5. grup ve 3. grup kombinasyonları major cerrahi prosedürler için uygun bulunurken 1. grup ve 2. grup anestezi protokolleri ise minor cerrahi prosedürler için daha uygun bulundu. Kalp atım sayısında 2. grupta ve 3. grupta başlangıç değerlerine göre azalma istatiksel olarak anlamlı bulundu. CRP ve cTn-I değerlerinde, gruplar arasında ve başlangıç değerlerine göre istatiksel olarak analizleri yapıldı; ancak anlamlı bir fark belirlenmedi. Sonuç olarak, kardiyak troponinler uzun süreli anesteziye bağlı oluşabilecek akut miyokardiyal hasarın erken belirlenmesinde kullanılabileceği kanısına varılmıştır.

The main concerns in appropriate anaesthetic administration are to have a safe and reliable anaesthetic effect with minimal dosage of anaesthetic agents and to minimize the hemodinamic and cardiovascular complications. Myocardial injuries are amongst common complications during anaesthesia or other ischemic disorders ². There is evidence that preconditioning heart for ischemic conditions can be a protector factor to the heart itself and others from severe ischemic situations ³. Indeed, there are anaesthetic agents with protective impacts on myocardial, renal and cerebral tissues during anaesthesia ⁴.

Volatile anaesthetic agents such as isoflurane and sevoflurane induce the repair of the myocardial tissue and protect it by increasing reperfusion. Decreasing heart rate and myocardial contractility may be favorable alterations for myocardium by diminishing its oxygen demands and the infarct size ⁵. On the other hand, the injectable anaesthetics like propofol and fentanyl are accepted as "less protective" for myocard ⁶. Although its degree varies according to myocardial ischemia or mechanism and anesthesia type, it is possible to preserve the myocardium during anaesthesia in many species ⁷.

There are several markers to determine the myocardial damage. Myoglobin, which is one of the such a marker is an indicator of myocyte damage however it is not specific for the tissue ⁸. Similar to myoglobin, creatine phosphokinase isoenzyme used for the diagnosis of myocardial infarction, also is not specific for myocardial damage as it is found in other tissues and organs such as skeletal muscle, vascular muscle, brain, uterus and placenta ⁹. It is now possible to determine myocardial damage by measuring highly specific cardiac structural proteins such as troponins ^10-12. Compared to other cardiac markers, cardiac troponins are more useful in determining heart damage. Cardiac troponin is a specific and highly sensitive marker of myocardial injury ¹³. These markers have made it possible to diagnose heart diseases more accurately and earlier. Early diagnosis and early treatment of myocardial injury is very important for the prognosis ^12,14. There are several studies on troponin (Tn) levels in cardiomyopathy patients and its relation to prognosis ¹¹.

The main objective of this study was to determine the possible effect(s) of long-time anesthesia by isoflurane, sevoflurane, propofol-fentanyl, medetomidine-midozolam-ketamine and xylazine combinations on myocardial tissue by measuring hemodynamic parameters, serum cTn-I and CRP concentrations in rabbits.

All experiments were approved by Adnan Menderes University Local Board of Ethics Committe for Animal Experiments (Number-64583101/2013/81).

Monitoring of the Reflexes: The main reflexes (standing, ear and finger) of each individual rabbit were evaluated in five minutes intervals before, during and after the administration processes and recorded. The durations of the surgical anesthesia and total sedation times were recorded. To avoid eye dryness, Viscotears® were used for the eyes to while they were sleeping.

Anesthesia Protocols: Thirty rabbits were randomly separated into five groups consisting of three males and three 3 females. Twenty-four hours before the examinations, weights, body temperatures, pulses and respiratoric rates of each animals were recorded. Rabbits showing abnormal physiological findings were excluded from the study.

