinical case plasma sodium level with saline fluid (Kan et al., 2014). information and the Research Clinic Unit, Office of In the amitriptyline intoxicated dog model, sodium Research Affairs, Chulalongkorn University, for bicarbonate could reverse the ventricular tachycardia English language editing. to a normal sinus rhythm and attenuate the hypotensive effect of amitriptyline within the References therapeutic duration that correlated with the plasma pH (Sasyniuk et al., 1986). However, the complications Abeyaratne DDK, Liyanapathirana C, Gamage A, associated with the sodium bicarbonate induced severe acid-base disturbance are serious, including Karunarathne P, Botheju M and Indrakumar J dysrhythmias. 2016. Survival after severe amitriptyline Although cardiovascular toxicity generally can be improved by correction of the hypotension and poisoning with prolonged ventricular acidosis, patients with serious arrhythmias may require administration of an antiarrhythmic drug. For tachycardia and cardiac arrest. BMC Res Notes. 9: tachyarrhythmia with QRS prolongation, class I and III antiarrhythmics should be avoided as they can 167. https://doi.org/10.1186/s13104-016-1963-0. Açikalin A, Satar S, Avc A, Topal M, Kuvandk G and Sebe A 2010. QTc intervals in drug poisoning patients with tricyclic antidepressants and

5242 Sutayatram S. et al. / Thai J Vet Med. 2018. 48(4): 515-528. selective serotonin reuptake inhibitors. Am J Clark L, Doyle RS and Shroeder S 2017. Skin lesions Ther. 17(1): 30-33. following pregabalin administration in a cat. Vet Adams MA and Dolan P 2012. Intervertebral disc Anaesth Analg. 44(2): 383-385. degeneration: evidence for two distinct phenotypes. J Anat. 221(6): 497-506. Corti L 2014. Nonpharmaceutical approaches to pain Baltzer W 2010. “Pain Management in Cats.” [Online]. management. Top Companion Anim Med. 29(1): Available: https://www.cliniciansbrief.com/ 24-28. article/pain-management-cats. Accessed March 29, 2018. Crociolli GC, Cassu RN, Barbero RC, Rocha TLA, Barth N, Manns M and Muscholl E 1975. Arrhythmias Gomes DR and Nicácio GM 2015. Gabapentin as and inhibition of noradrenalin uptake caused by an adjuvant for postoperative pain management tricyclic antidepressants and chlorpromazine on in dogs undergoing mastectomy. J Vet Med Sci. the isolated perfused rabbit heart. Naunyn 77(8): 1011-1015. Schmiedebergs Arch Pharmacol. 288(2-3): 215- 231. Cruccu G, Anand P, Attal N, Garcia-Larrea L, Haanpää Bendtsen L, Jensen R and Olesen J 1996. Amitriptyline, M, Jørum E, Serra J and Jensen TS 2004. EFNS a combined serotonin and noradrenaline re- guidelines on neuropathic pain assessment. Eur J uptake inhibitor, reduces exteroceptive Neurol. 11(3): 153-162. suppression of temporal muscle activity in patients with chronic tension-type headache. de Leon-Casasola OA 2007. Multimodal approaches to Electroencephalogr Clin Neurophysiol. 101(5): the management of neuropathic pain: the role of 418-422. topical analgesia. J Pain Symptom Manage. 33(3): Body R, Bartram T, Azam F and Mackway-Jones K 356-364. 2011. Guidelines in Emergency Medicine Network (GEMNet): guideline for the Deng Y, Luo L, Hu Y, Fang K and Liu J 2016. Clinical management of tricyclic antidepressant overdose. practice guidelines for the management of Emerg Med J. 28(4): 347-368. neuropathic pain: a systematic review. BMC Boeck V and Jørgensen A 1980. Electrocardiographic Anesthesiol. 18(16): 12. https://doi.org/ and cardiovascular changes in cats and dogs 10.1186/s12871-015-0150-5. caused by high doses of amitriptyline given as conventional tablets or a sustained release Dharmshaktu P, Tayal V and Kalra BS 2012. Efficacy of preparation. Acta Pharmacol Toxicol (Copenh). antidepressants as analgesics: a review. J Clin 46(3): 161-170. Pharmacol. 52(1): 6-17. Bolognesi R, Tsialtas D, Vasini P, Conti M and Manca C 1997. Abnormal ventricular repolarization Drugs.com 2018 “Amitriptyline: the Food and Drug mimicking myocardial infarction after Administration (FDA) prescribing information.” heterocyclic antidepressant overdose. Am J [Online]. Available: https://www.drugs.com/ Cardiol. 79(2): 242-245. pro/amitriptyline.html. Accessed February 18, Boothe DM 2011. Chapter 28: Pain Control: Locally- 2018. and Centrally-Acting Analgesics. In: Small Animal Clinical Pharmacology and Therapeutics Enslin A and Nikolić G 2005. Tricyclic alternans. Heart 2nd ed Missouri: Elsevier Saunders 994-1044. Lung. 34(4): 295-297. Brackenridge RG, Peters TJ and Watson JM 1968. Myocardial damage in amitriptyline and Epstein ME, Rodanm L, Griffenhagen G, Kadrlik J, nortriptyline poisoning. Scott Med J. 13(6): 208- Petty MC, Robertson SA and Simpson W 2015. 210. 2015 AAHA/AAFP pain management guidelines Burgess CD, Montgomery S, Wadsworth J and Turner for dogs and cats. J Feline Med Surg. 17(3): 251- P 1979. Cardiovascular effects of amitriptyline, 272. mianserin, zimelidine and nomifensine in depressed patients. Postgrad Med J. 55(648): 704- Evenson KR, Welch VL, Cascio WE and Simpson RJJr 708. 2000. Validation of a short rhythm strip compared Burrows GD, Vohra J, Hunt D, Sloman JG, Scoggins BA to ambulatory ECG monitoring for ventricular and Davies B 1976. Cardiac effects of different ectopy. J Clin Epidemiol. 53(5): 491-497. tricyclic antidepressant drugs. Br J Psychiatry. 129(4): 335-341. Feenstra J, Grobbee DE, Remme WJ and Stricker BHCh Cashmore RG, Harcourt-Brown TR, Freeman PM, 1999. Drug-induced heart failure. J Am Coll Jeffery ND and Granger N 2009. Clinical Cardiol. 33(5): 1152-1162. diagnosis and treatment of suspected neuropathic pain in three dogs. Aust Vet J. 87(1): Finnerup NB, Attal N, Haroutounian S, McNicol E, 45-50. Baron R, Dworkin RH, Gilron I, Haanpää M, Casis O and Sánchez-Chapula JA 1998. Mechanism of Hansson P, Jensen TS, Kamerman PR, Lund K, block of cardiac transient outward K+ current Moore A, Raja SN, Rice AS, Rowbotham M, Sena (Ito) by antidepressant drugs. J Cardiovasc E, Siddall P, Smith BH and Wallace M 2015. Pharmacol. 32(4): 527-534. Pharmacotherapy for neuropathic pain in adults: a systematic review and meta-analysis. Lancet Neurol. 14(2): 162-173. Fuxe K, Ögren SO, Agnati L, Gustafsson JA and Jonsson G 1977. On the mechanism of action of the antidepressant drugs amitriptyline and nortriptyline. Evidence for 5-hydroxytryptamine receptor blocking activity. Neurosci Lett. 6(4): 339-343. Galetta F, Lunardi M, Cini G, Stella MR and Giusti C 1993. Cardiovascular effects of amitriptyline in therapeutic dosages. Echocardiographic study. Clin Ter. 143(1): 29-34.

Sutayatram S. et al. / Thai J Vet Med. 2018. 48(4): 515-528. 5253 Garcia-Larrea L 2014. Chapter 43: The mianserin--a comparative study with amitriptyline and a placebo in healthy subjects. Pathophysiology of Neuropathic Pain: Critical Int J Clin Pharmacol Ther Toxicol. 18(3): 104-109. Kramarz E and Foryś M 2013. Blok obu odnóg pęczka Review of Models and Mechanisms. In: Pain 2014 Hisa w przebiegu leczenia amitriptyliną: Bilateral bundle branch block during treatment with Refresher Courses: 15th World Congress on Pain amitriptyline. Kardiol Pol. 71(6): 635-637. KuKanich B 2013. Outpatient oral analgesics in dogs SN Raja and CL Sommer (ed) Washington, DC: and cats beyond nonsteroidal antiinflammatory drugs: an evidence-based approach. Vet Clin IASP Press 453-475. North Am Small Anim Pract. 43(5): 1109-1125. Kukes VG, Kondratenko SN, Savelyeva MI, Glassman AH and Bigger JTJr 1981. Cardiovascular Starodubtsev AK and Gneushev ET 2009. Experimental and clinical pharmacokinetics of effects of therapeutic doses of tricyclic amitryptiline: comparative analysis. Bull Exp Biol Med. 147(4): 434-437. antidepressants. A review. Arch Gen Psychiatry. Lebre AT, Prado PP, Yonamine ES, Rasslan Z, Bonadia JC and Lima CA 1995. Severe bradyarrythmia 38(7): 815-820. induced by tricyclic antidepressants in an elderly patient. Rev Assoc Med Bras (1992). 41(4): 271- Glassman AH, Pardell R and Woodring S 1988. 273. Li W and Lamichhane J 2017. From heartbreak to left Cardiovascular effects of the standard tricyclic bundle branch block: a case of amitriptyline overdose. Am J Ther. 24(5): e621-e623. antidepressants. Clin Chem. 34(5): 856-858. https://doi.org/10.1097/MJT.0000000000000614 . Glassman AH, Roose SP and Bigger JTJr 1993. The Licht CMM, de Geus EJC, Seldenrijk A, van Hout HPJ, Zitman FG, van Dyck R and Penninx BWJH 2009. safety of tricyclic antidepressants in cardiac Depression is associated with decreased blood pressure, but antidepressant use increases the patients. Risk-benefit reconsidered. JAMA. risk for hypertension. Hypertension. 53(4): 631- 638. 269(20): 2673-2675. Louie AK, Louie EK and Lannon RA 1992. Systemic hypertension associated with tricyclic Grubb T 2010. Chronic neuropathic pain in veterinary antidepressant treatment in patients with panic disorder. Am J Cardiol. 70(15): 1306-1309. patients. Top Companion Anim Med. 25(1): 45- Lubna NJ, Wada T, Nakamura Y, Chiba K, Cao X, Izumi-Nakaseko H, Ando K, Naito AT, Satoh Y 52. and Sugiyama A 2018. Amitriptyline may have possibility to induce Brugada syndrome rather Hans G, Masquelier E and De Cock P 2007. The than long QT syndrome. Cardiovasc Toxicol. 18(1): 91-98. https://doi.org/10.1007/S12012- diagnosis and management of neuropathic pain 017-9417-Z. Mathews K, Kronen PW, Lascelles D, Nolan A, in daily practice in Belgium: an observational Robertson S, Steagall PVM, Wright B and Yamashita K 2014. Guidelines for recognition, study. BMC Public Health. 7: 170. https:// assessment and treatment of pain. J Small Anim Pract. 55(6): E10-68. https://doi.org/10.1111/ doi.org/10.1186/1471-2458-7-170. jsap.12200. Mathews KA 2008. Neuropathic pain in dogs and cats: Hayes C, Browne S, Lantry G and Burstal R 2002. if only they could tell us if they hurt. Vet Clin North Am Small Anim Pract. 38(6): 1365-1414. Neuropathic pain in the acute pain service: a Minoura Y, Di Diego JM, Barajas-Martínez H, Zygmunt AC, Hu D, Sicouri S and Antzelevitch C prospective survey. Acute Pain. 4(2): 45-48. 2012. Ionic and cellular mechanisms underlying the development of acquired Brugada syndrome Hefner G, Hahn M, Hohner M, Roll SC, Klimke A and in patients treated with antidepressants. J Cardiovasc Electrophysiol. 23(4): 423-432. Hiemke C 2018. QTc time correlates with Mizisin AP, Shelton GD, Burgers ML, Powell HC and Cuddon PA 2002. Neurological complications amitriptyline and venlafaxine serum levels in associated with spontaneously occurring feline diabetes mellitus. J Neuropathol Exp Neurol. elderly psychiatric inpatients. 61(10): 872-884. Montastruc G, Favreliere S, Sommet A, Pathak A, Pharmacopsychiatry. https://doi.org/10.1055/s- Lapeyre-Mestre M, Perault-Pochat MC and 0044-102009. Hessov IB 1971. Hypertension during chlorimipramine therapy. Br Med J. 1(5745): 406. Ikeda Y, Nishimoto M, Shimazu Y, Hashimoto H, Umemura K and Nakashima M 1996. Comparative electrophysiological effects of the antidepressants fluvoxamine and amitriptyline in the canine heart after myocardial infarction. Naunyn Schmiedebergs Arch Pharmacol. 354(1): 30-37. Jiménez-Yedra A, García-Herrera RA, Rivera-Notario M, Ramirez-Hernandez V, Santamaría-Mayo E, Zaragoza-Vera C, Zaragoza-Vera M, Arjona- Jimenez G and Avendaño-Carrillo H 2014. Neuropathic pain in dogs and cats: current evaluation and treatment perspectives: review. Int J Anim Vet Adv. 6(1): 8-14. Kan AA, de Lange DW, Donker DW and Meulenbelt J 2014. Management of prolonged QT interval and torsades de pointes in the intoxicated patient. Neth J Med. 72(3): 119-126. Kerr GW, McGuffie AC and Wilkie S 2001. Tricyclic antidepressant overdose: a review. Emerg Med J. 18: 236-241. Kessner SS, Bingel U and Thomalla G 2016. Somatosensory deficits after stroke: a scoping review. Top Stroke Rehabil. 23(2): 136-146. Kopera H 1978. Anticholinergic and blood pressure effects of mianserin, amitriptyline and placebo. Br J Clin Pharmacol. 5(Suppl 1): 29S-34S. Kopera H, Fluch N, Harpf H, Klein WW and Stulemeijer SM 1980. Cardiovascular effects of

5226 Sutayatram S. et al. / Thai J Vet Med. 2018. 48(4): 515-528. Montastruc JL 2010. Drugs and dilated Spain: a cross-sectional, multicentre, cardiomyopathies: a case/noncase study in the observational study. Clin Drug Investig. 29(7): French PharmacoVigilance Database. Br J Clin 441-450. https://doi.org/10.2165/00044011-2009 Pharmacol. 69(3): 287-294. 29070-00002. Moore SA 2016. Managing neuropathic pain in dogs. Plumb DC 2018. Amitriptyline HCl. In: Plumb's Front Vet Sci. 3: 12. https://doi.org/10.3389/ Veterinary Drug Handbook 9th ed New Jersey: fvets.2016.00012. Wiley-Blackwell 76-81. Moulin DE, Boulanger A, Clark AJ, Clarke H, Dao T, Rafiee L, Hajhashemi V and Javanmard SH 2017. In Finley GA, Furlan A, Gilron I, Gordon A, Morley- vitro and in vivo modulation of LPS and Forster PK, Sessle BJ, Squire P, Stinson J, Taenzer carrageenan-induced expression of inflammatory P, Velly A, Ware MA, Weinberg EL and genes by amitriptyline. J Pharm Pharmacogn Res. Williamson OD 2014. Pharmacological 5(3): 144-155. management of chronic neuropathic pain: revised Reant P, Dijos M, Donal E, Mignot A, Ritter P, consensus statement from the Canadian Pain Bordachar P, Dos Santos P, Leclercq C, Roudaut Society. Pain Res Manag. 19(6): 328-335. R, Habib G and Lafitte S 2010. Systolic time Muir WW, Wiese AJ and Wittum TE 2004. Prevalence intervals as simple echocardiographic parameters and characteristics of pain in dogs and cats of left ventricular systolic performance: examined as outpatients at a veterinary teaching correlation with ejection fraction and hospital. J Am Vet Med Assoc. 224(9): 1459-1463. longitudinal two-dimensional strain. Eur J National Institute for Health and Care Excellence 2017. Echocardiogr. 11(10): 834-844. “Neuropathic pain–pharmacological Reich MR, Ohad DG, Overall KL and Dunham AE management: The pharmacological management 2000. Electrocardiographic assessment of of neuropathic pain in adults in non-specialist antianxiety medication in dogs and correlation settings.” [Online]. Available: http://guidance. with serum drug concentration. J Am Vet Med nice.org.uk/CG173. Accessed February 18, 2018. Assoc. 216(10): 1571-1575. Nattel S 1985. Frequency-dependent effects of Rudorfer MV and Potter WZ 1999. Metabolism of amitriptyline on ventricular conduction and cardiac rhythm in dogs. Circulation. 72(4): 898- tricyclic antidepressants. Cell Mol Neurobiol. 906. 19(3): 373-409. Nattel S, Keable H and Sasyniuk BI 1984. Experimental amitriptyline intoxication: electrophysiologic Rusbridge C 2015. Canine chondrodystrophic manifestations and management. J Cardiovasc Pharmacol. 6(1): 83-89. intervertebral disc disease (Hansen type I disc Nishimoto M, Hashimoto H, Ozaki T, Nagashima S disease). BMC Musculoskelet Disord. 16(Suppl and Nakashima M 1990. Effects of antidepressants on the intraventricular 1): S11. https://doi.org/10.1186/1471-2474-16- conduction and the incidence of arrhythmias induced by programmed ventricular stimulation S1-S11. in the dog heart after myocardial infarction. Naunyn Schmiedebergs Arch Pharmacol. 341(3): Sasyniuk BI, Jhamandas V and Valois M 1986. 240-245. Experimental amitriptyline intoxication: Norkus C, Rankin D and KuKanich B 2015. Evaluation of the pharmacokinetics of oral amitriptyline and treatment of cardiac toxicity with sodium its active metabolite nortriptyline in fed and fasted Greyhound dogs. J Vet Pharmacol Ther. bicarbonate. Ann Emerg Med. 15(9): 1052-1059. 38(6): 619-622. Simpson GM, Pi EH and White K 1983. Plasma drug O'Hagan BJ 2006. Neuropathic pain in a cat post- amputation. Aust Vet J. 84(3): 83-86. levels and clinical response to antidepressants. J Ohtori S, Orita S, Yamashita M, Ishikawa T, Ito T, Clin Psychiatry. 44(5): 27-34. Shigemura T, Nishiyama H, Konno S, Ohta H, Takaso M, Inoue G, Eguchi Y, Ochiai N, Kishida Song JH, Ham SS, Shin YK and Lee CS 2000. S, Kuniyoshi K, Aoki Y, Arai G, Miyagi M, Kamoda H, Suzkuki M, Nakamura J, Furuya T, Amitriptyline modulation of Na+ channels in rat Kubota G, Sakuma Y, Oikawa Y, Suzuki M, Sasho T, Nakagawa K, Toyone T and Takahashi K 2012. dorsal root ganglion neurons. Eur J Pharmacol. Existence of a neuropathic pain component in patients with osteoarthritis of the knee. Yonsei 401(3): 297-305. Med J. 53(4): 801-805. Southall DP and Kilpatrick SM 1974. Imipramine Papich MG 2011. Amitriptyline hydrochloride. In: Saunders Handbook of Veterinary Drugs: Small poisoning: survival of a child after prolonged and Large Animal 3rd ed Missouri: Elsevier Saunders 33-34. cardiac massage. Br Med J. 4(5943): 508. Pérez C, Saldaña MT, Navarro A, Vilardaga I and Rejas Thanacoody HK and Thomas SH 2005. Tricyclic J 2009. Prevalence and characterization of neuropathic pain in a primary-care setting in antidepressant poisoning: cardiovascular toxicity. Toxicol Rev. 24(3): 205-214. Thorn CF 2017. “Amitriptyline and Nortriptyline Pathway, Pharmacokinetics.” [Online]. Available: https://www.pharmgkb.org/ pathway/PA166163647. Accessed March 29, 2018. Truini A, Garcia-Larrea L and Cruccu G 2013. Reappraising neuropathic pain in humans—how symptoms help disclose mechanisms. Nat Rev Neurol. 9(10): 572-582. Verbree FC, Reijnen TMAJ, Mitrov-Winkelmolen L, Overdiek JWPM and Dennesen PJW 2016. Tricyclic antidepressant poisoning: cardiovascular and neurological toxicity. Neth J Crit Care. 24(2): 16-19.

