Immunological Effects of Propofol, Ketamine and Isoflurane in Dexmedetomidine and Butorphanol Premedicated Dogs

General anaesthesia is indispensable in canine patients for safe and effective handling during various surgical, medical or diagnostic procedures. Production of general anaesthesia is warranted in dogs even for minor procedures which may be performed under local or regional anaesthesia in other species. Inhalation anaesthetics offer special advantage over injectable agents owing to their minimal effect on the body systems and ability to predict and adjust the anaesthetic depth during the surgical procedure. Total intravenous anaesthesia (TIVA) using rapid bolus injection or continuous rate infusion (CRI) makes anaesthetic management easy without any sophisticated instruments.  The general anaesthetic agents used for this purpose, apart from providing the basic qualities such as sedation, analgesia and muscle relaxation, should also have a wide margin of safety and should not cause any long-term alteration in body homeostasis. Unfortunately, no such agent has been found so far which is totally free from unwanted effects. Most of the anaesthetic agents caused immune suppression by influencing the functions of immunocompetent cells and inflammatory mediator gene expression and secretion (Kurosawa and Kato, 2008). Major surgeries also had effects on the immune system which were related to the extent of surgical trauma and the neuroendocrine stress response (Pirttikangas et al., 1995). Anaesthetic agents depressed the immune response by influencing phagocytic activity, lymphocyte transformation and cytokine production which can further increase the morbidity of procedures (Durlu et al., 2002). Therefore, the present study was carried out to evaluate the effect of Propofol, Ketamine and Isoflurane anaesthesia in Dexmedetomidine and Butorphanol premedicated dogs undergoing ovariohysterectomy in dogs.

Twenty-four female dogs between 1-6 years of age and irrespective of breeds presented to the Department of Veterinary Surgery and Radiology, CVSc and AH, CAU(I), Selesih, Aizawl for elective ovariohysterectomy were randomly divided into four groups viz. Group A, B, C and D comprising of six animals in each. The study was approved by the institutional animal ethics committee (IAEC/17-18/596). The animals were fasted for 12 hours and water was withheld for 8 hours prior to the procedure. The surgical site was prepared in all the animals and intravenous PTFE catheter with injection valve and luer lock plug was fixed and slow intravenous fluid (NSS) was administered throughout the procedure.
 
Anaesthetic protocol
 
All the animals were premedicated with Glycopyrrolate @ 0.01 mg/kg IM. Fifteen minutes later, the animals were injected with Dexmedetomidine@5 µg/kg and Butorphanol @ 0.1 mg/kg, IV. Induction of anaesthesia was done with Propofol, IV in group A and B and with Ketamine, IV in group C and D till effect. Anaesthesia was maintained using Isoflurane with fresh gas flow rate of 10 ml/kg/minute at a higher value of 3-5 per cent for initial 5 minutes to stabilize and then the vaporizer settings were changed accordingly to maintain surgical plane of anaesthesia in group A and C. In group B and D, anaesthesia was maintained with Propofol@ 0.2-0.5 mg/kg/min and Ketamine@ 0.002-0.02 mg/kg/min with continuous rate infusion.
       
Blood samples (1.5 ml) were collected in heparinized vials before anesthesia, after anesthesia at 2 hours, 1 day and 3 days. The cytokine mRNA expressions were studied out using real-time qPCR for IL-2, IL-4, IL-6, IL-10 and TGF-β at the aforesaid time intervals in all the groups.
 
RNA extraction and cDNA synthesis
 
200µl of heparinized blood samples were taken for RNA extraction. The total RNA was extracted using Gene JET RNA purification kit (Thermo Scientific, K0731) following the manufacturer’s recommendation. The quantity and purity (A₂₆₀ / A₂₈₀) of total RNA was estimated using a Nanodrop spectrophotometer (ND-2000C, Thermo Fisher Scientific, Wilmington, DE). The RNA samples with a ratio of 1.8 to 2 were only considered for further experiments. RNA (500ng) was reverse transcribed into cDNA in a 20 µl reaction mixture using Maxima H minus First Stand cDNA synthesis kit (Fermentus, K1651) according to the manufacturer’s instruction.
 