All examinations were performed between the hours of 9:00-13:00 in each day during the study. In group 1, induction was performed with isoflurane by using face mask. Induction was noted and endotracheal intubation of the airways was performed. The tube was connected to the anaesthesia machine and anesthesia was lasted for four hours. In group 2 the same procedure in group 1 was applied using sevoflurane instead. In group 3, midazolam (1.0 mg/kg), medetomidine (0.05 mg/kg) and ketamine (20 mg/kg) were administered intramuscularly. Anaesthesia protocols were lasted for four hours with additional injections. In some animals anesthesia was completely reversed by using the chemical antagonists, flumazenil (0.1 mg/kg) and atipamezole (0.5 mg/kg), injected subcutaneously. In group 4, anaesthesia was induced with fentanyl (0.0053 mg/kg) and propofol (4-8 mg/kg) and maintained with fentanyl (0.48 μ/kg/min) and propofol (0.6 mg/kg/min) for four hours. In group 5 xylazine (5 mg/kg) were used for premedication. General anesthesia was performed with ketamine (5 mg/kg). Four hours anesthesia was maintained using half dose of the first administration dose.

During the applications V. auricularis magna was used. It was catheterized with 22 g catheter (NovaCath®) and to avoid catheter obstruction %0.9 NaCl was administered with heparinized (Nevparin®) syrings to the catheter after each application.

Monitoring Clinic Parameters: Body temperatures observed with digital termometer (Nimo®), respiratory rates were noted and tail was clipped for placement of a pulse oximeter probe for measuring oxygen saturation (SpO2) with monitor (PETAS KMA® 275). Data were collected at preanesthesia, induction (0 min) and 5, 10, 15, 30, 45, 60, 120, 180, 240 mins after the induction.

Laboratory Tests: Blood samples (2 mL each) were collected from marginal ear vein before (0 time), during and after 6, 12 and 24 hours of induction for measurement of CRP and cTn-I. Blood samples were allowed to analysis of CRP and cTn-I.

Serum CRP: Serum CRP was measured by Rabbit CRP Elisa Kit, commercially available from CUSABIO Biotech Co., Ltd. (Cat No: CSB-E06847Rb). The assay was performed according to the manufacturer's instructions. ELISA plates were pre-coated with rabbit CRP. Colour was developed with TMB substrate and absorbance was measured at 450 nm against standard curves. Mean values were reported. The sensitivity of the assay was 2 pg/mL. The detection range was 7.8 pg/mL-2000 pg/mL. Intra-assay and inter-assay coefficient of variations were <8% and <10%, respectively.

Serum Cardiac Troponin I (cTn-I): Serum cTn-I was measured by rabbit cardiac troponin I (cTn-I) Elisa Kit, commercially available from CUSABIO Biotech Co., Ltd. (Cat No: CSB-E11286Rb). The assay was performed according to the manufacturer's instructions. ELISA plates were pre-coated with anti-rabbit cTn-I antibody. Colour was developed with TMB substrate and absorbance was measured at 450 nm against standard curves. Mean values were reported. The sensitivity of the assay was <11.75 pg/mL. The detection range was 47 pg/ml-3000 pg/mL. Intra-assay and inter-assay coefficient of variations were <8% and <10%, respectively.

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Tablo 1: The times for loss of the reflexes in the groups

When respiratory rates were evaluated between groups, the rate decreased significantly in propofol-fentanyl group at 5, 45 and 60 minutes compared to the others (Table 2). In group 4, the respiratory rates started to decrease at earlier time points in group 5 compared to the groups 1 and 2. The respiratory rates of animals in group 2 stayed highest at all time points during anaesthesia compared to the animals in other groups (Table 2). O2 saturation dropped below 80% in group 5 (Table 2). Only in group 3 the body temperature of the rabbits measured below 37 ⁰C after 120 min of anaesthesia (Table 2).

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Tablo 2: Effects of different anaesthesia groups on body temperature, respiatory rate, heart rate and haemoglobin oxygen saturation over time

When the changes in heart rate between groups were examined, there was a statistically significant decrease in group 3 compared to group 4 and 2 at 120, 180 and 240 minutes (Table 2). The most significant decrease was observed in ketamine-medetomidine-midazolam group at 180 mins (Table 2).

