Preconditioning with lidocaine and xylazine in experimental equine jejunal ischaemia

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2020 The Authors. Equine Veterinary Journal published by John Wiley & Sons Ltd on behalf of EVJ Ltd 1Clinic for Horses, University of Veterinary Medicine, Hannover, Germany 2Institute for Anatomy, University of Veterinary Medicine, Hannover, Germany 3Department of Biometry, University of Veterinary Medicine, Hannover, Germany


| INTRODUC TI ON
Small intestinal strangulation with concurrent ischaemia/reperfusion injury is a major cause of mortality in horses. 1 An increasingly popular mechanism for the treatment of ischaemic lesions in human medicine is ischaemic preconditioning. 2 This refers to the activation of intrinsic cell survival programs after exposure to mild ischaemic stimuli or pharmacologic agents, and several studies have demonstrated the beneficial effect on the survival of intestinal tissue. 3 Besides mechanical ischaemic preconditioning, many different pharmacological agents, like volatile anaesthetics and alpha-2 agonists, have been shown to activate these protective cell responses. 2 Some authors have reported the pharmacological preconditioning effect of the alpha-2 agonist dexmedetomidine on intestinal injury in rabbits and rats. 4,5 Moreover, a protective effect of dexmedetomidine was identified in an experimental model of equine small intestinal strangulation. 6 This beneficial effect may be due to its anti-inflammatory and antiapoptotic properties. 4 To the authors' knowledge, there are no reports on a preconditioning effect of xylazine, a more commonly used alpha-2 agonist in horses.
Lidocaine is the most commonly used prokinetic drug in the perioperative management of horses undergoing colic surgery. 7 In experimental models, it has been shown to have a beneficial effect on the intestine after ischaemia and reperfusion, by decreasing intestinal oedema, 8 decreasing COX-2 expression in intestinal mucosa 9 and by limiting the increased gut wall permeability compared to flunixin-meglumine administration alone. 10 In a more recent experimental study, the horses treated with lidocaine did not show a consistent decrease in intestinal neutrophil infiltration compared to the untreated horses. 11 Currently, the exact mechanism of these actions remains unclear. No studies have explored if there is a preconditioning effect of lidocaine in horses.
The aim of this study was to investigate whether xylazine and lidocaine, which are routinely used in the management of small intestinal colic, have a preconditioning effect on equine jejunal ischaemia. The objective was to describe histomorphology, apoptosis and inflammatory cell count in the intestinal tissue undergoing experimental ischaemia, and to compare these results between different treatment groups. We hypothesise that preconditioning with xylazine or lidocaine will ameliorate ischaemia/reperfusion injury. Identifying a preconditioning effect of either pharmacologic agent would support its use in sedation protocols and anaesthetic regimens for horses with colic.

| Animals
For this terminal in vivo experiment, 10 adult Warmblood horses were randomly assigned to a lidocaine (group L; n = 5) or a xylazine (group X; n = 5) group. Group L comprised of three mares, one gelding and one stallion, with an age range of 9-19 years (12 ± 4 years, mean ± SD) and the weight ranging between 540 and 619 kg (573 ± 31 kg). Group X comprised of four mares and one stallion, with an age range of 4-21 years (14 ± 8 years) and the weight between 520 and 705 kg (577 ± 76 kg). A historical control group consisting of five Warmblood horses, with an age range of 2-14 years (5.4 ± 4.9 years) and the weight between 464 and 610 kg (544 ± 53 kg), was used to limit the amount of horses needed. This control group was taken from our previous study evaluating the preconditioning effect of dexmedetomidine, 6 using the same experimental model, anaesthetic regime and sample preparation as the current study. The horses were acquired by the equine hospital for educational purposes and were subjected to euthanasia due to problems unrelated to the gastrointestinal tract, such as orthopaedic disease. All horses were systemically healthy without any signs of gastrointestinal disorders. At least 2 weeks prior to surgery, the horses were stabled and no medication was administered during this time.
The horses had free access to hay and water and were hand-walked daily. Feed but not water was withheld before surgery for six hours.