Quantification of cytokine gene expression by real-time qPCR
 
Expression level of transcript genes IL-2, IL-4, IL-6, IL-10 and TGF-β and GAPDH (housekeeping gene) were measured by Light cycler 48 real-time PCR instrument with software version 1.5. Each sample was tested in triplicate in Light cycler® Multiwell plate 96 (Roche Diagnostics, Mannheim, Germany). The oligonucleotides for qPCR (Table 1) were adopted from previously published report of Tomihari et al., 2015. Each reaction mixture (10 μl total volume) comprised of 2 μl cDNA, 5 μL 2 × SYBR green master mix (Thermo scientific, Lithuania, EU), 0.5 µM primer pairs and 2 μl PCR-grade water. A no template control reaction (NTC) was included in each assay. The thermo cycling conditions employed for all the genes were: pre-incubation at 95°C for 10 min followed by 40 cycles of 30 sec at 95°C, 30 sec at 59°C and 30 sec at 72°C. After the amplification, a melting peak analysis with a temperature gradient of 0.1°C sec^-1 from 65°C to 97°C was performed to confirm the PCR amplification specificity, contamination and absence of primer-dimers. Relative quantification of a target gene was done by comparing expression level of reference gene GAPDH as per the method of Livak and Schmittgen, (2001).
 
CMI response by nitrobluetetrazolium (NBT) test
 
The test was performed within one hour of blood sample collection as per the method described by Hudson and Hay (1980). The cells were stimulated with phytohemagglutinin P (Pha-P) by adding 15 µl to 1.5 ml of blood and incubated at 37°C for 10 minutes. Then 0.1 ml of freshly prepared NBT dye solution was added, mixed gently and incubated at 37°C for 20 minutes. The blood was added drop wise to a nylon wool column and once sample had entered the column, washed twice with 2 ml PBS and then 2 ml of distilled water was added to lyse the RBC. Dye reduction was checked by adding 2 drops of HCL to the column and washed again with 2 ml distilled water. The nylon wool was removed with forceps and placed in 5ml dioxinin a glass container and incubated at 70°C with occasional vigorous shaking until the nylon wool returns to its original white color. The dioxin extract was centrifuged and measured the extinction at 520 nm using spectrophotometer. Results were expressed as the corrected optical density (OD) by subtracting the dioxin control.
 
Data analysis
 
The data obtained were analyzed using statistical package SPSS version 16. One-way ANOVA based on Fisher’s Least Significant Difference method was used to determine the significant difference among the different treatments and time intervals. The significant values in ANOVA were further tested through Duncan multiple range test. Results are presented as mean ± SE and differences were considered significant when P<0.05.

The IL-2 level did not vary significantly at 0 min and at different observation periods among the groups (Fig 1). In all the groups, IL-2 decreased at 2 hours within each group and thereafter, it returned gradually toward the pre-induction level on day 3 with significant variation (P<0.05) except in group B. Interleukin 2 (IL-2) is secreted by CD4⁺ Lymphocytes (Th1) and it is necessary for the growth and function of T cells and it stimulates the growth, differentiation and survival of antigen-selected cytotoxic T cells CD4⁺ Lymphocytes (Th1). The suppression of IL-2 was observed initially at 2 hr. after anaesthesia which might be attributable to the effects of anaesthestic agents. All anaesthetics had direct suppressive effects on cellular and neuro-humoral immunity and influence the functions of immunocompetent cells and inflammatory mediator gene expression and secretion (Kurosawa and Kato, 2008). The initial IL-2 suppression observed in this study might be due toButorphanol, as opioids behave like cytokines and modulate the immune response by interaction with their receptors both in the central and peripheral nervous system (Ricardo et al., 2004). Use of preemptive analgesic before induction of anaesthesia resulted in suppression of IL-2 production and might prevent immune function alterations in the early postoperative period (Beilin et al., 2007). Opioid also caused immune suppression following sympathetic activation resulting in catecholamine and corticosteroid release by κ and µ receptor activation (Sanders et al., 2009). It might also be attributable to the effects of Dexmedetomidine. Due to its anti-inflammatory effect, Dexmedetomidine caused reduction in pro-inflammatory cytokine production (Anderson et al., 2014).  Helmy et al., (1999) also reported a significant reduction in IL-2 due to the effect of anaesthesia. In the present study, significant suppression of IL-2 in group A and C might also be due to the effects of Isoflurane as most of the newer volatile anaesthetics like Isoflurane, Sevoflurane and Desflurane attenuate the release of pro-inflammatory cytokines (Wanger et al., 2010). Faller et al., (2012) reported less lung damage, inflammation and stress protein expression caused by mechanical ventilation in Isoflurane anaesthetized mice. Ketamine had possible immunomodulatory and anti-inflammatory effects (Liu et al., 2012) and might add to the significant suppression of IL-2 in group D. The non-significant decrease in IL-2 level in group B might be attributable to Propofol as it is believed to have some immuno-protective effects and TIVA with Propofol might benefit immunological support in the perioperative period of dogs (Tomihari et al., 2015).


