Studies to Evaluate the Safety of Constant Rate Infusions of Dexmedetomidine, Ketamine and Lidocaine Alone or in Combination during Isoflurane Anesthesia in Horses

Horses mount a huge stress response to anesthesia compared to humans and other companion animals and hence are risky candidates for anesthesia (Taylor, 1989, Wagner et al., 2008).  Day-to-day equine clinics handle surgical diseases that require both short and long-term anesthetic procedures. Though TIVA is considered safer in horses for short-term surgery, inhalant agents are unavoidable for long-term procedures (Wagner, 2008). Newer sedatives and drug combinations are employed to reduce the side effects and dose rate of inhalant agents (Bettschart-wolfensberger and Larenza, 2007). The continuous rate infusion of Alpha 2 agonists helped to abolish many of the side effects apprehended when they were used as bolus infusions (Marcilla et al., 2012).  A newer alpha 2 agonist like Dexmedetomidine was found to reduce the MAC value of isoflurane significantly when used as continuous rate infusions in horses (Schauvliege et al., 2011). Gastrointestinal surgery, particularly colic case is a big challenge to both the surgeon and anesthetist. Safer anesthetic protocols can give hope to handle risky surgeries like colic in horses while avoiding economic loss to the owner. The present study explored the safety of balanced anesthetic protocols employing Dexmedetomidine, Ketamine and Lidocaine constant rate infusion alone or in combination with Isoflurane anesthesia for elective and gastrointestinal surgery in horses.

Animals
The client owned horses of either sex, with an average body weight of 253.75 (160kg to 410 kg) and an age of 4 to 9-year-old referred for elective surgery, soft tissue surgery, tumor removal, ESF, castration (ASA scale 1 and 2) and requiring approximately 1 hour of surgical procedures presented at the Referral Veterinary Polyclinic, IVRI from March 2013 to December 2015 were considered for this study. Permission to perform the study was sought from Institute Animal Ethics Committee (IVRI/SURG/12-15/012). Informed consent was taken from the owner for the collection of blood samples at required intervals.
 
Experimental Design
 
The study was conducted on 28 apparently healthy adult horses with the approved consent of the owner. Animals were assigned to groups S, D, DK, and DKL having seven animals in each. Individual horses of the assigned group were treated with CRI of the following drugs along with inhalant agent Isoflurane 2% in Oxygen (10ml/kg/min) as shown in (Table 1).


 
Technique of drug administration
 
The food was withheld 10-12 hours before anesthesia with limited access to water. A 14 gauge catheter was aseptically inserted into the jugular vein for IV administration of anesthetics and blood sample collection. Pre-anesthetics were administered after proper restraining of the horses. Xylazine1 (Xylaxin®, Indian Immunological Limited, Hyderabad, India) @ 1 mg/kg body weight and Butorphanol2 (Butodol-2®, Neon Laboratories Limited, Mumbai) @ 0.05mg/kg body weight were injected intravenously.  After 10 minutes, anesthetic induction was done with Ketamine3 (Ketamax- 50®, Troikaa Pharmaceuticals Ltd. Uttarakhand, India) (2mg/kg) and Midazolam4 (Mezolam®, Uttarakhand, India) (0.2 mg/kg) intravenously.  All the drugs were injected using separate syringes. Immediately after induction, an endotracheal tube was inserted and the CRI was started as mentioned in (Table 1). Thiopentone sodium5 (Thiosol* sodium, Neon Laboratories Limited, Mumbai (250 mg, 5%) bolus was administered whenever deeper anesthesia was required.
 
Physiological Parameters
 
Heart rate (beats/min), respiratory rate (breaths/min) and rectal temperature (0F) were recorded before administration of the drug(s) and at 0 (after induction), 15, 30, 45, and 60 min after administration of the drugs.

Biochemical observations
 
Blood samples (8 ml) were collected from the jugular vein in a disposable syringe before anesthesia (baseline) and at 0 (start of CRI), 15, 30, 45, and 60 min after injection of the anesthetic(s). The blood samples were centrifuged at 3000 rpm for 5 min and the serum was collected and stored at - 20^oC until assayed. The serum samples were subjected to the following estimations: Triglycerides: Triglycerides were estimated by using commercial kits supplied by Span Diagnostics Ltd., Surat, India. The values were expressed in mg/dl, Cholesterol: Cholesterol was estimated by using commercial kits supplied by Span Diagnostics Ltd., Surat, India. The values were expressed in mg/dl, Blood glucose: The blood glucose was estimated by using Blood Glucose Meter (Romo check insta, Acon Loboratiries, Inc. U.S.A). The values are expressed in mg/dl. Insulin, Cortisol, Adrenocorticotropic hormone (ACTH) were estimated through the RIA kit.
 
