Tiletamine/zolazepam and dexmedetomidine with tramadol provide effective general anesthesia in rats

Abstract Background Tiletamine/zolazepam is a dissociative anesthetic combination commonly used in small animals but information is limited in rats. The alpha‐2 agonist, dexmedetomidine, has gained popularity in laboratory animal anesthesia. Tramadol is a weak opioid mu agonist. The aim of this study was to assess whether the tiletamine/zolazepam/dexmedetomidine (ZD) combination effectively provides a surgical anesthesia plane comparable to tiletamine/zolazepam/dexmedetomidine with tramadol (ZDT) in a minor procedure in rats. Methods Rats were induced with ZD or ZDT. After the loss of paw withdrawal, a small incision was made on the rats’ left thighs as a surgical stimulus. Rats were maintained under a surgical anesthesia plane by assessing the loss of the paw withdrawal reflex for 45 minutes, then atipamezole was administered. Monitored anesthesia parameters included: (a) physiological parameters – pulse rate (PR), respiratory rate (RR), tissue oxygen saturation (%SpO2), and body temperature; (b) duration parameters – induction time, onset and duration of surgical anesthesia plane, onset of recovery, and recovery time. Results PR was significantly lower at 10 minutes in ZD and 5 minutes in ZDT groups. No difference was observed for RR, %SpO2, and body temperature. Likewise, there were no differences for duration parameters: induction time was less than 3 minutes; onset and duration of surgical anesthesia plane were approximately 5 and 45 minutes, respectively; onset of recovery (time to move) was 51 minutes; and recovery time was 52 minutes, respectively. Conclusion These data suggest the ZD combination provides a surgical anesthesia plane comparable to ZDT in a rat incisional pain model.

using smaller doses of each drug to provide surgical anesthesia.
Dexmedetomidine, due to its higher alpha-2 adrenergic receptor specificity 11 and longer duration of action compared to xylazine, has gained heightened popularity. There is limited information on the tiletamine/zolazepam/dexmedetomidine combination in rats.
Although both tiletamine and dexmedetomidine provide analgesia, due to pain pathway complexity, multimodal and pre-emptive analgesia is encouraged, when possible. Therefore, tramadol was included in the injectable anesthetic combination, ZDT, to provide multimodal analgesia through another analgesic class. Tramadol is a weak µ-opioid agonist, 12 recommended for minor painful procedures. 13 Although tramadol is controlled in North America, it is not considered as a controlled substance in some countries, ie Thailand, China (Hong Kong), and Philippines. There are few publications regarding tramadol's combination with tiletamine/zolazepam/dexmedetomidine. This study aimed to investigate whether tramadol added to a tiletamine/zolazepam/dexmedetomidine combination improves the surgical anesthesia plane in an incisional pain model. The null hypothesis was that there was no difference between tiletamine/zolazepam/dexmedetomidine and tiletamine/ zolazepam/dexmedetomidine with tramadol regarding measured parameters. This technique will be clinically significant because by using lower doses of each drug, it will cause fewer side effects and complications, yielding safer anesthesia while still providing a surgical anesthesia plane.

| Experimental groups
Rats were randomly assigned to 1 of 2 treatment groups (n = 9 each

| Anesthesia and surgery
After drug administration and observed loss of righting reflex, rats were placed on a heating pad (RightTemp ® Jr, Kent Scientific, Torrington, CT) and maintained with 100% oxygen (500 mL/min) via nose cone. Sterile ophthalmic ointment (ChlorOph ® , Seng Thai Company Ltd., Thailand) was applied to both eyes; pre-warmed 0.9% NaCl (10 mL/kg, SC) and cefazolin (30 mg/kg, SC, Cefazolin ® , Utopian Company Ltd., Thailand) were administered. Rats were placed in right lateral recumbency and the left thighs were shaved and aseptically prepared. Following loss of paw withdrawal reflex (T0) which was evaluated every 15 seconds, a 0.5-cm skin incision was made in the dorsoventral direction and immediately closed with 3-0 polypropylene suture (Prolene ® , Ethicon™, Johnson & Johnson, USA) in a simple interrupted pattern. Forty-five minutes after drug administration (T45), atipamezole (1 mg/kg, SC, Antisedan ® , Virbac, Thailand; 5 mg/mL), was administered to reverse dexmedetomidine.
Rats were individually placed sternally in a recovery box with a heating pad placed half-way underneath the recovery box. Rats were monitored continuously until fully recovered (no ataxia observed) before returning them to their home cages. Clinical assessments including pink skin color, gait, and quick recovery following atipamezole administration were observed.