Sutayatram S. et al. / Thai J Vet Med. 2018. 48(4): 515-528. 5273 Vollmer KO, von Hodenberg A and Kölle EU 1986. Pharmacokinetics and metabolism of gabapentin in rat, dog and man. Arzneimittelforschung. 36(5): 830-839. Wagner AE, Mich PM, Uhrig SR and Hellyer PW 2010. Clinical evaluation of perioperative administration of gabapentin as an adjunct for postoperative analgesia in dogs undergoing amputation of a forelimb. J Am Vet Med Assoc. 236(7): 751-756. Warrington SJ, Turner P and Skrumsager BK 1989. Cardiovascular (ECG and systolic time intervals) and anticholinergic effects of repeated doses of femoxetine--a comparison with amitriptyline and placebo in healthy men. Br J Clin Pharmacol. 27(3): 343-351. Watanabe Y, Saito H and Abe K 1993. Tricyclic antidepressants block NMDA receptor-mediated synaptic responses and induction of long-term potentiation in rat hippocampal slices. Neuropharmacology. 32(5): 479-486. Wenzel-Seifert K, Wittmann M and Haen E 2011. QTc prolongation by psychotropic drugs and the risk of Torsade de Pointes. Dtsch Arztebl Int. 108(41): 687-693. Western Australian Therapeutic Advisory Group 2017. “WATAG Advisory Note: Guidelines for the Pharmacological Treatment of Neuropathic Pain (2017).” [Online]. Available: http://www.watag. org.au/watag/docs/Advisor_Note_Neuropathi c_Pain_Guidelines.pdf. Accessed February 18, 2018. Williams RBJr and Sherter C 1971. Cardiac complications of tricyclic antidepressant therapy. Ann Intern Med. 74(3): 395-398. Wismer TA 2000. “Antidepressant drug overdoses in dogs.” [Online]. Available: http://aspcapro.org/ sites/pro/files/e-toxbrief_0700.pdf. Accessed February 18, 2018. Woolf CJ and Mannion RJ 1999. Neuropathic pain: aetiology, symptoms, mechanisms, and management. Lancet. 353(9168): 1959-1964. Xin H and Liu SF 1986. Effects of amitriptyline on ventricular fibrillation threshold, effective refractory period, diastolic threshold and sinus node function in dogs. Acta Pharmacol Sin. 7(6): 556-559. Yankelson L, Hochstadt A, Sadeh B, Pick B, Finkelstein A, Rosso R and Viskin S 2018. New formula for defining \"normal\" and \"prolonged\" QT in patients with bundle branch block. J Electrocardiol. 51(3): 481-486. Yekehtaz H, Farokhnia M and Akhondzadeh S 2013. Cardiovascular considerations in antidepressant therapy: an evidence-based review. J Tehran Heart Cent. 8(4): 169-176. Yokota S, Ishikura Y and Ono H 1987. Cardiovascular effects of paroxetine, a newly developed antidepressant, in anesthetized dogs in comparison with those of imipramine, amitriptyline and clomipramine. Jpn J Pharmacol. 45(3): 335-342. Ziegler VE and Biggs JT 1977. Electrocardiographic findings in patients undergoing amitriptyline treatment. Dis Nerv Syst. 38(9): 697-699.

5282 Sutayatram S. et al. / Thai J Vet Med. 2018. 48(4): 515-528. บทคัดยอ่ ความเปน็ พิษต่อหวั ใจและหลอดเลอื ดของยาอะมทิ ริปไทลนี ในการรักษาอาการปวดเหตปุ ระสาทในสุนัข ทรายแก้ว สัตยธรรม1* กมั ปนาท สนุ ทรวิภาต2 ปิยสริ ิ แกลโกศล3 ในภาวะปัจจุบนั อายุขัยของสตั ว์เลยี้ งยืนยาวขึ้นเนือ่ งจากการพัฒนาของการดแู ลสตั วเ์ ลีย้ งโดยสตั วแพทย์ ทาให้ปญั หาทเ่ี ก่ยี วขอ้ ง กบั ความเสอ่ื มตามอายจุ งึ เพม่ิ ขึ้นตามมา และเปน็ ท่ีร้กู นั วา่ ความผดิ ปกติของระบบประสาทเปน็ ปญั หาทเ่ี กิดไดท้ ั้งจากความเส่ือมตามอายุ และ รอยโรคจากสาเหตุตา่ ง ๆ ซง่ึ ในหลายกรณมี กั จะพบอาการปวดเหตปุ ระสาท (neuropathic pain) ตามมา ซ่ึงอาการน้ีส่งผลกระทบอย่างมาก ตอ่ ผลทางคลนิ ิกและคณุ ภาพชวี ิต แตก่ ารตรวจหาสาเหตุท่จี าเพาะของอาการน้ใี นผู้ปว่ ยแต่ละรายนน้ั ทาได้ยาก จึงทาให้การวางแผนการรักษา มักอาศยั ข้อมูลจากอาการที่ตรวจพบ และผลจากการทดลองรกั ษาทไ่ี ดร้ บั การยอมรบั โดยยาท่ีนิยมนามาใชใ้ นการรักษาอาการนเี้ ปน็ อันดบั แรก ๆ คือ ยาอะมทิ ริปไทลีน (amitriptyline) ท่ีเป็นยาแกซ้ มึ เศร้ากลุ่มไตรไซคลกิ (tricyclic antidepressant) ท่ีมผี ลหลายอย่างตอ่ ตัวรับของ สารสื่อประสาทและชอ่ งไอออนตา่ ง ๆ อยา่ งไรกต็ ามผลของยาตวั นี้ยงั ข้ึนกับหลายปัจจัย เชน่ ปริมาณยาทไี่ ดร้ บั ประสทิ ธิภาพของร่างกายด้าน เมแทบอลซิ ึมของยา โรคต่าง ๆ ทม่ี อี ยู่ และการตอบสนองของเซลล์ต่อยา นอกจากผลทางการรกั ษา ยาอะมทิ รปิ ไทลีนยงั สามารถกอ่ ผลขา้ งเคียงตา่ ง ๆ โดยเฉพาะความเป็นพิษต่อหัวใจและหลอดเลือด อยา่ งไรกต็ ามขอ้ มูลดา้ นความเปน็ พษิ ต่อหวั ใจและหลอดเลือดในทางการ สัตวแพทย์ยังมคี ่อนข้างนอ้ ย คณะผเู้ ขียนจึงได้ทาการรวบรวมและวจิ ารณข์ อ้ มูลเกย่ี วกับการใชย้ าอะมทิ ริปไทลนี เพื่อการรกั ษาอาการปวดเหตุ ประสาท และความเปน็ พษิ ต่อหวั ใจและหลอดเลอื ดทเ่ี กีย่ วข้องจากสิ่งตีพิมพ์ทงั้ ในของมนษุ ย์และสัตว์ไว้ในบทความวชิ าการน้ี โดยหวังว่าข้อมูล เหล่าน้จี ะช่วยเนน้ ถึงความสาคญั ของการตรวจประเมินระบบหวั ใจและหลอดเลือดเมื่อมีการใช้ยาอะมิทรปิ ไทลีน คาสาคัญ: อะมิทรปิ ไทลนี สุนขั ความเป็นพษิ ตอ่ หัวใจและหลอดเลอื ด อาการปวดเหตปุ ระสาท ภาควชิ าสรรี วิทยา1 ภาควชิ าศลั ยศาสตร2์ โรงพยาบาลสตั วเ์ ลก็ 3 คณะสตั วแพทยศาสตร์ จุฬาลงกรณม์ หาวิทยาลัย ปทุมวัน กรงุ เทพ ฯ 10330 *ผ้รู ับผิดชอบบทความ E-mail: [email protected]

Original Article Comparison of isolation techniques for bone marrow derived canine mesenchymal stem cells (MSCs) and the compatibility of MSCs loaded onto polycarpolactone hydroxyapatite Chalika Wangdee1 Sasithorn Panasophonkul5 Napaphat Thadavirul3 Kumpanart Soontornvipart1 Prasit Pavasant2 Pitt Supaphol3 Mongkol Techakumphu4 Theerawat Tharasanit4* Abstract Bone marrow was collected from 7 dogs and submitted to 3 different MSC isolation techniques (direct plating, red blood cell lysis and gradient density). The number of cells, expression of MSC markers and in vitro the osteogenic differentiation obtained from each technique were examined. In order to study in vivo the osteogenic capability of derived MSCs, non-union ulna lesions (n=3) were firstly induced by transplantation of a composite of polycarprolactone/ hydroxyapatite (PCL/HAp) scaffold experimentally on to a critical-size ulna bone defect. The MSCs were injected into the lesions. The non-union sites were examined by radiography, angiography and histology at 2, 4, 6, 8 and 12 weeks after MSC injection. Gradient density and RBC lysis techniques yielded higher numbers of putative MSCs on day 7 of culture compared with the direct plating technique. A large proportion of isolated MSCs, irrespective of the isolation techniques, expressed all MSC markers (CD 44 and CD 90). For all MSC transplanted dogs, neither radiological changes at scaffold-ulna interface nor callus formation was observed, although all donors used demonstrated in vitro osteogenesis. At 16 weeks after MSC injection, the angiogram indicated increased neovascularization. This was confirmed by the histological finding that there was an improvement of vascularization within the thick-fibrous tissue surrounding the scaffold. Gradient density and RBC lysis treatment are suitable MSC isolation techniques, in terms of the numbers of cells obtained and also their MSC properties. However, potential use of these MSCs following injection to a non-union bone site was compromised possibly because of a lack of osteogenic stimulation. Keywords: bone marrow, dogs, mesenchymal stem cells, non-union bone, polycarprolactone/ hydroxyapatite (PCL/HAp) scaffold 1Department of Veterinary Surgery, 4Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Henri-dunant road, Bangkok 10330, Thailand 2Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Henri dunant road, Bangkok 10330, Thailand 3The Petroleum and Petrochemical College, Chulalongkorn University, Phayathai road, Bangkok 10330, Thailand 5Department of Companion Animal and Wildlife Clinic, Faculty of Veterinary Medicine, Chiang Mai University, Mae Hia, Chiang Mai, 50100, Thailand. *Correspondence: [email protected] Thai J Vet Med. 2018. 48(4): 529-539.

530 Wangdee C. et al. / Thai J Vet Med. 2018. 48(4): 529-539. Introduction properties and provides osteoconductive and osteointegrative potential (Di Silvio et al., 2002; Wang, In dogs, incidence of non-union is 3.4% and 2006; Neuendorf et al., 2008). The objectives of this the most common site (40-60%) is the radius and/or study were to compare the effects of isolation ulna (Bartels, 1987). The etiology of non-union involves techniques on the derivation of bone marrow MSCs multiple factors including poor blood supply to the and also to examine the effect of MSC transplantation fracture site, poor apposition of the fracture end, on bone healing of an experimentally ‘induced’ non- fracture motion, large fracture gap, pathologic fracture, union ulna bone defect in dogs. a foreign body, necrotic bone, infection and non- justified corticosteroid therapy ( Vertenten et al., 2010; Materials and Methods Calori et al., 2011). Autologous bone graft is the gold standard for non-union treatment leading to bone All chemicals used in this study were union through osteogenesis, osteoconduction and purchased from Sigma-Aldrich, St. Louis, USA, unless osteoinduction. However, it causes a high risk of otherwise specified. fracture and infection, pain at the donor site and limited cancellous bone (Lee et al., 2009). Collection and isolation of canine bone marrow derived mesenchymal stem cells: This study was Stem cell therapy serves as an interesting tool approved by the Committee for the Ethical Care of for regenerative medicine in human and in veterinary Animals of the Chulalongkorn University (No. medicine (Barry and Murphy, 2004; Ribitsch et al., 0931055). Seven mixed-breed dogs, weighing between 2010). Mesenchymal stem cells (MSCs) are able to self- 10 to 15 kg were enrolled in this study. All dogs were renew and differentiate into bone, cartilage or adipose physically examined and remained healthy tissue (Zuk et al., 2002). MSCs have been used for the throughout the experiment. Routine blood analysis cell-based therapy of bone and soft tissue regeneration was performed preoperatively. Acepromazine 0.02 (Jang et al., 2008; Kraus and Kirker-Head, 2006; mg/kg and morphine 0.5 mg/kg were administered Zucconi et al., 2010). Although bone marrow are the intramuscularly and anesthesia was induced with major accessible and enriched source of MSCs, the propofol (Fresenius Kabi, Austria GmbH, Graz, numbers of MSCs obtained from bone marrow is Austria) and maintained with isoflurane in 100% generally insufficient for transplantation since the oxygen. Cefazolin (25 mg/kg) was administered population of MSCs has been reported to range about intravenously as a prophylactic antibiotic. Epidural 0.001 to 0.01% of bone marrow in rodents and felines anesthesia using 0.5% bupivacaine (1 mg/kg) (Kadiyala et al., 1997; Martin et al., 2002). This therefore combined with morphine (0.1 mg/kg) was highlights the importance of the isolation technique for additionally performed to relieve the pain sensation MSCs. The characterization of MSCs usually relies caused by the bone marrow aspiration procedure. upon plastic adherence, expression of some surface Fifteen milliliters of bone marrow were harvested from markers such as CD 90, CD 105 and Stro-1 while the iliac crest of each dog with a 1 ml heparinized lacking the expression of hemopoietic stem cell marker syringe (100IU/ml). (CD 34) ( Wagner et al., 2005; Dominici et al., 2006). In dogs, direct plating of bone marrow aspirate is The bone marrow aspirate (15 ml) was frequently used for bone marrow derived MSCs. equally divided into 3 aliquots, and each aliquot of 5 However, a lack of information about the comparison ml bone marrow was then submitted to one of the of techniques for MSC isolation has been reported in following isolation techniques: 1) direct plating, 2) red dogs. It is therefore important to standardize the MSC blood cell lysis treatment and 3) gradient density. isolation technique in order to increase the cell homogeneity of the MSCs population, thereby Direct plating: Direct plating was performed by improving efficiency for bone regeneration (Roberts et adding bone marrow aspirate directly into a 10 cm al., 2008). Petri-dish (BD-FalconTM, Franklin Lake, NJ, USA) containing 7 ml of MSC culture medium with A scaffold is a three-dimensional structure approximately 30-40% confluence. After 24 h of with interconnection between pores allowing cell culture, the attached cells were washed with attachment and replacing bone defects. Dulbecco’s phosphate buffered saline without calcium Polycaprolactone (PCL) is a biodegradable synthetic and magnesium (DPBS, Invitrogen, Carlsbad, CA, polymer ( Williams et al., 2005; Amato et al., 2007) with USA) 2-3 times and the fresh MSC medium was then advantages over other polymeric materials for the bone added. formation and remodeling phase including good biocompatibility, providing mechanical strength, Red blood cell lysis treatment: Red blood cell lysis giving more stability in ambient conditions and slow buffer (8.3 g/L ammonium chloride in 0.01 M Tris-HCl biodegradation rate (Gunatillake and Adhikari, 2003; buffer, pH 7.5 + 0.2) was used to eliminate the Shor et al., 2007). Hydroxyapatite (HAp) contaminated red blood cells (RBC) in the bone (Ca10[PO4]6[OH]2) is known as the ceramic of choice for marrow aspirate. The RBC lysis buffer was mixed at a bone tissue engineering because its chemical and ratio of 1: 1 with bone marrow aspirate and then crystal properties resemble the mineral component of incubated at room temperature (approximately 25- bone. In addition, it has an excellent biocompatibility, 26oC) for 5 min. The mixture was then centrifuged at osteoconductive capacity and ability to bind directly to 1000 rpm for 5 min. After the supernatant was host bone (Di Silvio et al., 2002; Wang, 2006; discarded, the pellet was resuspended with MSC Hutmacher et al., 2007; Neuendorf et al., 2008). The medium, and the isolated cells were cultured in MSC combination of PCL and HAp improves mechanical