| Anaesthetic protocol and monitoring
Before the procedure, a 12-gauge Teflon catheter (Intraflon, Vygon GmbH) was placed in the left jugular vein. In the control group (group C; n = 5), anaesthesia was induced without prior sedation.
The horses were infused with 5% guaifenesin (My-50 mg/mL, CP-Pharma GmbH) until ataxia was apparent. At this point, 0.05 mg/kg diazepam (Ziapam 5 mg/kg, Ecuphar GmbH) and 2.5 mg/kg ketamine (Narketan, Vétoquinol GmbH) were administered to induce general anaesthesia. Orotracheal intubation was performed and anaesthesia was maintained with isoflurane (Isofluran CP, CP-Pharma GmbH) in 100% oxygen. The horses in group L were anaesthetised according to the same protocol, and additionally received a loading dose of 1.3 mg kg BW lidocaine (Lidocain 2%, Bela-Pharm GmbH) over 10 minutes prior to induction. Subsequently, a continuous rate infusion (CRI) of lidocaine at a rate of 0.05 mg/kg/min was instituted within 5 minutes after induction. 12 Group X, received a loading dose to the absence of a concurrent reduction of histomorphological injury, the clinical significance remains uncertain.

K E Y W O R D S
horse, ischaemic, intestine, colic, anaesthesia, protective of 1 mg/kg xylazine (Xylavet 20 mg/mL, CP-Pharma GmbH) over 10 minutes prior to induction of anaesthesia, followed by a CRI of 1 mg/kg/h. 13 In group C, lactated Ringer's solution (Ringer-Laktat EcobagClick, B. Braun Melsungen AG) and dobutamine (Dobutaminratiopharm 250 mg, Ratiopharm GmbH) were given at a constant rate of 5 mL/kg/h and 0.5 µg/kg/min respectively. In groups L and X, lactated Ringer's solution was started at 5 mL/kg/h and increased stepwise in increments of 5 mL/kg/h to a maximum of 20 mL/kg/h to maintain a mean arterial blood pressure (MAP) above 60 mm Hg.
This was supplemented by a CRI of dobutamine at a rate of 0.3 µg/ kg/min if the initial increase in intravenous fluid rate did not have an effect, and subsequently titrated with increments of 0.3 µg/kg/min. This stepwise approach was continued until the MAP reached the desired level above 60 mm Hg. If the MAP rose above 80 mm Hg, the rates were decreased in the same manner. During the procedure, cardiovascular and respiratory values were monitored and documented every 10 minutes. Direct arterial blood pressure measurement and arterial and mixed venous blood gas analyses were performed, as well as cardiac output measurement by thermodilution as described previously. 14 The cardiac index (CI) was determined by dividing the cardiac output by the bodyweight, and the oxygen extraction ratio (OER) was calculated as the ratio of the difference between the arterial and mixed venous blood oxygen content to the arterial blood oxygen content.

| Sample preparation
The intestinal samples were fixed in 4% formaldehyde and embedded in paraffin. Four micrometre thick sections were cut and the slides were stained routinely with haematoxylin and eosin (H&E) for histomorphological examination. Immunohistochemical staining was performed for cleaved caspase-3 as marker for apoptosis, and for terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) as marker for late-apoptosis and cell necrosis. The staining for cleaved caspase-3 was performed using commercial antibodies (Cleaved Caspase-3Asp175 antibody, Cell Signaling Technology Europe B.V.). The complete staining protocol is provided in Data S1.

| Histomorphological and immunohistochemical examination
The histomorphological and immunohistochemical examination of all the slides including those of the historical control group was performed by the same observer, after being blinded to the sample type and group assignment. This observer (N.V.) was trained by an experienced histologist (C.P.). The histomorphological properties of the mucosa were assessed by light microscopy (AXIO Scope.A1, Carl Zeiss GmbH) in H&E-stained slides in 10 adjoined high-power fields (HPFs) at a 400-fold magnification using a modified Chiu score. 17,18 One slide per time point per horse was assessed. Each field of view was scored individually, and subsequently averaged to make up the final score of each slide. In this modified Chiu score, the villous morphology and the degree of haemorrhage in the tissue were scored separately ( Table 1).
The TUNEL-and caspase-3-positive cells in the mucosa were

| Data analyses
Prior to commencing the study, a power calculation was performed with free software (G*Power 3.1.9.2, Heinrich Heine Universität).
To detect a difference of 1 grade in the histomorphology score between the treatment groups with a standard deviation of 0.5, based on a power of 0.8 and alpha of 0.05, a total sample size of 10 horses was required.