Cardiovascular study
 
Systolic blood pressure (SBP), Diastolic blood pressure (DBP), Mean arterial pressure (MAP), and Central venous pressure (CVP) were recorded during pre, peri, and post-operative periods. CVP was measured with the help of a water column manometer (cm of H₂O) (C.V.P. Manometer®, Romsons Scientific and Surgical Industries Pvt. Ltd., Agra, India) before and 0 (after induction), 15, 30, 45, and 60 minutes of anesthesia. Pulse rate and SPO₂ were measured using a pulse oximeter (Model: MD300C26, pulse oximeter; Beijing Choice Electronic Tech. Co.Ltd. SN: 122526304335). The probe of the instrument was applied to the tongue of the animal. The recordings were made at the same intervals as for CVP and Oxygen saturation of hemoglobin (SpO₂).
 
Clinical observations
 
The quality of sedation, induction, muscle relaxation, analgesia, and recovery from anesthesia were recorded. Sedation was recorded after pre-anesthetic administration as 0: No sedation, 1: Mild sedation with slightly lowered head, 2: Moderate sedation with the head lowered below manubrium, but respond to audible stimuli, and 3: Intense sedation with head lowered below manubrium, but no response to audible stimuli. Ataxia score was evaluated before and after premedication. It was graded on 0 to 3 scales as; 0- No ataxia, 1- Animal was stable but slightly swaying, 2-Animal  was swaying and leaning against the stock, and 3- Animal was leaning against the stock and swaying with its hind limbs crossed and forelimbs buckling at the carpal joint. Relaxation of the trunk and limb muscles was taken as a measure of muscle relaxation during the study. It was evaluated by observing the muscular activity of the trunk and limbs. The subjective observation was graded 0 to 3 scale as follows; 0: Muscle relaxation present in trunk and limbs, 1: Muscle twitching present in some regions of trunk and limbs, 2: Muscle twitching present over the majority portions of trunk and limbs and 3: Muscle rigidity present over the majority portions of trunk and limbs. Muscle relaxation was recorded at 0 (after induction), 15, 30, 45, and 60 min intervals. The quality of intubation was recorded as the ease and attempts to intubate by the same observer throughout the anesthetic trials. Anesthetic depth was assessed by physical signs, including movement, the position of the eyeball, depression of the protective reflexes of the eye,  loss of the swallowing reflex,  rate and depth of breathing, and the horse’s response to surgical stimulation. The total dose of thiopentone (mg)/isoflurane (ml) required during the entire surgical procedure and the duration of the anesthesia for the surgical procedure were also recorded. Quality of recovery was scored as 1: One attempt to stand, no ataxia, 2: One or two attempts to stand, some ataxia, 3: >2 attempts to stand, but quiet recovery, 4 :> 2 attempts to stand, excitation, and 5: Severe excitation (Clark-prince, 2013).
 
Statistical analysis
 
Continuous data of parametric variables within and between groups at different time intervals were analyzed using repeated measures ANOVA and that of non-parameric variables (scores) at different intervals were compared using Kruskal-Wallis test. The data were analyzed by using SAS 9.3 software.

The cardiovascular and biochemical changes of the individual as well as the combination of short-acting anesthetic adjuvants as constant rate infusion during total intravenous and inhalant anesthetic (isoflurane) have been explored for anesthesia of horse. Though TIVA is considered safer in this species, inhalant agents are unavoidable for long-term procedures. Newer sedatives and drug combinations are being tested by various workers to reduce the side effects and dose rate of inhalant agents (Bettschart-Wolfensberger et al., 2011). The present study was designed to derive a safer anesthetic protocol with Dexmedetomidine, Ketamine, and Lidocaine as constant rate infusion along with Isoflurane anesthesia for elective surgery in horses. Different surgical procedures performed during the study are mentioned in (Table 2).  



Xylazine and butorphanol brought about sedation while induction of anesthesia with midazolam- Ketamine provided adequate muscle relaxation, and the horses attained recumbency easily with little assistance. Pharyngeal and laryngeal reflexes were abolished in most of the cases and freehand intubation was achieved easily. Lack of adequate laryngeal relaxation could also be due to pre-existing chronic inflammation of the larynx and trachea. Whenever freehand intubation was found difficult, a single fast bolus of 250 mg thiopentone sodium (5% solution) provided adequate muscle relaxation for intubation. Anaesthetic protocols in horses using xylaxine and ketamine alone as sedative and induction agent often required larger doses of Thiopentone sodium (1.5-2g, 5% solution as bolus) for facilitating intubation. In our study we have observed that the pre-anesthetic and induction protocols employed in our study spared the use of thiopentone, or if at all required, a very small amount of thiopentone aided in laryngeal relaxation. Manually assisted ventilation was provided whenever apnoea spanning for more than a few minutes was observed.