Wangdee C. et al. / Thai J Vet Med. 2018. 48(4): 529-539. 531 culture medium with approximately 30-40% (each dog represented one replicate). Osteogenic confluence. The non-attached cells were washed out differentiation was performed as essentially described the following day. by Bosch et al. (2006) with minor modifications. The MSCs were loaded into a petridish at a density of Gradient density: Bone marrow aspirate (5 ml) was 35,000 cells per cm2. The bone induction was gently layered onto a histopaque 1077 (density 1.077 performed when the cells were approximately 80% +0.001) in a 15 ml conical tube (BD-FalconTM, Franklin confluent, the osteogenic medium containing MSC Lake, NJ, USA). The centrifugation was set at 26oC and medium supplemented with 0.1 µM dexamethasone, 400g for 30 min, and the interface containing 50 µM ascorbic acid, 10 mM β-glycerophosphate mononuclear cells was then collected. The (Merck, Darmstadt, Germany) was then added into the presumptive MSCs were washed with MSC culture culture dish. The in vitro bone differentiation was medium two times prior to culture. performed for 21 days. Von Kossa staining was used to detect the deposition of calcium phosphate indicating Culture of bone marrow derived mesenchymal stem de novo bone formation. cells: Following MSC isolation (day 0) as previously described, the isolated cells were allowed to attach to Scaffold preparation and MSC transplantation: the culture plate for 24 h prior to washing with DPBS. PCL/HAp composite scaffolds were prepared as These cultured cells were assigned as primary cells at previously described (Wutticharoenmongkol et al., passage 0 (P0). MSC culture medium was composed of 2007). The scaffolds were cut into 10x5x25 millimeters a low-glucose Dulbecco’s modified Eagle’s medium and sterilized with 100% (v/v) absolute ethanol for 1 h. The (low glucose DMEM) supplemented with 10% (v/v) scaffolds were washed thoroughly with sterilized distilled fetal bovine serum (FBS, Invitrogen), 2 mM L- water and then DPBS to remove the ethanol. glutamine (Invitrogen, Carlsbad, USA), 100 unit/ml penicillin G, 100 µg/ml streptomycin, 40µg/ml Anesthesia and surgical procedures were gentamicin and 5 µg/ml amphotericin B. performed similar to previously described. Brachial plexus block using 0.5% bupivacaine (1.3 mg/kg) was A subculture of MSCs was performed at day additionally performed prior to bilateral ulnar 7 after MSC isolation by treating the cells with 0.05% osteotomy to reduce pain. A total of 3 dogs were (w/v) trypsin-EDTA (Invitrogen). The disaggregated induced to bilateral ulnar non-union (n=6) by cutting cells were then centrifuged and split into a new Petri- the mid-shaft of the ulnar bone (2.5 cm) using an dish at a ratio of 1:3. Putative MSCs at passages 3 (P3) oscillating saw. The ostectomy sites were implanted were used for flow cytometric analysis in order to with the PCL/HAp composite scaffold to improve examine the expression of cell-surface antigens and bone stability. After the operation, all forelimbs were also for in vitro differentiation. MSCs at P3 were applied with modified Robert Jones bandages subcultured to passage (P4) for in vivo transplantation. reinforced with thermoplastic splints (Vet-lite, In all cases, the culture condition was performed at Bangkok, Thailand). All dogs also received 5 mg/kg 37oC in a humidified condition of 5 % CO2 in air. enrofloxacin (Bayer, Bangkok, Thailand) and 4 mg/kg carprofen (Pfizer, Bangkok, Thailand) orally for 7 days Assessing the characteristics of canine mesenchymal after implantation. All dogs demonstrated a non-union stem cells of ostectomy site at 12 weeks after scaffold Flow cytometry: The examination of MSCs was implantation. For transplantation, autologous MSCs performed as essentially described by Tharasanit et al. were subcultured to passage 4 as previously described. (2011). Canine MSCs at the 3rd passages from a total of After trypsinization, approximately 1x107 cells/ml 5 dogs (Dog no. 1, 4, 6, 7 and 8) were immunologically were loaded into a syringe containing a minimum examined for surface markers. These markers included volume (0.5 ml) of DMEM supplemented with 1% the positive (CD 44, CD 90) and negative (CD 34) (v/v) FBS. Three MSC transplanted sites (proximal, markers. A total of 200,000 to 300,000 cells were used middle and distal parts of the PCL/HAp scaffold implant) for immunolabeling. Rat monoclonal anti-canine CD were injected via a 23 G indwelling intravenous 90 (AbD serotec, Kidlington, UK) with rabbit anti-rat catheter with MSCs under a fluoroscope (Philips FITC secondary antibodies and monoclonal anti- healthcare, Eindhoven, the Netherlands). canine CD44 antibody conjugated with allophycocyanin (APC) (R&D system, Minneapolis, Examination of bone formation USA) were used as MSC positive markers. A rat Radiography: Lateral radiographs were taken monoclonal anti-canine CD 34 antibody conjugated immediately and at 2, 4, 6, 8, 10 and 12 weeks post- with fluorescein isothiocyanate (FITC) was used as the operatively in all dogs. Bone healing was evaluated negative MSC marker. Fluorescently-labeled MSCs using the radiographic scoring system previously were washed and fixed with 1% (w/v) described (Johnson et al., 1996b) as shown in table 1. paraformaldehyde in PBS and stored at 4°C in the dark Bone union was justified grading from 0 to 3 at the until analysis. Non-staining MSCs and MSCs labeled proximal and distal of implanted material. In addition, with only the secondary antibody were used as the new bone formation was scored 0 to 4. controls. At least 20,000 MSCs were analyzed by flow cytometry. Angiography: Fluoroscopic angiography was performed in two experimental dogs at 12 weeks In vitro bone differentiation of MSCs: Canine bone before MSC injection (dog no. 1, 2) and 16 weeks after marrow derived MSCs at passage 3 from four dogs MSC injection (dog no.1) in order to observe the blood were simultaneously induced to osteogenic lineage

5320 Wangdee C. et al. / Thai J Vet Med. 2018. 48(4): 529-539. vessels at the implantation site. Anesthesia and G E healthcare, Buckinghamshire, UK) was slowly surgical procedures were performed as previously administrated in order to observe the presence of the described. A 23 G intravenous catheter connected with an blood vessel at the MSC transplanted site using extension tube was inserted into the axillary vein and the fluoroscopy. radiographic contrast medium (iohexol, Omnipaque®, Table 1 Radiographic scoring system for evaluation of bone healing (Johnson et al., 1996b). Bone formation Score Description Bone union 0 No new bone; graft approximates density of soft tissue 1 Minimal new bone composed mostly of noncontiguous Areas of minimal density 2 New bone present as mostly contiguous areas of normal Density and fills approximately 50% of the defect 3 New bone present as mostly contiguous areas of normal 4 Density and fills approximately 51-95% of the defect 0 1 New bone a solid contiguous mass that fills > 95% of the defect 2 3 No contact between new bone and noninvolved adjacent normal bone Partial bridge (< 50%) from new bone to adjacent normal bone Partial bridge (> 50%) from new bone to adjacent normal bone Complete bridge from new bone to adjacent normal bone Histological examination of MSCs loaded scaffold: and Mann-Whitney U statistical tests. In all cases, The biopsy was performed both before (n=2) and after statistical analysis was performed using the SPSS MSC transplantation (n=2) at 12 and 20 weeks post- statistical program (version 17.0). P values <0.05 were transplantation. The biopsy samples were fixed with considered statistically significant. 10% (v/v) buffered formalin, and the fixed samples were embedded in paraffin and processed following Results the guidelines for a routine histological procedure. The sections were stained with hematoxylin & eosin (H&E) Following MSC isolation, putative MSCs and examined for neovascularization and bone were attached on to a Petri-dish. These MSCs formation under a light microscope. Masson’s trichrome irrespective of the MSC isolation techniques staining was additionally performed to detect the demonstrated a typical MSC morphology (Fig. 1 A-C). presence of collagen fibers. There were several cell types including thin spindle shaped, typical fibroblast-like and mantle cells. On day Statistical analysis: Values are present as means ± 7 after MSC isolation, the total number of MSCs standard deviation (SD). The efficacy for different MSC obtained from a 5-ml of bone marrow aspirate was isolation techniques on the number of isolated MSCs, ranged from 0.35×106 to 2.8×106, 0.04×106 to 1.43×, viability and expression of MSC markers were 0.03×106 to 0.75×106 for gradient density, RBC lysis compared by one-way analysis of variance (ANOVA) treatment and direct plating, respectively (Table 2). and post-hoc analysis with the least significant The gradient density techniques significantly increased difference (LSD). Angiographs and histological the numbers of isolated MSCs when compared with findings were descriptively analyzed. The differences the direct plating technique (P<0.05), while cell yields in radiographic scores at 2, 4, 6 and 8 weeks post MSC obtained from this gradient density were efficiently transplantation were evaluated using Kruskal-Wallis comparable to RBC lysis treatment. Table 2 Mean ± SD (range) of the percentage of MSCS on day 7 and the proportion of MSCs (passage 3) positive to cell surface markers. The MSCs were isolated from bone marrow using three different techniques. Total cell number Cell surface markers (x106) CD 90 CD 44 CD 34 Whole aspiration 0.26 + 0.28a 98.74+1.73 95.84+3.05 0.018+0.03 RBC lysis treatment (0.03-0.75) (95.66-99.59) (91.93-99.15) (0-0.01) Gradient 0.94 + 0.55a,b 99.28+0.82 93.48+10.76 (0.04-1.43) (99.18-99.89) (74.3-99.21) 0.014+0.03 1.08 + 0.92b 93.62+6.65 (0-0.06) (0.35-2.8) 99.2+0.96 (82.53-98.88) (99.02-99.89) 0.012+0.027 (0-0.06) Cell-surface antigen profiles of canine MSCs of percentage of cells positive for CD 44, CD 90 and were ascertained after immunolabeling with canine- CD34 are shown in table 2. Canine MSCs at passage 3 specific/cross-reacted monoclonal antibodies and highly expressed CD 44 (95.66-99.89) and rarely examined with a flow cytometer. Ranges and averages expressed CD 34 in all cases (Table 2). Furthermore, the

Wangdee C. et al. / Thai J Vet Med. 2018. 48(4): 529-539. 533 cultured MSCs were demonstrated to have the when examined at 16 weeks after MSC injection (Fig. capability to differentiate into osteogenic lineage as 4B). This result was in an accordance with the histology they positively stained with Von Kossa following of the host-PCL/HAp transplanted sites that osteogenic induction (Fig. 2). neovascularization was observed in PCL/HAp both with and without MSC injection. However, no such Following MSC transplantation into non- osteoid formation was observed in the histological union sites, all three dogs could walk properly within 24 sections. The transplanted PCL/HAp scaffolds were hours of surgery. Neither radiological changes at the filled with loose and unorganized connective tissue (Fig. scaffold-ulna interface nor callus formation were 5A) but the thickness of the fibrous tissues and the presented at the implant sites of PCL/HAp alone (Fig. 3A) number of small blood vessels were likely to increase and PCL/HAp combined with MSCs (Fig. 3B) at 2, 4, 6, 8 at 20 weeks after MSCs injection (Fig. 5B). The fibrous and 12 weeks post-operatively. The density of the implant tissue mainly comprised of the spindle cells with extensive grafts was comparable to soft tissue density. The collagen deposition which was confirmed by Masson’s radiographic scores were 0 in all cases. trichrome staining (Fig. 5B). Interestingly, we also found a number of multinucleated gaint cells and lymphocytes Fluoroscopic angiography indicated the within the scaffolds indicating an inflammatory presence of blood vessels within the PCL/HAp response. scaffolds at 12 weeks after implantation (Fig. 4A). These blood vessels descriptively appeared to increase Figure 1 Morphology of canine MSCs derived from bone marrow using direct plating (A), RBC lysis treatment (B) and gradient density (C). Figure 2 An example of canine bone marrow MSCs that were induced to differentiate into osteogenic lineage as they were positive to Von Kossa staining.

534 Wangdee C. et al. / Thai J Vet Med. 2018. 48(4): 529-539. Figure 3 Radiographs demonstrating canine ulnar segment defect with PCL/HAp alone (A) or PCL/HAp combined with MSCs at 12 weeks (B) postoperatively. Note that no de novo bone formation was observed. Figure 4 Fluoroscopic angiography at 12 weeks (A) after PCL/HAp scaffold implant and at 16 weeks after MSC injection (B). The increased blood vessels (black and gray lines) were observed at the implantation site after BMSC injection. Figure 5 Histology of PCL/HAp implanted site at 12 weeks after surgery (A) and at 20 weeks after MSC injection (B) indicates the newly formation of capillaries and arterioles. Masson’s trichrome staining (B) indicates the thickening of fibrous tissue mainly constructed by an extensive collagen fibers.

Wangdee C. et al. / Thai J Vet Med. 2018. 48(4): 529-539. 535 Discussion vascular destruction (McCartney and MacDonald, 2006). A combination of osteopotential cell and In this study, we demonstrated that bone biolodegradable scaffold is one of the most successful marrow derived canine MSCs could be enriched by strategies in bone tissue engineering. Our previous treating the bone marrow aspirate prior to culture, and data suggested that PLC/HAp composite scaffold the harvested cells were highly expressed MSC demonstrated a promising approach for promoting markers (CD 44 and CD 90) and had the potential to new bone formation when transplanted to calvarial differentiate into osteogenic lineage in vitro. However, defect in mice (Chuenjitkuntaworn et al., 2010). In the de novo bone formation following transplantation addition, previous study suggested that of the MSCs into PCL/HAp scaffold was polycaprolactone-tricalcium phosphate for loading compromised. MSCs presented a high amount of lamellar bone in dog mandibles (Khojasteh et al., 2013). In order to minimize Bone marrow is considered as a primary the risk of ischemic lesion, we therefore first source of MSCs but only 0.001 to 0.01% of bone marrow transplanted the PCL/HAp scaffold to the large bone mononuclear cells in rodents and felines were lesion allowing neovascularization within the scaffold identified as MSCs (Kadiyala et al., 1997; Martin et al., before MSC transplantation. After 12 weeks of 2002). We first examined the different techniques to PCL/HAp scaffold transplant, no bone formation was enrich MSCs from bone marrow aspirate and found found but fluoroscopy indicated the that gradient density significantly increased the neovascularization within the scaffold (Fig. 4A). numbers of isolated MSC on day 7 of culture when However, no osteogenic differentiation was observed compared with the direct plating technique (P<0.05, following MSC transplant into the vascularized table 2). It had become clear that the presence of RBC scaffolds. From this result, it has become clear that the in the culture dish negatively affected the number of implanted site of the scaffold and species employed harvested MSCs probably because the RBC may plays an important role for bone regeneration (i.e. compromise the MSCs to settle and adhere to the Petri- cortical versus cancellous bone) (Dickson et al., 2007). dish (Horn et al., 2011). In addition, excessive RBC lysis Furthermore, a large size and slow degradable PCL- during culture may cause the release of free HAp scaffold may also create an unfavorable hemoglobin that can induce cellular stress and environment for bone formation ( Bostman et al., 1990; apoptosis ( Meguro et al., 2001; Kumar and Bergsma et al., 1993; Prokop et al., 2004). In this study, Bandyopadhyay, 2005). Although the efficacy of the several multinucleated giant cells in the PLC/HAp gradient density was similar to that of RBC lysis scaffold indicated chronic reactions due to foreign treatment (P>0.05), the RBC lysis buffer treatment is body response. Similar tissue reaction was previously preferable for clinical use since it requires less time and reported (Linder and Lundskog, 1975; Volker et al., is inexpensive. This technique may also be beneficial 1997; Tindel et al., 2001). Although the scaffolds used for the initial growth of the isolated MSCs due to the in this study were contaminated with copper (probably platelet derived growth factor secreted from the from hydroxyapatite ceramic) as we further analyzed the remaining platelets following RBC lysis (Horn et al., scaffold using energy dispersive x-ray (SEM/EDX) 2008). In addition to the effect of isolation techniques analysis (unpublished data), the failure for osteogenesis on MSC derivation, the numbers of MSCs isolated were considerably variable among donors, irrespective of transplanted MSCs in the scaffold was probably not of the isolation techniques used. This result is in an caused by the cell cytotoxicity of the contaminated agreement to the facts that the number of bone marrow copper. There was good evidence of fibrovascular MSCs significantly decreases with increased age and tissue infiltration with an extent matrix deposition poor health status ( Egrise et al., 1992; Dodson et al., throughout the entire PCL/HAp scaffold, and injection 1996; Rao and Mattson, 2001; Bobis et al., 2006), while of MSCs into the scaffold seemed to increase the the cells obtained from young donors grew more thickening size of the fibroblastic mass and rapidly (Musina et al., 2005). However, there was no vascularization (Fig. 5B). In fact, though several factors effect of isolation techniques on the expression of MSC are critical for bone tissue engineering, the main makers (CD 40 and CD 90) and also in vitro hallmark remains to be the sufficient osteogenic factors differentiation, suggesting that MSC properties could to stimulate de novo bone differentiation of the be maintained using the current culture system. transplanted MSCs (Calori et al., 2011). Several growth However, study of the effect of long term culture of factors and pathways have been demonstrated to be these MSCs on MSC proliferation activity and also necessary for bone development (Lin and Hankenson, differentiation capability remains elusive. This aspect 2011). For example, bone morphologic protein-2 (BMP- is important given that the large numbers of cells 2) increased osteocalcin release from MSCs promoting would be required for cell/tissue engineering (Barry and healing response and induced chondrogenic and and Murphy, 2004). osteogenic differentiation of human bone marrow MSCs ( Schmitt et al., 1999; Cheng et al., 2003; Shen et To date, several attempts have been made on al., 2010) and also promoted the new bone formation in transplantation of MSC into long-bone defects femoral defects in rats (Burastero et al., 2010) and (Johnson et al., 1996b; Kadiyala et al., 1997; Burastero increased the local population of cells and the et al., 2010; Vertenten et al., 2010). However, the de connective tissue progenitors in a canine femur defect novo bone formation following transplant has been model with the combination of MSCs (Takigami et al., unsuccessful (Kadiyala et al., 1997) especially in dogs 2007). Moreover, early markers of osteogenesis were that have a slower bone turnover rate compared with induced in canine MSCs by a combination of BMP-2 mice (Cook et al., 1994). Furthermore, the injured bone and ascorbate (Volk et al., 2005). site often becomes ischemic due to the excessive