When we measured the CRP and cTn-I serum levels before, during and after 6, 12 and 24 hours of induction no significant change in values observed in any of the groups (Table 3).

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Table 3: Changes in CRP and cTnI over time

Mazaheri-Khameneh et al. ¹⁷ reported that the duration of disappearance of the righting reflex after induction of propofol (12.5 mg/kg induction; 1 mg/kg/min, 30 min) was 9±0.81 secs in rabbits and the total anesthesia duration was 37±0.96 mins.

In our study ketamine-xylazine administration decreased respiratory rates of the animals at earlier time points compared to the other groups. Ketamine can cause temporary apnea in animals due to administration dosage. At higher doses, apneic, superficial and irregular respiration occurs ¹⁹.

Pal et al. ²⁰ reported that respiratory rates of rabbits decreased significantly after 10-20 minutes, following the administration of combination of ketamine with xylazine, acepromazine or medetomidine. Similar findings were found in a study ²¹ in which propofol was administered intravenously in rabbits. According to Ponte and Sadler ^22, propofol reduces the respiratory rate and tidal volume, and is a chemodepressor that decreases the ventilation response to hypoxia with increasing infusion rate. In our study, oxygen saturation stayed same in propofol-fentanyl group at all time points whereas it decreased in xylazine-ketamine group and at 180 and 240 mins (Table 2). There was a statistically significant decrease in ketamine compared to other anesthesia groups.

Hess et al. ²³ administered medetomidine and naphthalene analog naftilmedetomidine, intravenously to rabbits and found that oxygen saturation dropped significantly in the rabbits given medetomidine for 3 minutes after administration. Grint and Pamela ²⁴ compared ketamine-midazolam and ketamine-medetomidine anesthesia in a study where they mixed the drugs in the same injector and after applying them intramuscullary they entubated the rabbits and examined some parameters. Although the rabbits were entubated, the oxygen saturation of the animals in the medetomidine-ketamine group was 93-99% (average 96). In our study, oxygen saturation after ketamine and xylazine injections fell below 85%, indicating the necessity of endotracheal entubation in prolonged anesthesia. Cruz et al. ²⁵ observed a similar decrease in oxygen saturation in animals where they were given a propofol infusion in a similar study in rabbits, and a parallel increase in saturation was observed at forthcoming. Wiese et al. ¹⁸ reported that in a study performed in cats, propofol was significantly reduced at higher doses compared to baseline oxygen saturation as a result of infusion and then returned to baseline values.

One of the most important complications in prolonged anesthesia is hypothermia. In our study, the duration of anesthesia was one hour. Body temperatures of rabbits stayed stable with a slight increase at all groups but Ketamine-medetomidine-midazolam where they decreased significantly below 37 ⁰C after 120 mins (Table 2).

The heart rates in the group 3 at 120 (P<0.05), 180 (P<0.05) and 240 mins (P<0.05) compared to group’s baseline values were statistically significant. Similar findings observed with group 5 for 240 mins vs group’s baseline values.

When serum cTnI and CRP levels were evaluated against in-group baseline values we observed no significant change at 24 hours period (during and after sedation) in any of the animals.

Effects on serum cTn-I concentrations of anesthesia in horses during MR exposures was investigated by Slack at al. (26) in a clinical trial. Similar to our findings they reported no significant change in cTn-I level in the first postoperative 24 hours. Singletary et al. ²⁷ showed similar results in dogs sedated intravenously with medetomidine (10 μg/kg) and butorphenol (0.2 mg/kg) combination. On the other hand, a study by Cilli at al. ²⁸ comparing serum cTn-I levels after various anesthesia protocols in 107 dogs with different races and different health status reported that cTn-I levels increased in 14% of the dogs in the postanaesthetic period whichmay be caused by bleeding, some hypoxic periods during the surgical procedures and excessive changes occasionally occurring in the pulse and blood pressure.

In conclusion, we suggest that cTn-I can be used both in experimental and clinical studies to detect early myocardial damage associated with anesthesia. We hope that these markers can be used clinically in different animal species.

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