 
IL-4 did not vary significantly at 0 min and at different observation periods among the groups (Fig 2). Within the groups, the IL-4 increased non-significantly in all the groups at 2 hours and there after, it returned gradually toward the pre-induction level on day 1. The anti-inflammatory cytokine IL-4 is produced by Th2 cells, NK lymphocytes and mast cells. IL-4 enhances IgE production and inhibits IL-4 liberation. Elevated level of IL-4 following Thiopentone and Ketamine administration and to a lesser extent by Propofol was also reported by Kumar et al., (2002). Helmy et al., (1999) observed no significant effect on IL-4 production in response to total intravenous anaesthesia with Propofol, Sufentanil and Atracurium. Similarly, Tomihari et al., (2015) also did not observe any significant changes in IL-4 with Propofol and Isoflurane for maintenance of anaesthesia in healthy dogs. The non-significant elevation in IL-4 level in the present study might be attributable to the effect of different anaesthetic agents used as Dexmedetomidine, Butorphanol, Ketamine and Isoflurane are known to possess anti-inflammatory effect (Anderson et al., 2014).
 

       
The values of IL-6 did not vary significantly at 0 min and at different observation periods among the groups (Fig 3). Within the groups, the IL-6 decreased significantly (P<0.01) in all the groups at 2 hours. Thereafter, it increased gradually and the values were beyond the pre-induction level on day 3. Interleukin 6 (IL-6) was produced mainly bypro-infiammatory cytokines including IL-6. These cytokines can induce peripheral and central sensitization leading to hyperalgesia. Use of preemptive analgesia reduced pain and caused attenuation of pro-inflammatory cytokine production (Beilin et al., 2003). The reduced magnitude of IL-6 response during major abdominal surgery was also observed by Crozier et al., (1994) with Propofol, Fentanyl as compared with Isoflurane anaesthesia and opined that the reduction was not due to Propofol but probably due to opioid by acting on its receptors leading to a reduction in intracellular cyclic AMP (cAMP) which was an important secondary messenger in triggering the release of IL-6. The effects of opioids on monocytes might reduce the concentrations of IL-6. Kumar et al., (2002) also stated that a reduction in cAMP was associated with inhibition of IL-6 synthesis. Corcoran et al., (2006) commented that Propofol might have attenuated IL-6 level during cardiopulmonary bypass surgery in human, initially which increased later at 24 hr. Similarly, Takaono et al., (2002) also demonstrated that Propofol inhibited IL-6 production by lipopolysaccharide-stimulated mononuclear cells. However, Fekkes et al., (2016) concluded that plasma IL-6 levels were not altered by Propofol but by the duration and type of surgery, which was the most important factor influencing IL-6 synthesis. Ketamine directly inhibited the production of pro-inflammatory cytokines IL-6 in human whole blood (Kawasaki et al., 1999). Ketamine exerted its anti-inflammatory effects by suppression of neutrophil chemotaxis and superoxide formation and by reducing production of IL-6 and oxidative burst in macrophages (Anderson et al., 2014). Ketamine inhibits IL-6 mRNA synthesis by LPS-activated macrophages. Beilin et al., (2007) reported that addition of small doses of Ketamine before induction of anaesthesia resulted in attenuation of secretion of the pro-infiammatory cytokines IL-6. Roytblat et al., (1998) also observed that Ketamine with opioid-based anaesthesia suppressed the increase of serum IL-6 during and after cardiac surgery. Dexmedetomidine also caused a reduction in IL-6 cytokine concentrations (Anderson et al., 2014).