536 Wangdee C. et al. / Thai J Vet Med. 2018. 48(4): 529-539. In conclusion, this study revealed that MSCs with BMP-7 improves bone regeneration of can be derived from bone marrow. These MSCs could critical-size segmental bone defects in athymic be further enriched by treating bone marrow aspirate rats. Bone 47(1): 117-126. with gradient density and red blood cell lysis Calori GM, Mazza E, Colombo M, Ripamonti C and treatment. Although these MSCs highly expressed the Tagliabue L 2011. Treatment of long bone non- MSC markers and also retained the differentiation unions with polytherapy: indications and clinical potential, the bone differentiation following results. Injury 42(6): 587-590. implantation was compromised. Further novel Cheng H, Jiang W, Phillips FM, Haydon RC, Peng Y, strategies in particular the combination of tissue Zhou L, Luu HH, An N, Breyer B, Vanichakarn P, engineering and osteogenic substances that can create Szatkowski JP, Park JY and He TC 2003. a proper environment for de novo bone formation Osteogenic activity of the fourteen types of remain to be studied. human bone morphogenetic proteins (BMPs). J Bone Joint Surg Am 85-A(8): 1544-1552. Conflict of interest statement: None of the authors of Chuenjitkuntaworn B, Inrung W, Damrongsri D, this paper has a financial or personal relationship with Mekaapiruk K, Supaphol P and Pavasant P 2010. other people or organizations that could Polycaprolactone/hydroxyapatite composite inappropriately influence or bias the content of the scaffolds: preparation, characterization, and in paper. vitro and in vivo biological responses of human primary bone cells. J Biomed Mater Res A 94(1): Acknowledgements 241-251. Cook SD, Baffes GC, Wolfe MW, Sampath TK and This project was financially supported by the Rueger DC 1994. Recombinant human bone Thailand Research Fund (DBG 5280015). We also morphogenetic protein-7 induces healing in a would like to thank Supranee Buranapraditkun for canine long-bone segmental defect model. Clin technical support on flow cytometry analysis. Orthop Relat Res (301): 302-312. Di Silvio L, Dalby MJ and Bonfield W 2002. Osteoblast References behaviour on HA/PE composite surfaces with different HA volumes. Biomaterials 23(1): 101- Alaminos M, Perez-Kohler B, Garzon I, Garcia- 107. Honduvilla N, Romero B, Campos A and Bujan J Dickson G, Buchanan F, Marsh D, Harkin-Jones E, 2010. Transdifferentiation potentiality of human Little U and McCaigue M 2007. Orthopaedic Wharton's jelly stem cells towards vascular tissue engineering and bone regeneration. endothelial cells. J Cell Physiol 223(3): 640-647. Technol Health Care 15(1): 57-67. Dodson SA, Bernard GW, Kenney EB and Carranza FA Amato I, Ciapetti G, Pagani S, Marletta G, Satriano C, 1996. In vitro comparison of aged and young Baldini N and Granchi D 2007. Expression of cell osteogenic and hemopoietic bone marrow stem adhesion receptors in human osteoblasts cultured cells and their derivative colonies. J Periodontol on biofunctionalized poly-(epsilon-caprolactone) 67(3): 184-196. surfaces. Biomaterials 28(25): 3668-3678. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Barry FP and Murphy JM 2004. Mesenchymal stem Prockop D and Horwitz E 2006. Minimal criteria cells: clinical applications and biological for defining multipotent mesenchymal stromal characterization. Int J Biochem Cell Biol 36(4): cells. The International Society for Cellular 568-584. Therapy position statement. Cytotherapy 8(4): 315-317. Bartels KE 1987. Nonunion. Vet Clin North Am. Small Egrise D, Martin D, Vienne A, Neve P and Schoutens Anim Pract 17(4): 799-809. A 1992. The number of fibroblastic colonies formed from bone marrow is decreased and the Bergsma EJ, Rozema FR, Bos RR and de Bruijn WC in vitro proliferation rate of trabecular bone cells 1993. Foreign body reactions to resorbable increased in aged rats. Bone 13(5): 355-361. poly(L-lactide) bone plates and screws used for Gunatillake PA and Adhikari R 2003. Biodegradable the fixation of unstable zygomatic fractures. J synthetic polymers for tissue engineering. Eur Oral Maxillofac Surg 51(6): 666-670. Cell Mater 20(5): 1-16. Horn P, Bork S, Diehlmann A, Walenda T, Eckstein V, Bobis S, Jarocha D and Majka M 2006. Mesenchymal Ho AD and Wagner W 2008. Isolation of human stem cells: characteristics and clinical mesenchymal stromal cells is more efficient by applications. Folia Histochem Cytobiol 44(4): 215- red blood cell lysis. Cytotherapy 10(7): 676-685. 230. Horn P, Bork S and Wagner W 2011. Standardized isolation of human mesenchymal stromal cells Bosch P, Pratt SL and Stice SL 2006. Isolation, with red blood cell lysis. Methods Mol Biol 698: characterization, gene modification, and nuclear 23-35. reprogramming of porcine mesenchymal stem Hutmacher DW, Schantz JT, Lam CX, Tan KC and Lim cells. Biol Reprod 74(1): 46-57. TC 2007. State of the art and future directions of scaffold-based bone engineering from a Bostman O, Hirvensalo E, Makinen J and Rokkanen P 1990. Foreign-body reactions to fracture fixation implants of biodegradable synthetic polymers. Journal Bone Joint Surg Br 72(4): 592-596. Burastero G, Scarfi S, Ferraris C, Fresia C, Sessarego N, Fruscione F, Monetti F, Scarfo F, Schupbach P, Podesta M, Grappiolo G and Zocchi E 2010. The association of human mesenchymal stem cells

Wangdee C. et al. / Thai J Vet Med. 2018. 48(4): 529-539. 5375 biomaterials perspective. J Tissue Eng Regen Med materials and tissue engineering scaffolds. Acta 1(4): 245-260. Jang BJ, Byeon YE, Lim JH, Ryu HH, Kim WH, Koyama biomat 4(5): 1288-1296. Y, Kikuchi M, Kang KS and Kweon OK 2008. Implantation of canine umbilical cord blood- Prokop A, Jubel A, Helling HJ, Eibach T, Peters C, derived mesenchymal stem cells mixed with beta- tricalcium phosphate enhances osteogenesis in Baldus SE and Rehm KE 2004. Soft tissue bone defect model dogs. J Vet Sci 9(4): 387-393. Johnson KD, Frierson KE, Keller TS, Cook C, reactions of different biodegradable polylactide Scheinberg R, Zerwekh J, Meyers L and Sciadini MF 1996b. Porous ceramics as bone graft implants. Biomaterials 25(2): 259-267. substitutes in long bone defects: a biomechanical, histological, and radiographic analysis. J Orthop Rao MS and Mattson MP 2001. Stem cells and aging: Res 14(3): 351-369. Kadiyala S, Jaiswal N and Bruder SP 1997. Culture- expanding the possibilities. Mech Ageing Dev Expanded, Bone Marrow-Derived Mesenchymal Stem Cells Can Regenerate a Critical-Sized 122(7): 713-734. Segmental Bone Defect. Tissue Eng 3(2): 173-185. Khojasteh A, Behnia H, Hosseini FS, Dehghan MM, Ribitsch I, Burk J, Delling U, Geissler C, Gittel C, Julke Abbasnia P and Abbas FM 2013. The effect of PLC-TCP scaffold loaded with mesenchymal H and Brehm W 2010. Basic Science and Clinical stem cells on vertical bone augmentation in dog mandible: A prelimimary report. J Biomed Mater Application of Stem Cells in Veterinary Medicine. Res B Appl Biomater 101(5): 848-854. Krabbe C, Zimmer J and Meyer M 2005. Neural Adv Biochem Eng Biotechnol 123: 219-263. transdifferentiation of mesenchymal stem cells--a critical review. APMIS 113(11-12): 831-844. Roberts SJ, Howard D, Buttery LD and Shakesheff KM Kraus KH and Kirker-Head C 2006. Mesenchymal stem cells and bone regeneration. Vet Surg 35(3): 232- 2008. Clinical applications of musculoskeletal 242. Kumar S and Bandyopadhyay U 2005. Free heme tissue engineering. Br Med Bull 86: 7-22. toxicity and its detoxification systems in human. Toxicol Lett 157(3): 175-188. Schmitt JM, Hwang K, Winn SR and Hollinger JO 1999. Langer R and Vacanti JP 1993. Tissue engineering. Science 260(5110): 920-926. Bone morphogenetic proteins: an update on basic Lee HB, Chung YS, Heo SY and Kim NS 2009. Augmentation of bone healing of nonunion biology and clinical relevance. J Orthop Res 17(2): fracture using stem cell based tissue engineering in a dog: a case report. Veterinarni Medicina 269-278. 54(4): 198-203. Lin GL and Hankenson KD 2011. Integration of BMP, Shen B, Wei A, Whittaker S, Williams LA, Tao H, Ma Wnt, and notch signaling pathways in osteoblast differentiation. J Cell Biochem 112(12): 3491-3501. DD and Diwan AD 2010. The role of BMP-7 in Linder L and Lundskog J 1975. Incorporation of stainless steel, titanium and Vitallium in bone. chondrogenic and osteogenic differentiation of Injury 6(4): 277-285. Martin DR, Cox NR, Hathcock TL, Niemeyer GP and human bone marrow multipotent mesenchymal Baker HJ 2002. Isolation and characterization of multipotential mesenchymal stem cells from stromal cells in vitro. J Cell Biochem 109(2): 406- feline bone marrow. Exp Hematol 30(8): 879-886. McCartney WT and MacDonald JB 2006. Incidence of 416. non-union in long bone fracture in 233 cats. Int J Appl Res Vet Med 4(3): 209-212. Shor L, Guceri S, Wen X, Gandhi M and Sun W 2007. Meguro T, Chen B, Parent AD and Zhang JH 2001. Caspase inhibitors attenuate oxyhemoglobin- Fabrication of three-dimensional induced apoptosis in endothelial cells. Stroke 32(2): 561-566. polycaprolactone/hydroxyapatite tissue Musina RA, Bekchanova ES and Sukhikh GT 2005. Comparison of mesenchymal stem cells obtained scaffolds and osteoblast-scaffold interactions in from different human tissues. Bull Exp Bio Med 139(4): 504-509. vitro. Biomaterials 28(35): 5291-5297. Neuendorf RE, Saiz E, Tomsia AP and Ritchie RO 2008. Adhesion between biodegradable polymers and Takigami H, Kumagai K, Latson L, Togawa D, Bauer T, hydroxyapatite: Relevance to synthetic bone-like Powell K, Butler RS and Muschler GF 2007. Bone formation following OP-1 implantation is improved by addition of autogenous bone marrow cells in a canine femur defect model. J Orthop Res 25: 1333-1342. Tharasanit T, Phutikanit N, Wangdee C, Soontornvipart K,Tantrajak S, Kaewamatawong T, Suwimonteerabutr J, Supaphol P and Techakumphu 2011. Differentiation potentials of canine bone marrow mesenchymal stem cells. Thai J Vet Med 41(1): 79-86. Tindel NL, Marcillo AE, Tay BK, Bunge RP and Eismont FJ 2001. The effect of surg9ically implanted bullet fragments on the spinal cord in a rabbit model. J Bone Joint Surg 83-A(6): 884-890. Vertenten G, Gasthuys F, Cornelissen M, Schacht E and Vlaminck L 2010. Enhancing bone healing and regeneration: present and future perspectives in veterinary orthopaedics. VCOT 23(3): 153-162. Volk SW, Diefenderfer DL, Christopher SA, Haskins ME and Leboy PS 2005. Effects of osteogenic inducers on cultures of canine mesenchymal stem cells. Am J Vet Res 66(10): 1729-1737. Volker W, Dorszewski A, Unruh V, Robenek H, Breithardt G and Buddecke E 1997. Copper- induced inflammatory reactions of rat carotid arteries mimic restenosis/arteriosclerosis-like neointima formation. Atherosclerosis 130,(1-2): 29-36. Wagner W, Wein F, Seckinger A, Frankhauser M, Wirkner U, Krause U, Blake J, Schwager C, Eckstein V, Ansorge W and Ho AD 2005. Comparative characteristics of mesenchymal

5368 Wangdee C. et al. / Thai J Vet Med. 2018. 48(4): 529-539. stem cells from human bone marrow, adipose tissue, and umbilical cord blood. Exp Hematol 33(11): 1402-1416. Wang M 2006. Composite scaffolds for bone tissue engineering. Am J Biochem Biotech 2(2): 80-84. Williams JM, Adewunmi A, Schek RM, Flanagan CL, Krebsbach PH, Feinberg SE, Hollister SJ and Das S 2005. Bone tissue engineering using polycaprolactone scaffolds fabricated via selective laser sintering. Biomaterials 26(23): 4817-4827. Wutticharoenmongkol P, Pavasant P and Supaphol P 2007. Osteoblastic phenotype expression of MC3T3-E1 cultured on electrospun polycaprolactone fiber mats filled with hydroxyapatite nanoparticles. Biomacromolecules 8(8): 2602-2610. Zucconi E, Vieira NM, Bueno DF, Secco M, Jazedje T, Ambrosio CE, Passos-Bueno MR, Miglino MA and Zatz M 2010. Mesenchymal stem cells derived from canine umbilical cord vein--a novel source for cell therapy studies. Stem Cells Dev 19(3): 395-402. Zuk PA, Zhu M, Ashjian P, De Ugarte DA, Huang JI, Mizuno H, Alfonso ZC, Fraser JK, Benhaim P and Hedrick MH 2002. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 13(12): 4279-4295.