There was a significant decline in rectal temperature in all the groups till the completion of the observations (45 to 60 min) (Fig 1). The decrease in RT as recorded in groups might be due to a decrease in the skeletal muscle tone, reduced metabolic rate, and muscle relaxation along with depression of the thermoregulatory center of the brain. Alpha-2 adrenergic agonists have been reported to induce prolonged depression of thermoregulation (Ponder and Clarke, 1980). These agents have also been found to depress hypothalamic noradrenergic α-2 receptors to cause hypothermia (Macdonald et al., 1988). Heart rate fluctuated near the baseline values during these intervals in all the groups. A decreased heart rate in groups DK and DKL might be due to the overriding effect of dexmedetomidine over ketamine (Fig 1). The incidence of postoperative bradycardia has been reported as high as 40% in healthy surgical patients who received dexmedetomidine, especially in high doses (Aho et al., 1993). Respiration rate also showed a significant decline from baseline values in groups DK and DKL (Fig 1). Suppression of hypothalamic center by dexmedetomidine and profound relaxation of intercostal muscles might have influenced in lowering respiration rate in these groups. Thiopentone administration can also cause depression in the respiratory center of the brain (Rawling and Kolata, 1983). At clinically effective doses, dexmedetomidine has been shown to cause much less respiratory depression than other sedatives (Belleville et al., 1992). However, co-administration of dexmedetomidine with anesthetic agents is likely to cause additive effects (Aantaa et al., 1990). Cardiovascular parameters showed a significant decline in mean arterial pressure in groups DKL as compared to other groups (Fig 2). CRI   administration of lidocaine might have reduced the MAP in DKL group animals. Similarly, a lower MAP was observed after CRI administration of Lidocaine in horses (Feary et al., 2005). Fielding et al. (2006) also reported significant bradycardia and decreased mean arterial blood pressure six hours after CRI of ketamine in horses. Contrary to the present study findings, CRI of ketamine used for short- to medium-term infusion did not show significant cardiovascular depressing effects in horses (Sharma et al., 2019).





The changes were non-significant in Central Venous Pressure and Capillary Oxygen Saturation (SpO₂) in different groups (Fig 3). The variation in CVP in the present study may probably be attributed to the pooling of blood in the venous circulation because of the low heart rate caused by the administration of multiple drugs in this study. High levels of plasma dexmedetomidine stimulate Alpha-2B adrenoceptors in smooth muscles of blood vessels producing vasoconstriction and consequently hypertension (Macmillan et al., 1996). A non-significant decrease in SpO₂ has been reported following administration of butorphanol–dexmedetomidine in propofol anesthetized dogs (Surbhi et al., 2010) which is quite similar to results obtained in this study.



Blood glucose was significantly higher in group DK as compared to base value during 45-60 min and the Insulin levels showed a decline from base values during these periods (Fig 4). The changes in biochemical parameters indicating stress like total cholesterol and triglycerides differed non-significantly in different groups (Fig 5). There was a significant increase in cortisol in group S at 60 min intervals compared to the base value. ACTH values increased in Group DKL at 15, 30, and 45 minutes intervals compared to baseline (Fig 6). Overall sedation and muscle relaxation score did not show any significant difference in all the groups. Quality of induction, recovery, and response to the stimulus was better (p≤0.05) in the DKL group as compared to groups D, S, and  DK. Muscle relaxation was better in groups D, DK, and DKL, when compared with group S. Ataxia, was significantly more in group D compared to groups DK and DKL (Table 3). There was no significant change in downtime and duration of anesthesia among groups. Time for first head lift, sternal recumbency time, and standing time was more in group DK and DKL. Isoflurane and thiopentone requirement were significantly less in groups D, DK, and DKL than in group S. Attempts of intubations were non-significantly less in group DK than in other groups (Table 4).











Response to surgical stimuli was significantly low in group DKL as compared to Group D. Ataxia score was more in group D as compared to DKL (p≤0.05). Attempts for intubation and standing time did not have any significant change in different groups. Isoflurane and thiopentone requirement during the observation period was found significantly high in group S as compared to groups D, DK, and DKL. Results of our study showed that constant rate infusion of multiple anesthetic adjuvants reduced the stress response to anesthesia, facilitated adequate muscle relaxation for smooth performance of surgery with lower response to surgical stimuli with a significant reduction in Isoflurane and thiopentone requirement. However, a drastic reduction in rectal temperature and respiration rate needs to be addressed with amenities for maintaining body temperature along with manual or machine-assisted ventilation.

Constant rate infusion of short-acting anaesthetic adjuvants facilitated peri-operative muscle relaxation, reduced response to surgical stimuli, aided smooth recovery from anaesthesia while reducing the requirement for Isoflurane and thiopentone. Careful monitoring of vital parameters like rectal temperature, respiration rate, and adequate external support to manage these parameters is warranted.

The authors acknowledge the scholars, scientific and technical staff of Division of Surgery and Director of ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India for providing necessary support and help at various levels for the study.

Authors report no conflict of interests.