| Statistical analysis
For physiological parameters, data were presented as means ± SEM, and F test in ANOVA for repeated measures with Bonferroni for multiple comparisons (SPSS, IBM, Somers, NY) was used (data were also tested for normality). For duration parameters, data were presented as medians and interquartile ranges (IQR: Q1, Q3), and the Mann-Whitney U test was used. A P value of less than .05 was considered significant. Sample size (n = 9/group) was calculated achieving 83.4% power of the test. F I G U R E 1 Pulse rate (bpm; mean ± SEM) in ZD and ZDT groups. *Significantly different value (P < .05) from 5 min after drug administration (baseline) value for the same treatment group  (Table 1). For the onset of surgical anesthesia plane, the time to loss of paw withdrawal reflexes (T0) did not significantly differ (P > .541) for ZD [5.

| D ISCUSS I ON
This study demonstrates that ZD and ZDT effectively provide a surgical anesthesia plane (general anesthesia) in rats for at least 45 minutes.
In ZD and ZDT groups, PR was only below the baseline (5 minutes after drug administration) at 20-25 and 30 minutes, respectively. A statistically nonsignificant trend of decreased RR relative to baseline (5 minutes after drug administration) was observable. %SpO 2 and body temperature did not differ between groups. There was no difference between ZD and ZDT for the time to loss of righting reflex, paw withdrawal reflex, and time to move, stand, and walk. All rats recovered approximately 50 minutes following atipamezole administration. These data support our hypothesis that a combination of ZD effectively provides a surgical anesthesia plane comparable to ZDT.   NMDA, 25 and opioid receptors. 26 A common dexmedetomidine side effect is bradycardia 22,27,28 ; therefore, we selected tramadol as it does not have a strong effect on the cardiovascular system and tramadol is fast-acting opioid with an onset of action within 30-60 minutes, which is appropriate for minor procedures. 13 33 Although tiletamine and dexmedetomidine may provide analgesia during the pre-operative period, analgesia may be insufficient during the post-operative period. Therefore, to provide post-operative analgesia, supplementing with another class of analgesic, tramadol, was warranted. Used alone, tramadol did not attenuate thermal hypersensitivity, but when combined with other drugs, it attenuated thermal hypersensitivity in rats. 13 In this current study, we combined tramadol with tiletamine/zolazepam and dexmedetomidine (ZDT). Tramadol's addition did not adversely impact the parameters used to assess anesthesia in this current study. Future studies assessing analgesia are needed. Because tiletamine/zolazepam (20 or 40 mg/kg, IM/IP) was reported to provide analgesia in rats, 34 it is possible the nociceptive stimulation used (paw clamp or skin incision) in the current study was less intense than expected. Therefore, analgesia provided by ZD alone could be sufficient to avoid a response. Rats showed no clinical signs of pain and distress (rough hair coat, porphyrin staining, lack of grooming etc) throughout the studies.
This study evaluated an alternative, effective injectable anesthetic option when gas anesthesia cannot be used. Both ZD and ZDT provide sufficient surgical anesthesia lasting at least 45 minutes in a modified rat model of incisional pain. Atipamezole can help to provide uneventful recovery from anesthesia in rats, following either the ZD or ZDT anesthesia protocol. Addition of Tramadol is likely to provide continued pain control following atipamezole's reversal of dexmedetomidine-mediated pain control.

ACK N OWLED G EM ENTS
We thank Ms Intuorn Chaisit for her assistance at the Laboratory Animal Unit, Faculty of Pharmaceutical Sciences, Chulalongkorn University, and Janis Atuk-Jones for her kind assistance in formatting and editing the manuscript. We also thank Virbac (Thailand) Ltd.
for providing anesthetics.