Wangdee C. et al. / Thai J Vet Med. 2018. 48(4): 529-539. 5395 บทคดั ย่อ การเปรียบเทียบเทคนคิ การคดั แยกเซลลต์ ้นกาเนิดมเี ซนไคมจ์ ากไขกระดกู สุนัขและความเขา้ กันได้ ของเซลลต์ อ่ โครงสรา้ งชนดิ โพลคี าร์โปแลตโตน ไฮดรอกซอี ะพาไทต์ ชาลกิ า หวังดี1 ศศธิ ร พนโสภณกลุ 5 ณปภชั ธาดาวริ ุฬห์3 กมั ปนาท สนุ ทรวิภาต1 ประสทิ ธิ์ ภวสันต์2 พชิ ญ์ ศุภผล3 มงคล เตชะกาพุ4 ธรี วฒั น์ ธาราศานิต4* ไขกระดกู จากสนุ ัขจานวน 7 ตัว นามาคัดแยกเซลลม์ ีเซนไคมด์ ้วยการใชว้ ิธีการคัดแยก 3 วธิ ีการ (direct plating, red blood cell lysis และ gradient density) ทาการตรวจหาจานวนเซลลท์ แ่ี ยกได้ การแสดงออกของ marker ท่ีจาเพาะตอ่ เซลล์มเี ซนไคม์และความสามารถ ในการกลายเป็นเซลล์กระดกู บนจานเพาะเล้ียงจากเซลลท์ แี่ ยกได้ทง้ั 3 วธิ ีก่อนการนามาปลกู ถา่ ยในกระดกู ulna ท่ถี ูกกระตุ้นใหเ้ กิดภาวะ กระดูกไม่ติดกันจานวน 3 ตวั เพอื่ ดปู ระสทิ ธภิ าพการสรา้ งกระดูกจากเซลล์มเี ซนไคม์ โดยทาการฝงั โครงรา่ งเลย้ี งเซลลช์ นดิ โพลคี าร์โปแลตโตน ไฮดรอกซอี ะพาไทต์ ไปในตาแหน่งกระดกู ท่ตี ดั ไปก่อนทาการฉดี เซลลช์ นดิ มีเซนไคมเ์ ข้าไปในโครงรา่ งเลย้ี งเซลล์ ทาการถา่ ยภาพเอกซเรย์ การ บันทกึ ภาพรังสีหลอดเลือดและตรวจดจู ลุ กายวภิ าคของเนอ้ื เย่ือในสัปดาห์ ที่ 2, 4, 6, 8 และ 12 หลงั การฉดี จานวนเซลลม์ ีเซนไคม์ทแ่ี ยกได้ จากวิธี gradient density และ RBC lysis มีมากกว่าจานวนเซลลม์ เี ซนไคมท์ ีแ่ ยกได้จากวธิ ี direct plating ในวนั ท่ี 7 ของการเล้ยี ง เซลล์มี เซนไคม์ทแี่ ยกได้จากทกุ ๆ วธิ มี กี ารแสดงออกของ marker ที่จาเพาะตอ่ เซลลม์ ีเซนไคม์ ไดแ้ ก่ CD 44 and CD 90 ไมพ่ บการเปลย่ี นแปลงจาก ภาพถา่ ยเอกซเรยห์ รอื การเกดิ แคลลสั บนโครงรา่ งเลยี้ งเซลลใ์ นสนขั ทุกตัวท่ไี ดร้ บั การ ปลกู ถา่ ยเซลลช์ นดิ มีเซนไคม์ ถงึ แมว้ า่ เซลล์มเี ซนไคมจ์ าก สนุ ขั ทุกตัวจะมีการสรา้ งกระดูกบนจานเพาะเลี้ยง จากภาพรังสีหลอดเลอื ดแสดงการสรา้ งหลอดเลือดใหม่เพม่ิ ข้นึ ในสปั ดาหท์ ่ี 16 หลงั ฉดี เซลล์ มีเซนไคม์ การตรวจทางจลุ กายวิภาคของเน้อื เยือ่ ได้ยนื ยนั การสรา้ งหลอดเลอื ดเพมิ่ ข้นึ และพบเนอ้ื เย่ือไฟบรสั อยรู่ อบๆ โครงรา่ งเล้ียงเซลล์ จาก การศึกษาครงั้ นี้สรุปได้ว่าการคดั แยกเซลล์ชนดิ มีเซนไคมท์ ่เี หมาะสมได้แกว่ ธิ ี gradient density และ RBC lysis โดยจะไดเ้ ซลล์ทีม่ จี านวน เยอะกว่าและมีคุณสมบตั ิของเซลลช์ นิดมีเซนไคม์ อย่างไรก็ตามศักยภาพของการใช้เซลลช์ นดิ มีเซนไคม์ในการฉดี เข้าไปยังกระดกู ท่ีมภี าวะ กระดูกไมต่ ดิ ยังต้องอาศัย ปัจจยั อื่นๆ เชน่ สารกระต้นุ การสร้างกระดูก คาสาคัญ: ไขกระดกู สุนัข เซลล์มีเซนไคม์ กระดูกไมต่ ิดกนั โครงร่างเลยี้ งเซลล์ชนิด โครงสรา้ งชนดิ โพลีคาร์โปแลตโตน ไฮดรอกซอี ะพาไทต์ 1ภาควชิ าศลั ยศาสตร์ 4ภาควชิ าสตู ิศาสตร์-เธนุเวชวิทยาและวทิ ยาการสบื พนั ธุ์ คณะสตั วแพทยศาสตร์ จฬุ าลงกรณ์มหาวิทยาลยั ถนนองั รดี นู งั ต์ เขตปทมุ วัน กรุงเทพฯ 10330 ประเทศไทย 2ภาควชิ ากายวิภาคศาสตร์ คณะทนั ตแพทยศาสตร์ จฬุ าลงกรณม์ หาวทิ ยาลยั ถนนองั รดี นู งั ต์ เขตปทุมวัน กรุงเทพฯ 10330 ประเทศไทย 3วทิ ยาลัยปโิ ตรเลียมและปิโตรเคมี จฬุ าลงกรณม์ หาวทิ ยาลยั ถนนพญาไท เขตปทุมวนั กรุงเทพฯ 10330 ประเทศไทย 5ภาควชิ าคลนิ ิกสตั วเ์ ลยี้ งและสตั วป์ า่ คณะสัตวแพทยศาสตร์ มหาวทิ ยาลัยเชยี งใหม่ แมเ่ หียะ เชยี งใหม่ 50100 ประเทศไทย *ผ้รู ับผิดชอบบทความ E-mail: [email protected]



Original Article Anti-proliferative and total ERK1/2 inhibitory effects of plant flavonols on Human cervical cancer (HeLa) cells Sookruetai Boonmasawai1* Ladawan Sariya2 Arpron Leesombun1 Kridsada Chaichoun1 Jarupha Taowan2 Orathai Thongjuy3 Abstract Kaempferol, myricetin and quercetin are abundant flavonols in edible fruit and vegetables. Much previous data has shown the beneficial effects of these flavonols in cancer treatments. Thus, the cytotoxic effects on rhesus monkey kidney epithelial cells ( LLC- MK2) and the anti- proliferative effects on human cervical cancer ( HeLa) cells through total ERK protein expression of kaempferol, myricetin and quercetin have been investigated. The cytotoxic assay at 24h revealed the high safety of three flavonols on LLC-MK2 cells. Kaempferol (1-1000 µM) did not have any significant effect on the viability of these normal epithelial cells. The cytotoxicity of myricetin and quercetin was 5 µM and 50 µM, respectively. No flavonols could suppress total ERK1/2 protein expression in LLC-MK2 cells. In HeLa cells, kaempferol (5 µM) and quercetin (1 µM) significantly inhibited cell proliferations and total ERK1/2 protein expression. Myricetin significantly reduced cancer cell proliferations at 1 µM without any effect on total ERK1/2 protein expression. In conclusion, kaempferol and quercetin had an inhibitory effect on HeLa cells via reduction of total ERK1/2 protein at 24h. But the anti-proliferative effects of myricetin did not exert via total ERK1/2 protein expression. This study affirms the potency of three flavonols as future chemotherapeutic agents and herbal supplements. Keywords: ERK, HeLa, kaempferol, myricetin, quercetin 1Department of Pre-clinic and Applied Animal Science 2The Monitoring and Surveillance Center for Zoonotic Diseases in Wildlife and Exotic Animals 3The Center for Veterinary Diagnosis Faculty of Veterinary Science, Mahidol University 999 Phuttamonthon 4 Road, Salaya, Phuttamonthon, Nakhon Pathom, Thailand 73170 *Correspondence: [email protected] Thai J Vet Med. 2018. 48(4): 541-549.

542 Boonmasawai S. et al. / Thai J Vet Med. 2018. 48(4): 541-549. Introduction proliferations. LLC-MK2 and HeLa cells were cultured with Dulbecco’s modified Eagle’s medium (DMEM) at Flavonols belong to a class of flavonoids that 37°C. The culture media contained 10% fetal bovine are abundantly found in fruit and vegetables (Yao et al., serum (FBS), 100 unit/ml penicillin G sodium, and 100 2004; George et al., 2017). The flavonoids with a ketone µg/ml streptomycin. After the cells were confluent in T- group and 3’-hydroxyl group on the C ring structure 75 flasks, they were sub-cultured into 96-well plates at are called flavonols (Kumar and Pandey, 2013). The a density of 5×103 cells/well for 24h before treatment, three important flavonols; kaempferol, myricetin and followed by determination of the epithelial cell quercetin are major flavonoids in many edible tropical cytotoxicity and the anti-proliferative effects on plant extracts (Miean and Mohamed, 2001). They are cervical cancer. mostly found in black and green tea, onions, broccoli, apples, grapes, coffee and beans (Somerset and Evaluation of cytotoxicity and anti-proliferative Johannot, 2008) with beneficial effects in cancer treatment (Panche et al., 2016; George et al., 2017). activity: After treatment with various concentrations Kaempferol exhibits anti-tumor properties (Xie et al., of flavonols for 24h, the epithelial cell cytotoxicity and 2013; Qiu et al., 2017) in hepatocellular carcinoma (Guo the cancer cell proliferations were determined by both et al., 2016) and breast cancer (Lee et al., 2017). The calorimetric MTT and SRB assays. The MTT assay has apoptotic effects of myricetin have been reported in been used as the gold standard to evaluate the pancreatic (Phillips et al., 2011), colon (Kim et al., 2014) cytotoxicity but the results had some variation when and thyroid cancer (Jo et al., 2017). As well as quercetin, detected by the variation observed in the linear range. this ubiquitous flavonol may be used as a potent systemic chemo-therapeutic agent (Mendoza and Burd, Thus, the SRB assay was also performed because this 2011; Carvalho et al., 2017) in breast (Su et al., 2016), assay is suitable for screening the effects of novel gastric (Li and Chen, 2017), and bladder cancer (Tao et therapeutic compounds (van Tonder et al., 2015). The al., 2017). anticancer drug (5-Fluorouracil: 5-FU) and the mitogen- The extracellular signal-regulated kinase activated protein kinase (MEK) inhibitor (PD-98059) (ERK) signalling pathway plays a crucial role in cancer cell proliferations and apoptosis (Cagnol et al., 2010). were used as positive control. The MTT assay was The limitation of total ERK1/2 and phosphorylated modified from Mosmann’s method (Mosmann, 1983). ERK activities enhance the death of HeLa cells by down-regulation of Bcl-2 expression and the collapse of After 24h treatment with various reagents, 2 mg/ml 3- mitochondrial membrane potential (MMP) (Chang et al., 2017). Most previous studies have revealed the anti- (4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium cancer effects of kaempferol (Jeong et al., 2009; Chen et bromide (MTT) was incubated for 3h at 37°C with the al., 2013) and myricetin (Lee et al., 2007) by reducing the normal epithelial cells and cancer cells in 96-well plates. phosphorylated ERK more than total ERK protein expression. The ERK signalling pathway is also the DMSO was used to solubilize the intracellular MTT- direct molecular target of quercetin (Lee et al., 2008). formazan for 5 mins at room temperature. Then a However, the direct mechanism of these flavonols on HeLa cells through total ERK protein expression has quantity of formazan that was directly proportional to not been investigated. Thus, this study aims to the viable cell number was determined by using a plate determine the cytotoxic effect on the normal epithelial reading spectrophotometer: Synergy™ HT Multi- cells and the anti-proliferative effect on cervical cancer cells via total ERK1/2 expression. This type of cancer is Detection Microplate Reader (BioTek, USA) at 570 nm one of the most common malignancies with high wavelength. The cell proliferations were confirmed by mortality among Asian woman (Patra et al., 2017; Zeng et al., 2017). Knowledge about the efficacy and the ERK SRB calorimetric assay (Vichai and Kirtikara, 2006; signalling mechanism of plant flavonols may be useful for developing new alternative anticancer agents from Orellana and Kasinski, 2016). After the incubation the nature. period, 50 µl ice-cold 50% trichloroacetic acid (TCA) in Materials and Methods sterile distilled water were incubated with the cells at 4°C for 60 min. The fixed cells in plates were washed Cell cultures and treatments: Rhesus monkey (Macaca mulatta) kidney epithelial cells (LLC-MK2) were used gently 5 times with distilled water and dried at room to evaluate the cytotoxicity of three flavonols: temperature. The epithelial cells and cancer cells were kaempferol, myricetin and quercetin (Sigma, USA). Various concentrations of flavonols (1, 5, 10, 50, 100, stained with 50 µl of 0.4% w/v sulphorhodamine B (SRB) 250, 500 and 1000 µM) in 0.001 % Dimethyl sulfoxide (DMSO) were incubated with epithelial cells for 24h. dissolved in 1% acetic acid solution for 30 min. Then 1% Human cervical adenocarcinoma (HeLa) cells were acetic acid was used to wash unbound the SRB solution used to evaluate the effects of flavonols on cancer cell 3-5 times and the plates left to air-dry. The culture plates were shaken on a plate shaker for 5 mins at room temperature. The absorbance of SRB dye in cells that was solubilized by 10 mM un-buffered Tris-based solution (pH 10.5) 200 µl /well was measured at 540 nm wavelength by Microplate Reader. The anti-proliferative activity was calculated as a percentage of cell proliferation using the following formula: Percentage of cell proliferation = (Absorbance of treatment/ Absorbance of control) × 100, the absorbance of non-treatment control and flavonol treatment coming from triplicate wells in each experiment.

Boonmasawai S. et al. / Thai J Vet Med. 2018. 48(4): 541-549. 543 Immunoblotting: The cells were incubated with Statistical analysis: The percentage of cell various reagents for 24h and washed by cold proliferations compared with non-treatment cells are phosphate buffer saline (PBS) containing 138 mM NaCl, statistically shown as mean ± SD. All statistical analyses were performed using SPSS 21. The half maximal 2.7 mM KCl, 8 mM Na2HPO4, 1.46 mM KH2PO4 before inhibitory concentration (IC50) value was calculated by being lysed with cold extract buffer (50 mM Tris base; GraphPad Prism 6. The Image J program was used to determine the density of the protein band on blotting 10 mM EDTA; 1% (V/V) Triton X-100; 0.57 mM PMSF; 1.5 paper. The mean values of data among treatment µM pepstatin A; 2 µM leupeptin). Protein groups were compared using one-way analysis of concentrations in each sample were determined by Bio- variance (ANOVA) and the t-test. The P-value < 0.05 was regarded as statistically significant. Rad protein assay kit (Life Science, Thailand). Equal Results amounts of protein were loaded into each lane of 12% separating SDS/PAGE gel and electrophorised for 2h at The cytotoxic effects of kaempferol, myricetin and quercetin on LLC-MK2 cells: After 24h incubation, 120 constant voltages. The separated protein bands various concentrations of kaempferol (1, 5, 10, 50, 100, 250, 500 and 1000 µM) had no effect on the viability of were transferred onto nitrocellulose membrane and LLC-MK2 cells (Fig.1a). Thus, IC50 of kaempferol could blocked by 5% low fat milk in Tris buffer saline (20 mm not be calculated. Myricetin at 5 µM significantly inhibited normal epithelial cells by MTT assay (77.3  Tris (pH 7.6) and 250 mm NaCl). The nitrocellulose 5.6 %; IC50 = 81.55 µM). While the inhibitory concentration by SRB assay was 10 µM (IC50 = 707.10 membranes were incubated with primary monoclonal µM) (Fig.1b). By both calorimetric assays, the inhibitory antibody for 90 min (Anti-total ERK [dilution 1:500], concentration of quercetin was 50 µM (86.2  0.8%; IC50 = Anti-beta-actin [dilution 1:5,000]) (Cell Signaling, USA) 2,322 µM and 60.2  5.4%; IC50=797.20 µM, respectively) (Fig.1c). followed by incubation with anti-rabbit IgG- conjugated horse radish peroxidase (dilution 1:2,000) (Cell Signaling, USA) for 60 mins. The p44/42 MAPK (ERK1/2) antibody was used to detect endogenous levels of total ERK1/2 protein. Immunoblots were developed with 3,3’-Diaminobenzidine; DAB substrate kit (Sigma, USA). Figure 1 Cytotoxicity of kaempferol (a), myricetin (b) and quercetin (c) on rhesus monkey kidney cells (LLC-MK2) by MTT (Black color bar) and SRB (Gray color bar) assays. Percentage of cell proliferation compared with non-treatment group (control) is represented as mean ± SD (n = 6). * P < 0.001 when compared with the control. Note: Figs.1-4, DMEM: free serum Dulbecco’s modified Eagle’s medium, DMSO: 0.001 % dimethyl sulfoxide, 5-FU: 20 µM 5-fluorouracil, PD-98059: 20 µM PD-98059.