The values of IL-10 did not vary significantly at 0 min and at different observation periods among the groups (Fig 4). Within the groups, the IL-10 increased non-significantly in all the groups at 2 hours. Thereafter, it decreased gradually and the values were beyond the pre-induction level on day 3. IL-10 was produced by monocytes, macrophages, T-regulatory cells and B lymphocytes. It was the main anti-inflammatory cytokine because it inhibited the synthesis of IFN-γ, TNF-α, IL-2 and IL-12 and induced synthesis of Ig G. Omera (2006) recorded no significant change in IL-10 after surgery with halothane, Isoflurane and sevoflurane premedicated with fentanyl and induced with Thiopentone for inguinal hernia repair in human patients. Tomihari et al., (2015) reported that the IL-10 showed no increase with Propofol like with Isoflurane and maintained same level until 2 hours. Ketamine, Propofol, Isoflurane Sevoflurane increased IL-10 levels (Colucci et al., 2013). Dexmedetomidine, Butorphanol, Ketamine and Isoflurane were known to possess anti-inflammatory effect (Anderson et al., 2014). The anti-inflammatory effects of Isoflurane might be mediated through inhibition of the nuclear transcription pathways. Isoflurane also caused production of a small amount reactive oxygen species (ROS) by the cells due to exposure to Isoflurane and results in negative feedback signal on protein kinase C-mediated ROS production (Anderson et al., 2014). The non-significant elevation in IL-10level in the present study might be attributable the effect of anaesthetic agents used during the study.
 

       
The values of TGF-β did not vary significantly at 0 min and at different observation periods among the groups (Fig 5). Within the groups, the TGF-β increased in all the groups at 2 hours. Thereafter, it decreased gradually and the values were beyond the pre-induction level on day 3. The changes in TGF-β were non-significant in group B, however, it was significant in group A (P<0.01), C and D (P<0.05). TGF-β is produced by activated T-regulatory lymphocytes and mac­rophages. TGF-β has immunosup­pressive effects because it inhibited the synthesis of IFN-γ, TNF-α, IL-1 and IL-2 and it also inhibited NK and CD8 cell cytotoxic activity (Colucci et al., 2013). The elevation in TGF-β level in the present study might be attributable to the combined effect of the anaesthetic agents used during the study as Dexmedetomidine, Butorphanol, Ketamine and Isoflurane were known to possess anti-inflammatory effect (Anderson et al., 2014).
 

       
The optical density (OD) values in NBT assay did not vary significantly at 0 min and at different observation periods among the groups (Fig 6). Within the groups, it decreased non-significantly in all the groups at 2 hours. Thereafter, it returned to the pre-induction level on day 3.  A decrease in OD value indicated less activation of neutrophils hence less uptake of the dye compound to generate formazan granule. Activated neutrophils in blood circulation get stimulated markedly and phagocytose more dye compound which was indicated by high OD value. Similar observations were also reported by Khan et al., (1995) with halothane and Costa et al., (2013) with Propofol anaesthesia. A decreased phagocytic activity of neutrophil was also reported by Inoue et al., 1992 after surgery. Prabhu et al., (2014) reported that the proportion of neutrophils and its phagocytic activity as assessed by NBT test were unaffected during minor surgery with Thiopentone, Ketamine, Isoflurane and epidural Bupivacaine. The immunomodulating effects of different anaesthetic agents were multidirectional and might be due to impairment of various functions of the immune system either directly, by disturbing the numbers and functions of immune-competent cells or indirectly by modulating the stress response (Scholl et al., 2012). Opioid administration led to suppression of cellular immune responses including antibody production, natural killer cell activity, cytokine expression and phagocytic activity (Ricardo et al., 2004). Ketamine can suppress macrophage function of phagocytosis, its oxidative ability and inflammatory cytokine production possibly via reduction of the mitochondrial membrane potential instead of direct cellular toxicity (Chang et al., 2005). In the present study, the effect of the anaesthetic on the expression levels of cytokines also supported the findings of NBT assay.
 

Transient immune suppressions were exhibited in all the anaesthetic combinations with least suppression in Propofol constant rate infusion.