544 Boonmasawai S. et al. / Thai J Vet Med. 2018. 48(4): 541-549. Anti-proliferative effects of kaempferol, myricetin and on HeLa cell proliferations at 5 µM by MTT method (69.5  3.1%; IC50 > 1000 µM). Myricetin significant quercetin on HeLa cells: The 0.001 % DMSO that was used as a solvent in flavonol solution did not have any concentration was 1 µM by both colorimetric assays cytotoxic effect on either both LLC-MK2 on HeLa cells. (MTT-IC50= 9.06 µM, SRB-IC50 = 3.27 µM). The results of The positive control, 5-FU (20 µM) and PD-98059 (20 µM) quercetin were not similar for the two methods. By had inhibitory effects on the proliferations of normal MTT assay, quercetin could significantly inhibit HeLa and cancer cells. Fig.2 represents the effects of three cells proliferation at 50 µM (47.3  1.9%; IC50 = 43.57 µM). flavonols on cervical cancer cell proliferations By SRB assay, the inhibitory concentration was 10 µM compared with non-treatment control. After 24h (61.7  7.5%; IC50 = 27.97 µM). treatment, kaempferol initially had a significant effect Figure 2 Effects of kaempferol (a), myricetin (b) and quercetin (c) on Human cervical adenocarcinoma (HeLa) cell proliferations by MTT (Black color bar) and SRB (Gray color bar) assays. Percentage of cell proliferation compared with non-treatment group (control) was represented as mean  SD (n = 6). * P < 0.001 when compared with the control. The effects of three flavonoids on Total ERK1/2 protein effectively than kaempferol (0.36, 0.46, 0.40 and 0.32 times, respectively). The phosphorylated ERK protein expression in LLC-MK2 and HeLa cells: LLC-MK2 and expression at 24h was also evaluated but the protein band did not express at this incubation time (data not HeLa cells were sub-cultured into 6-well plates with shown). free-bovine serum DMEM before treatment with various reagents for 24h. According to Western blot Discussion analysis of total ERK1/2 protein expression, kaempferol, myricetin and quercetin (1, 5, 10 and 50 The assays of cytotoxic effects revealed the µM) did not effect normal epithelial cells after high safety of kaempferol on normal epithelial cells. incubation (Fig.3). On HeLa cells, myricetin could not Compared with the epithelial cells from the human inhibit total ERK1/2 protein expression. However, two umbilical vein, the IC50 of kaempferol, myricetin and other flavonols: kaempferol and quercetin had obvious quercetin were 20, 100 and 50 µM, respectively (Kim et inhibitory effects on cancer cells (Fig.4). According to al., 2006). Myricetin and quercetin exhibited more protein density measurement, all concentrations of cytotoxicity than kaempferol on enterocytes of guinea kaempferol (1, 5, 10 and 50 µM) decreased the density pigs (Canada et al., 1989). of protein expression more then 0.57, 0.60, 0.70 and 0.66 times, respectively when compared with non- Starting from 5 µM, kaempferol significantly treatment control. After 24h treatment, 1, 5, 10 and 50 affected HeLa cell proliferations via reduction of total µM quercetin could supress total ERK1/2 less ERK1/2 protein at 24h. Compared with the kaempferol

Boonmasawai S. et al. / Thai J Vet Med. 2018. 48(4): 541-549. 545 extracted from Ginkgo Biloba, this natural active proliferation activities without any effects on the ERK protein. Hence, the mechanism of kaempferol may not ingredient inhibited pancreatic cancer cells at a higher usually inhibit cancer cell proliferations by reducing concentration (70 µM) and for longer periods (4 days) the MEK/ERK pathway (Sonoki et al., 2017). (Zhang et al., 2008). In osteosarcoma and oral cancer, Kaempferol had many alternative pathways including the anti-cancer activities of kaempferol related to the suppression of claudin-2 protein (Ikari et al., 2012) or inhibition of phosphorylated ERK (Lin et al, 2013) but angiogenesis-derived VEGF expression (Luo et al., did not effect on total ERK1/2 protein. Differently from 2012) after MEK and ERK activations. lung cancer (Ikari et al., 2012) and cholangiocarcinoma (Qin et al., 2016), kaempferol exhibited anti- Figure 3 Effects of kaempferol (a), myricetin and quercetin (b) on total ERK protein expression in LLC-MK2 cells. Figure 4 Effects of kaempferol (a), myricetin and quercetin (b) on total ERK protein expression in HeLa cells.

546 Boonmasawai S. et al. / Thai J Vet Med. 2018. 48(4): 541-549. Myricetin, the very common flavonols in Conflict of interest: The authors have no conflict of berries, vegetables, teas and wines possess anti- interest to declare proliferative activities against many types of cancer (Semwal et al., 2016). The molecular mechanism of Acknowledgements myricetin on cancer cells is related to the restriction of Akt/PKB (protein kinase B), MEK, and Janus kinase– This research project was supported by a signal transducer and activator of transcription (JAK- grant from Mahidol University, Thailand.. STAT) oncoproteins (Devi et al., 2015). The synthetic myricetin derivatives had potency on telomerase References inhibition and could down-regulate p65 and telomerase reverse transcriptase (TERT) expression in Boonmasawai S, Leesombun A, Chaichoun K, Taowan breast cancer (Xue et al., 2015). Moreover, the J, Sariya L and Thongjuy O. 2017. Effects of the suppression of phosphorylated ERK by myricetin three flavonoids; kaempferol, quercetin, and could depress cancer cell invasion and migration (Shih myricetin on Baby hamster kidney (BHK-21) cells et al., 2009). Our data showed the anti-cancer potency and Human hepatocellular carcinoma cell of myricetin on human cervical cancer cells at 5 times (HepG2) cells proliferations and total Erk1/2 lower than normal epithelial cells. However, these anti- protein expression. J Appl Anim Sci. 10(2): 23-34. proliferative effects did not exert via ERK1/2 protein reduction. Previous studies demonstrated that the anti- Cagnol S and Chambard JC. 2010. ERK and cell death: cancer mechanism of myricetin on human cervical mechanisms of ERK-induced cell death-apoptosis, cancer cells could occur by inducing caspase-3 activity autophagy and senescence. The FEBS journal. (Yi et al., 2015) and a p53-dependent apoptotic 277(1): 2-21. pathway (Huang et al., 2015). Canada AT, Watkins WD and Nguyen TD. 1989. The Quercetin had anti-proliferative effects on toxicity of flavonoids to guinea pig enterocytes. HeLa cells as well as other nine human cancer cells Toxicol Appl Pharmacol. 99(2): 357-61. including colon, prostate, pheocromocytoma, breast, lymphoblastic leukemia, myeloma, lymphoid and Cao J, Xia X, Chen X, Xiao J and Wang Q. 2013. ovarian cancer cells (Hashemzaei et al., 2017), with Characterization of flavonoids from Dryopteris harmless effects on normal fibroblasts (Mier-Giraldo et erythrosora and evaluation of their antioxidant, al., 2017). Quercetin could exert anti-tumor effects via anticancer and acetylcholinesterase inhibition total ERK1/2 inhibition in HeLa cells and Human activities. Food Chem Toxicol. 51: 242-50. hepatocellular carcinoma (HepG2) (Boonmasawai et al., 2017). In addition, quercetin also down-regulated Carvalho D, Paulino M, Polticelli F, Arredondo F, lipopolysaccharides (LPS)-induced JNK and ERK Williams RJ and Abin-Carriquiry JA. 2017. phosphorylation in macrophage (Park et al., 2016). In Structural evidence of quercetin multi-target human neuroglioma cells, quercetin induced bioactivity: A reverse virtual screening strategy. autophagy and apoptosis of cancer cells downstream Eur J Pharm Sci. 106: 393-403. after ERK activation (Lou et al., 2016). Autophagy had antitumor effects under certain conditions (Lei et al., Chang HK, Kim DS, Chae JJ, Kim M, Myong JP, Lee 2017). The quercetin-induced autophagy could be KH, Lee MW and Park TC. 2017. Inhibition of reduced by ERK phosphorylation inhibitor (Zhao et al., ERK activity enhances the cytotoxic effect of 2017). Quercetin could inhibit cell proliferations and peroxisome proliferator-activated receptor induce protective autophagy at low concentrations in gamma (PPARgamma) agonists in HeLa cells. HeLa cells (Wang et al., 2016). Biochem Biophys Res Commun. 482(4): 843-8. In conclusion, kaempferol (5 µM) and Chen HJ, Lin CM, Lee CY, Shih NC, Peng SF, Tsuzuki quercetin (1 µM) could significantly inhibit HeLa cell M, Amagaya S, Huang WW and Yang JS. 2013. proliferations through reduction of total ERK protein Kaempferol suppresses cell metastasis via at 24h. The anti-proliferative effects of myricetin (1 µM) inhibition of the ERK-p38-JNK and AP-1 signaling on these human cervical carcinoma cells did not exert pathways in U-2 OS human osteosarcoma cells. via total ERK1/2 suppression. While the three flavonols Oncol Rep. 30(2): 925-32. also had significant antiproliferative effects on HepG2, kaempferol did not have the inhibitory effects via Devi KP, Rajavel T, Habtemariam S, Nabavi SF and ERK1/2 protein reduction at 24h (Boonmasawai et al., Nabavi SM. 2015. Molecular mechanisms 2017). Thus, these flavonols have a future in cancer cell underlying anticancer effects of myricetin. Life therapeutic applications as chemotherapeutic agents Sci. 142: 19-25. and herbal supplements. Many edible plants naturally contain all three flavonols in single trees such as George VC, Dellaire G and Rupasinghe HPV. 2017. Dryopteris erythrosora (Cao et al., 2013), Matricaria Plant flavonoids in cancer chemoprevention: role chamomilla L. (Viapiana et al., 2016) and Camellia in genome stability. J Nutr Biochem. 45: 1-14. sinensis (Zhao et al., 2017). Kaempferol, myricetin and Guo H, Ren F, Zhang L, Zhang X, Yang R, Xie B, Li Z, quercetin can totally produce the useful effects from Hu Z, Duan Z and Zhang J. 2016. Kaempferol some single herb consumption. Therefore, the induces apoptosis in HepG2 cells via activation of synergistic effects of three flavonoids should be further the endoplasmic reticulum stress pathway. Mol investigated. Med Rep. 13(3): 2791-800. Hashemzaei M, Delarami Far A, Yari A, Heravi RE, Tabrizian K, Taghdisi SM, Sadegh SE, Tsarouhas K, Kouretas D, Tzanakakis G, Nikitovic D, Anisimov NY, Spandidos DA, Tsatsakis AM and

Boonmasawai S. et al. / Thai J Vet Med. 2018. 48(4): 541-549. 547 Rezaee R. 2017. Anticancer and apoptosis matrix metalloproteinase-2 expression by down- inducing effects of quercetin in vitro and in vivo. regulating ERK1/2 and the activator protein-1 Oncol Rep. 38(2): 819-28. signaling pathways in oral cancer cells. PLoS One. Huang H, Chen AY, Ye X, Li B, Rojanasakul Y, Rankin 8(11): e80883. GO and Chen YC. 2015. Myricetin inhibits Lou M, Zhang LN, Ji PG, Feng FQ, Liu JH, Yang C, Li proliferation of cisplatin-resistant cancer cells BF and Wang L. 2016. Quercetin nanoparticles through a p53-dependent apoptotic pathway. Int J induced autophagy and apoptosis through AKT/ERK/Caspase-3 signaling pathway in human Oncol. 47(4): 1494-502. neuroglioma cells: In vitro and in vivo. Biomed Ikari A, Sato T, Watanabe R, Yamazaki Y and Sugatani J. 2012. Increase in claudin-2 expression by an Pharmacother. 84: 1-9. EGFR/MEK/ERK/c-Fos pathway in lung Luo H, Rankin GO, Juliano N, Jiang BH and Chen YC. adenocarcinoma A549 cells. Biochim Biophys 2012. Kaempferol inhibits VEGF expression and Acta. 1823(6): 1110-8. in vitro angiogenesis through a novel ERK- Jeong JC, Kim MS, Kim TH and Kim YK. 2009. NFkappaB-cMyc-p21 pathway. Food Chem. 130(2): Kaempferol induces cell death through ERK and 321-8. Akt-dependent down-regulation of XIAP and survivin in human glioma cells. Neurochem Res. Mendoza EE and Burd R. 2011. Quercetin as a systemic chemopreventative agent: structural and 34(5): 991-1001. functional mechanisms. Mini Rev Med Chem. Jo S, Ha TK, Han SH, Kim ME, Jung I, Lee HW, Bae SK and Lee JS. 2017. Myricetin induces apoptosis of 11(14): 1216-21. human anaplastic thyroid cancer cells via Miean KH and Mohamed S. 2001. Flavonoid (myricetin, mitochondria dysfunction. Anticancer Res. 37(4): quercetin, kaempferol, luteolin, and apigenin) 1705-10. content of edible tropical plants. J Agric Food Kim JD, Liu L, Guo W and Meydani M. 2006. Chemical Chem. 49(6): 3106-12. structure of flavonols in relation to modulation of angiogenesis and immune-endothelial cell Mier-Giraldo H, Diaz-Barrera LE, Delgado-Murcia LG, adhesion. J Nutr Biochem. 17(3): 165-76. Valero-Valdivieso MF and Caez-Ramirez G. 2017. Cytotoxic and immunomodulatory potential Kim ME, Ha TK, Yoon JH and Lee JS. 2014. Myricetin activity of Physalis peruviana fruit extracts on induces cell death of human colon cancer cells via Cervical cancer (HeLa) and Fibroblast (L929) cells. BAX/BCL2-dependent pathway. Anticancer Res. J Evid Based Complementary Altern Med. 22(4): 34(2): 701-6. Kumar S and Pandey AK. 2013. Chemistry and 777-787. Mosmann T. 1983. Rapid colorimetric assay for cellular biological activities of flavonoids: an overview. growth and survival: application to proliferation ScientificWorldJournal. 2013: 162750. Lee GA, Choi KC and Hwang KA. 2017. Kaempferol, a and cytotoxicity assays. J Immunol Methods. 65(1- phytoestrogen, suppressed triclosan-induced 2): 55-63. Orellana EA and Kasinski AL. 2016. Sulforhodamine B epithelial-mesenchymal transition and metastatic- related behaviors of MCF-7 breast cancer cells. (SRB) assay in cell culture to investigate cell Environ Toxicol Pharmacol. 49: 48-57. proliferation. Bio Protoc. 5: 6(21). Panche AN, Diwan AD and Chandra SR. 2016. Lee KW, Kang NJ, Heo YS, Rogozin EA, Pugliese A, Hwang MK, Bowden GT, Bode AM, Lee HJ and Flavonoids: an overview. J Nutr Sci. 5: e47. Dong Z. 2008. Raf and MEK protein kinases are direct molecular targets for the chemopreventive Park JY, Lim MS, Kim SI, Lee HJ, Kim SS, Kwon YS and effect of quercetin, a major flavonol in red wine. Chun W. 2016. Quercetin-3-O-beta-D-glucuronide Cancer Res. 68(3): 946-55. suppresses lipopolysaccharide-induced JNK and Lee KW, Kang NJ, Rogozin EA, Kim HG, Cho YY, Bode ERK phosphorylation in LPS-challenged AM, Lee HJ, Surh YJ, Bowden GT and Dong Z. RAW264.7 Cells. Biomol Ther (Seoul). 24(6): 610-5. 2007. Myricetin is a novel natural inhibitor of Patra S, Upadhyay M and Chhabra P. 2017. Awareness neoplastic cell transformation and MEK1. of cervical cancer and willingness to participate in Carcinogenesis. 28(9): 1918-27. screening program: Public health policy Lei Y, Zhang D, Yu J, Dong H, Zhang J and Yang S. implications. J Cancer Res Ther. 13(2): 318-23. 2017. Targeting autophagy in cancer stem cells as Phillips P, Sangwan V, Borja-Cacho D, Dudeja V, an anticancer therapy. Cancer Lett. 393: 33-9. Vickers S and Saluja A. 2011. Myricetin induces pancreatic cancer cell death via the induction of Li H and Chen C. 2017. Quercetin has antimetastatic apoptosis and inhibition of the effects on gastric cancer cells via the interruption phosphatidylinositol 3-kinase (PI3K) signaling of uPA/uPAR function by modulating NF- pathway. Cancer Lett. 308(2): 181-8. kappab, PKC-delta, ERK1/2, and AMPKalpha. Qin Y, Cui W, Yang X and Tong B. 2016. Kaempferol Integr Cancer Ther. 1534735417696702. inhibits the growth and metastasis of Lin CW, Chen PN, Chen MK, Yang WE, Tang CH, cholangiocarcinoma in vitro and in vivo. Acta Yang SF and Hsieh YS. 2013. Kaempferol reduces Biochim Biophys Sin (Shanghai). 48(3): 238-45. Qiu W, Lin J, Zhu Y, Zhang J, Zeng L, Su M and Tian Y. 2017. Kaempferol modulates DNA methylation

5468 Boonmasawai S. et al. / Thai J Vet Med. 2018. 48(4): 541-549. and downregulates DNMT3B in bladder cancer. Yi JL, Shi S, Shen YL, Wang L, Chen HY, Zhu J and Ding Y. 2015. Myricetin and methyl eugenol Cell Physiol Biochem. 41(4): 1325-35. combination enhances the anticancer activity, cell Semwal DK, Semwal RB, Combrinck S and Viljoen A. cycle arrest and apoptosis induction of cis-platin 2016. Myricetin: A dietary molecule with diverse against HeLa cervical cancer cell lines. Int J Clin biological activities. Nutrients. 8(2): 90. Exp Pathol. 8(2): 1116-27. Shih YW, Wu PF, Lee YC, Shi MD and Chiang TA. 2009. Zeng Y, Cheng AS, Liu X and Chan CC. 2017. Cervical Myricetin suppresses invasion and migration of cancer survivors' perceived cognitive complaints and supportive care needs in mainland China: a human lung adenocarcinoma A549 cells: possible qualitative study. BMJ Open. 7(6): e014078. mediation by blocking the ERK signaling Zhang Y, Chen AY, Li M, Chen C and Yao Q. 2008. pathway. J Agric Food Chem. 57(9): 3490-9. Ginkgo biloba extract kaempferol inhibits cell Somerset SM and Johannot L. 2008. Dietary flavonoid proliferation and induces apoptosis in pancreatic cancer cells. J Surg Res. 148(1): 17-23. sources in Australian adults. Nutr Cancer. 60(4): Zhao X, Wang P, Li M, Wang Y, Jiang X, Cui L, Qian Y, 442-9. Zhuang J, Gao L and Xia T. 2017. Functional Sonoki H, Tanimae A, Endo S, Matsunaga T, Furuta T, characterization of a new tea (Camellia sinensis) Ichihara K and Ikari A. 2017. Kaempherol and flavonoid glycosyltransferase. J Agric Food Chem. luteolin decrease claudin-2 expression mediated 65(10): 2074-83. by inhibition of STAT3 in lung adenocarcinoma Zhao Y, Fan D, Zheng ZP, Li ET, Chen F, Cheng KW and Wang M. 2017. 8-C-(E-phenylethenyl) A549 Cells. Nutrients. 9(6): E597. quercetin from onion/beef soup induces Su Q, Peng M, Zhang Y, Xu W, Darko KO, Tao T, autophagic cell death in colon cancer cells Huang Y, Tao X and Yang X. 2016. Quercetin through ERK activation. Mol Nutr Food Res. 61(2): induces bladder cancer cells apoptosis by 1600437. activation of AMPK signaling pathway. Am J Cancer Res. 6(2): 498-508. Tao T, He C, Deng J, Huang Y, Su Q, Peng M, Yi M, Darko KO, Zou H and Yang X. 2017. A novel synthetic derivative of quercetin, 8- trifluoromethyl- 3,5,7,3',4'-O-pentamethyl- quercetin, inhibits bladder cancer growth by targeting the AMPK/mTOR signaling pathway. Oncotarget. 8(42): 71657-71671. van Tonder A, Joubert AM and Cromarty AD. 2015. Limitations of the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl-2H-tetrazolium bromide (MTT) assay when compared to three commonly used cell enumeration assays. BMC Res Notes. 20(8): 47. Viapiana A, Struck-Lewicka W, Konieczynski P, Wesolowski M and Kaliszan R. 2016. An Approach based on HPLC-fingerprint and chemometrics to quality consistency evaluation of Matricaria chamomilla L. commercial samples. Front Plant Sci. 7: 1561. Vichai V and Kirtikara K. 2006. Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat Protoc. 1(3): 1112-6. Wang Y, Zhang W, Lv Q, Zhang J and Zhu D. 2016. The critical role of quercetin in autophagy and apoptosis in HeLa cells. Tumour Biol. 37(1): 925-9. Xie F, Su M, Qiu W, Zhang M, Guo Z, Su B, Liu J, Li X and Zhou L. 2013. Kaempferol promotes apoptosis in human bladder cancer cells by inducing the tumor suppressor, PTEN. Int J Mol Sci. 14(11): 21215-26. Xue W, Song B-A, Zhao HJ, Qi XB, Huang YJ and Liu XH. 2015. Novel myricetin derivatives: Design, synthesis and anticancer activity. Eur J Med Chem. 97: 155-63. Yao LH, Jiang YM, Shi J, Tomás-Barberán FA, Datta N, Singanusong R and Chen SS. 2004. Flavonoids in food and their health benefits. Plant Foods Hum Nutr. 59(3): 113-22.

Boonmasawai S. et al. / Thai J Vet Med. 2018. 48(4): 541-549. 55497 บทคัดยอ่ การยบั ย้งั การเพิ่มจานวนของเซลล์ และการแสดงออกของโปรตีน total ERK1/2 ของสาร กล่มุ ฟลาโวนอลจากพืช ที่มตี ่อเซลลม์ ะเรง็ ปากมดลูก ชนิด Human cervical cancer (HeLa) สุขฤทยั บญุ มาไสว1* ลดาวลั ย์ สาริยา2 อาภรณ์ ล้สี มบญุ 1 กฤษฎา ใจชืน้ 1 จารุภา เถาวลั ย์2 อรทัย ทองจุย้ 3 kaempferol myricetin และ quercetin เป็นสารกลุ่มฟลาโวนอล (flavonols) ทีพ่ บมากในผักและผลไม้ท่ีรับประทานได้ มี รายงานจานวนมากก่อนหน้านี้ แสดงถงึ ประสิทธภิ าพของฟลาโวนอลดงั กลา่ ว ว่ามคี ุณสมบัติอันเป็นประโยชน์ ในการรกั ษามะเรง็ หลายชนดิ การทดลองครง้ั นี้ จึงได้ทาการทดสอบความเปน็ พษิ ตอ่ เซลล์ (cytotoxic effects) ของ kaempferol myricetin และ quercetin ทีม่ ีตอ่ เซลล์ ชนดิ rhesus monkey kidney epithelial cells (LLC-MK2) และการยับยงั้ การเพิ่มจานวนของเซลล์มะเร็ง (anti-proliferative effects) ผ่านการแสดงออกของโปรตีน total ERK ในเซลลม์ ะเรง็ ปากมดลกู (HeLa cells) ผลการทดลองท่ี 24 ช่วั โมง แสดงวา่ ฟลาโวนอลทงั้ สามชนิด มคี วามปลอดภยั สงู ต่อเซลลช์ นดิ LLC-MK2 ส่วน kaempferol ท่คี วามเข้มข้น 1-1000 µM ไมม่ ีผลตอ่ viability ของ epithelial cells ความ เข้มขน้ ของ myricetin ทเ่ี ปน็ พิษต่อเซลลป์ กติ มีคา่ เท่ากบั 5 µM สาร quercetin มผี ลต่อเซลล์ปกตอิ ยา่ งมนี ยั สาคัญ ทคี่ วามเขม้ ขน้ 50 µM สารฟลาโวนอลท้งั หมด ไมส่ ามารถลดการแสดงออกของโปรตีน total ERK1/2 ในเซลล์ LLC-MK2 ได้ สว่ นการทดลองในเซลล์ HeLa พบวา่ kaempferol (5 µM) และ quercetin (1 µM) ยับยง้ั ทง้ั การเพมิ่ จานวนของเซลล์ HeLa และการแสดงออกของโปรตีน total ERK1/2 สว่ น myricetin ยับย้ังเซลลม์ ะเร็งอยา่ งมีนัยสาคัญทางสถิติ ที่ 1 µM แตไ่ มย่ บั ยัง้ การแสดงออกของโปรตนี total ERK1/2 สรปุ ได้วา่ kaempferol และ quercetin สามารถยบั ยงั้ การเพิ่มจานวนของเซลล์มะเรง็ ผ่านการแสดงออกของโปรตนี total ERK1/2 ที่ 24 ชั่วโมง แตผ่ ลการยบั ย้ังของ myricetin ไม่ไดก้ ระตุ้นผ่านกลไกดังกล่าว การทดลองน้ียนื ยันผลของฟลาโวนอลท้งั สามชนดิ ว่า มแี นวโน้มท่ีจะเป็นไดท้ งั้ ยารกั ษาโรคมะเร็ง และอาหารเสริมสมุนไพรในอนาคต คาสาคัญ: ฟลาโวนอล เซลล์มะเรง็ ปากมดลกู kaempferol myricetin quercetin 1ภาควชิ าปรคี ลนิ ิกและสัตวศาสตร์ประยกุ ต์ 2ศนู ยเ์ ฝ้าระวงั และติดตามโรคจากสตั วป์ า่ สัตวต์ า่ งถิน่ และสตั ว์อพยพ 3ศูนยต์ รวจวินจิ ฉยั ทางการ สัตวแพทย์ คณะสตั วแพทยศาสตร์ มหาวทิ ยาลยั มหดิ ล 999 ถนนพทุ ธมณฑลสายส่ี ตาบลศาลายา อาเภอพทุ ธมณฑล จงั หวดั นครปฐม ประเทศไทย 73170 *ผ้รู บั ผดิ ชอบบทความ E-mail: [email protected]



Original Article High infection rate of Zika virus in mosquitoes collected from an area of active Zika virus transmission in eastern Thailand Apiwat Tawatsin1 Atchara Phumee2,5 Usavadee Thavara1 Patcharawan Sirisopa1 Wanapa Ritthison3 Kitsanaphong Thammakosol4 Proawpilart Intayot4 Yutthana Joyjinda2 Supaporn Wacharapluesadee2 Thiravat Hemachudha2 Padet Siriyasatien5* Abstract Zika virus (ZIKV), chikungunya virus (CHIKV) and dengue virus (DENV) are emerging and re-emerging arboviral diseases. These viruses are transmitted to humans through the bites of Aedes mosquitoes. Recently, ZIKV infection has been described as an emerging disease in Thailand and many countries, especially in tropical and sub- tropical areas. Specific drugs and vaccines against these infections are unavailable; therefore, effective disease control relies on vector control measures only. To understand the transmission cycle of these viruses and mosquito vectors, this study is designed to investigate the natural infection of ZIKV, CHIKV and DENV in field-caught mosquitoes by molecular techniques. Adults and larvae of mosquitoes were collected in and around the patients’ homes in the Klaeng District, Rayong Province, Thailand. CHIKV and DENV were detected by Multiplex Real-time RT-PCR and ZIKV was detected by Hemi-nested RT-PCR. ZIKV RNA was detected in 8 (10.3%) samples (5 (6.4%) females and 2 (2.6%) males of Aedes aegypti and 1 (1.3%) female Armigeres subalbatus) and CHIKV RNA in 5 (6.4%) (3 (3.8%) females and 2 (2.6%) larvae of Ae. aegypti), while DENV RNA was not detected in any samples. The Maximum Likelihood tree of nucleotide sequences of positive samples showed that ZIKV in mosquitoes were cladded within the Asian lineage. This study was a preliminary survey of the potential vectors of ZIKV, CHIKV and DENV in an affected area. Information obtained from this study helps to understand the natural infection rates in mosquitoes with ZIKV, CHIKV and DENV and may be valuable in creating the most effective mosquito vector control strategies in the future. Keywords: Zika, Chikungunya, Dengue, Mosquitoes, Thailand 1National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Nonthaburi, Thailand 2Thai Red Cross Emerging Infectious Health Science Centre, Neuroscience Center for Research and Development & WHO-CC for Research and Training on Viral Zoonoses, King Chulalongkorn Memorial Hospital, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand 3The Office of Disease Prevention and Control 6 Chonburi, Department of Disease Control, Ministry of Public Health, Chonburi, Thailand 4Medical Science Program, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand 5Vector Biology and Vector Borne Disease Research Unit, Department of Parasitology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand *Correspondence: [email protected] Thai J Vet Med. 2018. 48(4): 551-558.

552 Tawatsin A. et al. / Thai J Vet Med. 2018. 48(4): 551-558. Introduction Medicine, Chulalongkorn University, Bangkok, Thailand (COA No. 023/2560). Zika virus (ZIKV), dengue virus (DENV), and chikungunya virus (CHIKV) are arthropod-borne Sample collection: Larval or adult mosquitoes were viruses or arboviruses (Waggoner and Pinsky, 2016). collected from inside and outside patients’ homes in These viruses are primarily transmitted to humans by the Klaeng District, Rayong Province, eastern Aedes mosquitoes, and patients infected with these Thailand. These samples were randomized and viruses present similar clinical symptoms (Vasconcelos separated individually, kept in liquid nitrogen and and Calisher, 2016). Dengue (DEN) fever and transferred to the laboratory of Entomology, chikungunya (CHIK) fever are commonly found in Department of Parasitology, Faculty of Medicine, most tropical regions, whereas Zika (ZIK) fever is an Chulalongkorn University for viral detection. emerging infectious disease in areas of Africa, Asia, the Americas, and the Pacific Islands (Pastula et al., 2016). Viral RNA extraction: Individual mosquito specimens ZIKV infections were first described in Thailand in were extracted for viral RNA using a viral RNA 1954 in sera of indigenous residents by using extraction kit, Invisorb® Spin Virus RNA Mini kit neutralizing antibodies against several arthropod- (STRATEC molecular GmbH, Germany) following the borne viruses, such as ZIKV, West Nile virus (WNV), manufacturer’s instructions. RNA concentration and and Japanese encephalitis (JE) (Pond, 1963). Recently, purity were quantified by a Nano Drop 2000c several ZIK cases were reported among some travelers spectrophotometer (Thermo scientific, USA). (Canadian, German, and Japanese) returning from Thailand (Fonseca et al., 2014; Tappe et al., 2014; Multiplex Real-time RT-PCR for the Detection of Shinohara, 2014). Buathong et al. (2015) described CHIKV and DENV: CHIKV and 4 serotypes of DENV seven cases (3 patients from the Ratchaburi province, 2 were detected in samples in a single reaction tube [Cy5 patients from the Phetchabun province, 1 patient from (DEN1), FAM (DEN2), Texas Red (DEN3), Quasar 705 the Sisaket province, and 1 patient from the Lamphun (DEN4), and HEX (CHIKV)] using a commercial kit. province) of autochthonous acute ZIKV infections. One-step Multiplex Real-time RT-PCR was performed This diagnosis was confirmed by molecular or using an abTES™ DEN 5 qPCR I Kit (AIT biotech Pte. serological testing. The phylogenetic tree showed that Ltd, Singapore) on a CFX96 Real-Time System (Bio- these ZIKV were of Asian lineage (Buathong et al., Rad Laboratories, Inc, USA). The total volume of the 2015). On the other hand, the outbreak of CHIK fever reaction mixture was 25 μl, including 5.4 μl of nuclease- in Thailand was first reported in 1958 (Hammon et al., free water, 12.5 μl of 2x RT-PCR reaction mix, 0.1 μl of 1960), and it re-emerged in the southern region in PCR enhancer mix, 1.5 μl of DEN 5 primer/probe mix, 2008–2009 with more than 50,000 patients (Ungchusak, 0.5 μl RT/Taq enzyme mix, and 5 μl of viral RNA 2008; Suangto and Uppapong, 2009; Thavara et al., template. The cDNA synthesis was performed for 10 2009). In 2013, CHIK fever had become established in mins at 53°C, the Taq activation was performed for 2 BuengKan Province in northeastern Thailand min 30 sec at 95°C, followed by 41 cycles of 95°C for 17 (Wanlapakorn et al., 2014). For DENV in Thailand, the sec, 59°C for 31 sec and 68°C for 32 sec, with a final first major outbreak was in Bangkok in 1958 (Gubler, cycle of 68°C for 7 mins and a final holding at 4°C. 1997). Dengue disease cases in Thailand include four Samples were considered as having a positive result if DENV serotypes (DENV-1, -2, -3 or -4) (Musso et al., the specific fluorescent signals were a level higher than 2015). In 2016, the Bureau of Epidemiology (BoE) of the the threshold value. A threshold cycle of 38 was Thai Ministry of Public Health (MoPH) revealed that considered negative. approximately 63,310 cases of dengue had been found and had resulted in 61 deaths. Mosquitoes are the main ZIKV detection and sequencing: The RNA samples vectors of these three viruses (ZIKV, CHIKV and extracted from the mosquitoes were amplified by DENV) to humans; therefore, it may be expected that primer sets which were modified from Moureau et al. possible coinfection with two or three arboviruses may (2007) for detecting ZIKV RNA at the nonstructural occur in mosquitoes. In this study, we determined the protein 5 (NS5) gene region using hn-RT-PCR. The RT- natural infection of ZIKV, CHIKV, and DENV in PCR amplification reaction was set up in a final volume mosquitoes collected around patients’ homes in the of 25 μl using the Superscript III one-step RT-PCR kit. Klaeng District, Rayong Province in Thailand. CHIKV The RT-PCR conditions as follows; reverse and DENV were detected using Multiplex Real-time transcription at 50 °C for 30 mins, denaturation at 95 °C RT-PCR. Hemi-nested RT-PCR (hn-RT-PCR) for 15 mins, followed by 45 cycles of 94 °C for 20 sec, 55 developed from this study was able to detect ZIKV in °C for 20 sec, and 72°C for 30 sec and the final extension the mosquitoes effectively. Information obtained from at 72°C for 5 mins. The nested PCR was performed this study provides fundamental data for the with 2 μl from the first reaction using 1 unit of Taq understanding of natural transmission between ZIKV, DNA polymerase (Fermentas, USA) with conditions as CHIKV, and DENV and mosquitoes. Data of mosquito follows; denaturation at 94 °C for 2 mins, followed by vectors and ZIKV infection in the mosquitoes will be 45 cycles of 94 °C for 15 sec, 52 °C for 15 sec, and 72°C valuable in developing effective control strategies of for 30 sec and the final extension at 72°C for 5 min. The ZIKV, CHIKV, and DENV infections in Thailand. PCR products were analyzed via 2% agarose gel electrophoresis, stained with ethidium bromide and Materials and Methods visualized with Quantity One Quantification Analysis Software Version 4.5.2 (Gel DocEQ System; Bio-Rad, Ethics statement: The study was approved by the Hercules, CA). Positive PCR products were recovered animal research ethics committee of the Faculty of

Tawatsin A. et al. / Thai J Vet Med. 2018. 48(4): 551-558. 553 from the gel and purified using an Agarose Gel DNA Republic/2014), KF268950 (ARB7701/Central African Purification Kit: Invisorb® Fragment CleanUp Republic/2014), and KF268948 (ARB13565/Central (STRATEC Molecular GmbH, Germany) following the African Republic/1976)). manufacturer’s instructions. The purified DNA was sent for direct DNA sequencing to Macrogen Inc. Results (Macrogen, South Korea) to confirm the species identification. Nucleotide sequences were analyzed by A total of 78 mosquito samples (males (n=20), comparison with the GenBank database using a BLAST females (n=42) and larvae (n=16)) were used for ZIKV, search (https://blast.ncbi.nlm.nih. gov/Blast.cgi). CHIKV, and DENV RNA detection (Table 1). The adult mosquitoes belonged to three genera, which consist of Phylogenetic tree construction: The sequences were Aedes aegypti 69.23% (54/78), Culex quinquefasciatus aligned using BioEdit Sequence Alignment Editor 1.28% (1/78) and Armigeres subalbatus 8.98% (7/78), Version 7.1.9. The phylogenetic trees were constructed while larvae were identified as Aedes spp 20.51% using the Maximum Likelihood with Kimura’s 2- (16/78). The results of virus detection showed that parameter and bootstrap analysis with 1,000 ZIKV RNA was detected in 6 (7.69%) females of Ae. replications in MEGA Version 7.0. The sequences aegypti and Ar. subalbatus and 2 (2.56%) male of Ae. obtained from this study were analyzed against 7 aegypti using hn-RT-PCR (Figure 1A). Interestingly, reference strains of Asian lineage (GenBank accession ZIKV RNA was found in a female of Ar. subalbatus, and nos. JN860885 (FSS13025/Cambodia/2010), EU545988 the sequences of the NS5 genes of ZIKV amplified in (Yap/Micronesia/2007), KU312312 (Z1106033/ this study were 282-284 base pairs. The ZIKV Suriname/2015), KX694532 (THA_PLCal_ZV_2013/ sequences showed 99-100% sequence identity to the Thailand/2013), KF993678 (PLCal_ZV/Canada/2013), partial NS5 genes of ZIKV available in the GenBank KU321639 (ZikaSPH2015/Brazil/2015), and HQ234499 database. The nucleotide sequences of the NS5 of ZIKV (P6-740/Malaysia/1966)) and 7 reference strains from were submitted to the GenBank database, accession no African lineage (NC012532 (MR_766/Uganda/2005), MH306209-MH306216. The phylogenetic tree analysis LC002520 (MR766-NIID/Uganda/2014), HQ234501 clearly showed separate African and Asian lineages (ArD_41519/Senegal/1984), HQ234500 (IbH_30656/ and all ZIKV sequences in mosquitoes in this study Nigeria/1968), KF268949 (ARB15076/Central African were claded within the Asian lineage (Figure 1B). Table 1 Mosquito sample collection and molecular detection results Area of Species stage Sex Total Sample CHIKV Results ZIKV patients’ (n) DENV homes Ae. aegypti Adult Male 0 1 Cx. quinquefasciatus Adult Female 19 3 4 serotypes 4 Indoor Larva Male 31 N/A 0 N/A Aedes spp. Adult Female N/A 0 0 0 Outdoor Ae. aegypti Adult N/A 1 0 0 Ar. subalbatus Larva Male 1 0 N/A 1 Aedes spp. Female 0 0 1 Male 1 N/A N/A 3 0 Female N/A 0 1 N/A 2 0 0 7 0 15 N/A 0 0 78 5 08 N/A: not available In addition, 5 samples (3 females and 2 larvae) Discussion in Aedes spp. were positive for CHIKV RNA by one-step Multiplex Real-time RT-PCR, abTES™ DEN 5 qPCR I In the present study, ZIKV RNA was mostly detected in Ae. aegypti mosquitoes and also in a Ae. Kit (Figures 2A and 2B). Samples reported as positive aegypti male. Several reports revealed that Aedes had cycle threshold (Ct) values ≤38. In this study, 5 mosquitoes such as Ae. africanus, Ae. apicocoargenteus, samples were positive with Ct values of 32.19, 34.45, Ae. furcifer, Ae. luteocephalus, Ae. vitattus and Ae. aegypti and 30.32 in adult mosquitoes and 34.7 and 36.7 in are the principle vectors of ZIKV in Africa (Weinbren larvae. However, DENV showed negative results in all and Williams, 1958; Haddow et al., 1964; Fagbami et samples. al., 1979; McCrae and Kirya, 1982). In Southeast Asia, ZIKV was isolated from wild caught Ae. aegypti mosquitoes in Malaysia (Marchette et al., 1969); moreover, Ae. aegypti (Li et al., 2012) and Ae. albopictus

554 Tawatsin A. et al. / Thai J Vet Med. 2018. 48(4): 551-558. (Wong et al., 2013) mosquitoes were also reported as These findings determined that ZIKV may have a potential vectors for ZIKV transmission in Singapore. wider range of vectors. However, vectors for ZIKV A few reports found that ZIKV can be detected in transmission in Thailand have never been investigated. fields, including a collected male Ae. aegypti from Rio This is the first report that ZIKV can be found in female de Janeiro, Brazil (Ferreira-de-Brito et al., 2016) and Ar. subalbatus and in male Ae. aegypti mosquitoes in male Ae. furcifer mosquitoes collected from Senegal Thailand. This is the first study to detect ZIKV RNA in (Diallo et al., 2014). These findings are supported by Ar. subalbatus, and this mosquito species is commonly the recent experiment reported by Thangamani et al. found in tropical and subtropical areas. Currently, (2016), in which they demonstrated the occurrence of there is no study investigating Ar. subalbatus as a vector vertical transmission of ZIKV in Aedes mosquitoes in for ZIKV. Our results indicated that Ar. subalbatus F1 adult progeny. Surprisingly, we were able to detect could be another potential vector for ZIKV ZIKV RNA in a female Ar. subalbatus mosquito. Several transmission in this region. Further studies to reports mentioned that other mosquito genera could investigate the potential vectors for ZIKV transmission transmit ZIKV. Diallo et al. (2014) reported that ZIKV are required. ZIKV has been identified within two was detected in Cx. perfuscus, Mansonia uniformis and major lineages, African and Asian through Anopheles coustani collected from Southeastern Senegal phylogenetic analyses. The phylogenetic tree indicated by virus isolation and RT-PCR (Diallo et al., 2014). that the ZIKV in this study belong to the Asian lineage. Recently, there have been reported that Cx. pipiens The Asian lineage of ZIKV had been historically quinquefasciatus is a potential vector to transmit ZIKV reported in Malaysia, the Philippines, Pakistan, in southern China (Thangamani et al., 2016), and field- Cambodia and Thailand and caused an outbreak on caught Cx. quinquefasciatus showed positive ZIKV by Yap Island in 2007 (Gudo et al., 2016). RT-qPCR (Guo et al., 2016; Guedes DRD et al., 2016). Figure 1 A) The 2% agarose gel images showing the amplified product of size 282 bp of ZIKV from hn-RT-PCR (Lane M: molecular mass marker (100 base pairs [bp]); lane P: positive control, lane N: negative control (no DNA template: double-distilled water); lane N1: uninfected adult mosquito; lane N2: uninfected larval mosquito: Lane S1-12: mosquito samples). (B) Phylogenetic tree of a NS5 gene of ZIKV from mosquitoes (indicated in a red circle) compared with reference isolates obtained from GenBank. The tree was derived using the Maximum Likelihood method based on the Kimura 2-parameter model (bootstrap 1,000 times).

Tawatsin A. et al. / Thai J Vet Med. 2018. 48(4): 551-558. 555 Figure 2 (A) Showing positive of CHIKV in mosquitoes using Multiplex Real-time RT-PCR for CHIKV and DENV [Cy5 (DEN1), FAM (DEN2), Texas Red (DEN3), Quasar 705 (DEN4), and HEX (CHIKV)]. (B) Mosquito sample showed positive with CHIKV. CHIKV has three distinct genotypes based on Competing interests: The authors declare that they the E1 envelope glycoprotein sequences, including the have no competing interests. West African genotype, the East, Central and South African (ECSA) genotypes, and the Asian genotype Acknowledgements (Sudeep and Parashar, 2008). We found CHIKV RNA in 3 samples of female Ae. aegypti and 2 samples of This study was supported by the National Aedes spp. larva. Previous reports found that CHIKV Research Council of Thailand and Health Systems RNA present in field-caught male Ae. albopictus and Ae. Research Institute (Grant No. 61-003), National Science aegypti from Madagascar (Ratsitorahina et al., 2008) and Technology Development Agency (Thailand) (Grant No. P-16-50702) and for the Research Chair and Thailand (Thavara et al., 2009) by CHIK-specific Grant, Rachadapisek Sompote Fund for Postdoctoral RT-PCR. CHIKV transmission has been studied in Fellowship Grant No. RA/MF 11/61, Chulalongkorn various species of Aedes mosquitoes such as Ae. aegypti University. and Ae. albopictus from India (Mourya, 1987) and Ae. aegypti formosus and Ae. furcifer from South Africa References (Jupp et al., 1981); however, these experiments did not Agarwal A, Dash PK, Singh AK, Sharma S, Gopalan N, isolate CHIKV. Moreover, Ae. aegypti from India Rao PV, Parida MM and Reiter P 2014. Evidence of experimental vertical transmission of emerging showed the evidence of vertical transmission of novel ECSA genotype of Chikungunya Virus in emerging novel Indian Ocean lineage (IOL) of the Aedes aegypti. PLoS Negl Trop Dis. 8(7): e2990. ECSA genotype of CHIKV in both natural and experimental settings (Agarwal et al., 2014). In 2016, Buathong R, Hermann L, Thaisomboonsuk B, the infection of CHIKV IOL in laboratory strains of Ae. Rutvisuttinunt W, Klungthong C, aegypti and Ae. albopictus mosquitoes revealed that the Chinnawirotpisan P, Manasatienkij W, Nisalak A, Fernandez S, Yoon IK, Akrasewi P and Plipat T viruses were transmitted vertically to F5 and F6 2015. Detection of Zika Virus Infection in progenies in both Ae. aegypti and Ae. albopictus Thailand, 2012-2014. Am J Trop Med Hyg. 93(2):380-383. mosquitoes, respectively (Chompoosri et al., 2016). Our present study may support that it is possible that Chompoosri J, Thavara U, Tawatsin A, Boonserm R, there is transovarian transmission of CHIKV in Ae. Phumee A, Sangkitporn S and Siriyasatien P aegypti mosquitoes in the field. 2016. Vertical transmission of Indian Ocean Lineage of chikungunya virus in Aedes aegypti Literature suggests that virus transmission and Aedes albopictus mosquitoes. Parasit Vectors. depends on the mosquito species, geographical 9:227. location and virus type. However, extensive survey and more precise studies of ZIKV, CHIKV and DENV Diallo D, Sall AA, Diagne CT, Faye O, Faye O, Ba Y, infection in mosquito covering more areas and larger Hanley KA, Buenemann M, Weaver SC and sample sizes must be performed in order to understand Diallo M 2014. Zika virus emergence in virus-vector interaction. Although we did not find mosquitoes in southeastern Senegal, 2011. PLoS evidence of co-infecting viruses in this study, the effect ONE. 9(10): e10944. of co-infection between viruses (ZIKV, CHIKV and DENV) should be investigated. Competitive Fagbami AH 1979. Zika virus infections in Nigeria: suppression between DENV and CHIKV has been virological and seroepidemiological demonstrated by Potiwat et al. (2011). Moreover, investigations in Oyo State. J Hyg (Lond) 83(2): demonstration of the viability of viruses in mosquitoes 213–219. collected from the field is essential to confirm that the mosquitoes are vectors of ZIKV and CHIKV. Ferreira-de-Brito A, Ribeiro IP, Miranda RM, Fernandes RS, Campos SS, Silva KA, Castro MG, Bonaldo MC, Brasil P and Lourenço-de-Oliveira

556 Tawatsin A. et al. / Thai J Vet Med. 2018. 48(4): 551-558. R 2016. First detection of natural infection of Musso D. Cao-Lormeau, VM and Gubler DJ 2015. Zika Aedes aegypti with Zika virus in Brazil and virus: following the path of dengue and chikungunya? Lancet. 386(9990): 243–244. throughout South America. Mem Inst Oswaldo Cruz. 111(10): 655-658. Pastula DM, Smith DE, Beckham JD and Tyler KL 2016. Fonseca K, Meatherall B, Zarra D, Drebot M, An Overview of Zika Virus Disease. MacDonald J, Pabbaraju K, Wong S, Webster P, Neurohospitalist. 6(3): 93-94. Lindsay R and Tellier R 2014. First case of Zika virus infection in a returning Canadian traveler. Pond WL 1963. Arthropod-borne virus anti bodies in Am J Trop Med Hyg. 91(5): 1035-1038. sera from residents of South-east Asia. Trans Roy Gubler DJ 1997. Dengue and dengue hemorrhagic Soc Trop Med Hyg. 57(5): 364-371. fever: its history and resurgence as a global public health problem. In: Gubler DJ, Kuno G, editors. Potiwat R, Komalamisra N, Thavara U, Tawatsin A Dengue and dengue hemorrhagic fever. Oxford: and Siriyasatien P 2011. Competitive suppression CAB International, p. 1–22. between chikungunya and dengue virus in Aedes Gudo ES, Falk KI, Ali S, Muianga AF, Monteiro V and albopictus c6/36 cell line. Southeast Asian J Trop Cliff J 2016. A Historic Report of Zika in Mozambique: Implications for Assessing Current Med Public Health. 42(6): 1388-1394. Risk. PLoS Negl Trop Dis. 10(12): e0005052. Ratsitorahina M, Harisoa J, Ratovonjato J, Biacabe S, Guedes DR, Paiva MH, Donato MM, Barbosa PP, Krokovsky L, Rocha SWDS, Saraiva K, Crespo Reynes J-M, Zeller H, Raoelina Y, Talarmin A, MM, Rezende TM, Wallau GL, Barbosa RM, Richard V and Soares JL 2008. Outbreak of Oliveira CM, Melo-Santos MA, Pena L, Cordeiro dengue and chikungunya fevers, Toamasina, MT, Franca RFO, Oliveira AL, Peixoto CA, Leal Madagascar, 2006. Emerg Infect Dis. 14(7): 1135– WS and Ayres CF 2017. Zika virus replication in 1137. the mosquito Culex quinquefasciatus in Brazil. Shinohara K. Zika Virus - Japan ex Thailand. ProMed; 2014. http://www.promedmail.org Archive Emerg Microbes Infect. 6(8):e69. no. 20140823.2716731. Guo XX, Li CX, Deng YQ, Xing D, Liu QM, Wu Q, Sun Suangto P and Uppapong T. Chikungunya fever. Annual epidemiological surveillance report 2009, AJ, Dong YD, Cao WC, Qin CF and Zhao TY 2016. Bureau of Epidemiology, Department of Disease Culex pipiens quinquefasciatus: a potential vector to Control, Ministry of Public Health. [cited 2014 Mar 18]. http://www.boe.moph.go.th/ transmit Zika virus. Emerg Microbes Infect. Annual/Annual%202552/Main.html. 5(6):e102. Sudeep AB and Parashar D 2008. Chikungunya: an Haddow AJ, Williams MC, Woodall JP, Simpson DI overview. J Biosci. 33: 443-449. and Goma LK 1964. Twelve Isolations of Zika Tappe D, Rissland J, Gabriel M, Emmerich P, Gunther Virus from Aedes (Stegomyia) africanus (Theobald) S, Held G, Smola S and Schmidt-Chanasit J 2014. First case of laboratory-confirmed Zika virus Taken in and above a Uganda Forest. Bull World infection imported into Europe, November 2013. Health Organ. 31(1): 57–69. 19(4): pii.20685. Hammon W McD, Rudnick A and Sather GE 1960. Thangamani S, Huang J, Hart CE, Guzman H and Tesh Viruses associated with epidemic haemorrhagic RB 2016. Vertical Transmission of Zika Virus in fevers of the Philippines and Thailand. Science. Aedes aegypti Mosquitoes. Am J Trop Med Hyg. 131(3407): 1102-1103. Jupp PG, McIntosh BM and Dos Santos 1981. 95(5): 1169–1173. Laboratory vector studies on six mosquito and Thavara U, Tawatsin A, Pengsakul T, Bhakdeenuan P, one tick species with chikungunya virus. Trans R Soc Trop Med. 75(1): 15-19. Chanama S, Anantapreecha S, Molito C, Li MI, Wong PS, Ng LC and Tan CH 2012. Oral Chompoosri J, Thammapalo S, Sawanpanyalert P susceptibility of Singapore Aedes (Stegomyia) and Siriyasatien P 2009. Outbreak of aegypti (Linnaeus) to Zika virus. PLoS Negl Trop chikungunya fever in Thailand and virus detection in field population of vector mosquito Dis. 6(8): e1792. Aedes aegypti and Aedes albopictus. Southeast Marchette NJ, Garcia R and Rudnick A 1969. Isolation Asian J Trop Med Public Health. 40(5): 951– of Zika virus from Aedes aegypti mosquitoes in 962. Ungchusak K 2008. Chikungunya: Thailand confirmed. Malaysia. Am J Trop Med Hyg. 18(3): 411–415. Pro Med 2008 Oct 28. Available from McCrae AW and Kirya BG 1982. Yellow fever and Zika http://www.promedmail.org,archiveno.2008102 8.3407. virus epizootics and enzootics in Uganda. Trans Vasconcelos PF and Calisher CH 2016. Emergence of R Soc Trop Med Hyg. 76(4): 552–562. human arboviral diseases in the americas, 2000– Moureau G, Temmam S, Gonzalez JP, Charrel RN, 2016. Vector Borne Zoonotic Dis. 16(5): 295–301. Grard G and de Lamballerie X 2007. A real-time Waggoner JJ and Pinsky BA 2016. Zika virus: RT-PCR method for the universal detection and diagnostics for an emerging pandemic threat. J identification of flaviviruses. Vector Borne Clin Microbiol. 54(4): 860–867. Zoonotic Dis. 7(4): 467-477. Wanlapakorn N, Thongmee T, Linsuwanon P, Mourya DT 1987. Absence of transovarial transmission Chattakul P, Vongpunsawad S, Payungporn S of chikungunya virus in Aedes aegypti & Ae. and Poovorawan Y 2014. Chikungunya outbreak albopictus mosquitoes. Indian J Med Res. 85:593- in BuengKan Province, Thailand, 2013 [letter]. Emerg Infect Dis. 20(8): 1404-1406. 595.

Tawatsin A. et al. / Thai J Vet Med. 2018. 48(4): 551-558. 557 Weinbren MP and Williams MC 1958. Zika virus: further isolations in the Zika area, and some studies on the strains isolated. Trans R Soc Trop Med Hyg. 52(3): 263–268. Wong PS, Li MZ, Chong CS, Ng LC and Tan CH 2013. Aedes (Stegomyia) albopictus (Skuse): a potential vector of Zika virus in Singapore. PLoS Negl Trop Dis. 7(8): e2348.