| Anaesthetic parameters
The mean end-tidal concentrations of isoflurane (ET iso) to main-  Table S1.

| Histopathological evaluation
Compared to the pre-ischaemia sample, the villous score was significantly increased in all groups after ischaemia and reperfusion (P = .02 for groups C and L, P = .01 in group X), but no difference could be detected between ischaemia and reperfusion ( Figure 1). One horse from group X had a very low score for villous morphology at both ischaemia and reperfusion, with an average score of 1.2 ± 0.4 SD and 1.1 ± 0.3 SD respectively. The haemorrhage score was 0 or 1 for all horses in the pre-ischaemia samples, and there was a significant increase of 2-3 grades in the ischaemia and reperfusion samples compared to the pre-ischaemia sample in group L (P = .02 and P = .02) and group X (P = .03 and P = .02) (Figure 1). There was no statistically significant difference for either score between any of the experimental groups.
Calprotectin-positive cells were seen in the mucosa of all slides.
Compared to pre-ischaemia, all groups had a higher mucosal cell count after ischaemia (P = .02, P = .02 and P = .03 for groups C, L and X respectively) and reperfusion (P = .02, P = .02 and P = .03 for groups C, L and X respectively) ( Figure 2). In group X, the cell count was significantly lower during reperfusion compared to the control group with a median difference of 6.8 cells/mm 2 (P = .02). In the submucosal venules, a range of 0-4 calprotectin-positive cells per slide was found in the pre-ischaemia sample. In groups L and X, the cell count had increased significantly after ischaemia and reperfusion compared to pre-ischaemia (P = .02 and .03 for group L and P = .02 and .02 for group X). There were no statistically significant differences at any time point between the groups. In the serosa, no calprotectin-positive cells were found in the pre-ischaemia samples.
After ischaemia, only one horse in group L and one horse in group X had positive cells (two and eight cells respectively). In the reperfusion sample, the cell count was 94 (86-117), 10 (1-105) and 12  for groups C, L and X respectively (Figure 3). This was a statistically significant increase compared to both the pre-ischaemia (P = .02, P = .02 and P = .03 for groups C, L and X respectively) and ischaemia (P = .03, P = .03 and P = .02 for groups C, L and X respectively).
Group X had a significantly lower serosal cell count after reperfusion compared to group C (P = .05). No significant difference could be detected between group L and the two other groups (P > .99 and P = .07 for comparison with groups X and C respectively).
The pre-ischaemic samples all revealed caspase-3-and TUNELpositive cells (Table 2). After ischaemia, the caspase cell count increased significantly in groups C and L (P = .02 and P = .007 respectively) (Figure 4). Group X had a significantly lower relative cell count compared to the control group at this time point, with a median difference of 227% (P = .01) (Figure 4). During reperfusion, the caspase-positive cell counts increased significantly in all groups (P = .02) ( Figure 5). For the TUNEL-positive cells, a significant increase between pre-ischaemia and ischaemia was noted in all groups (P = .03, P = .02 and P = .03 for groups C, L and X respectively), as was a significant decrease between ischaemia and reperfusion (P = .02, P = .03 and P = .02 for groups C, L and X respectively) ( Figure 6).
There were no significant differences between the groups at any

F I G U R E 1
Box-plot diagram of the separated Chiu score for villous histomorphology and haemorrhage. The horizontal bar displays the median, the interquartile range is represented by the box and the minimum and maximum by the whisker plots. There were no significant differences between the groups. Significant differences (P < .05) between different time points within the groups are marked with an asterisk. P, pre-ischaemia; I, ischaemia; R, reperfusion These results might indicate a protective effect of xylazine on ischaemia/reperfusion injury, even though this was not supported by fewer histomorphological injury. A protective effect has also been described for the more selective alpha-2 agonist dexmedetomidine. 6 The use of xylazine for clinical cases may be more feasible, as this drug is licensed for horses and already part of many established anaesthetic regimens. Other alpha-2 agonists, like detomidine and Note: The positive cell count for the cleaved caspase-3 and TUNEL immunohistochemistry of the different groups and time points, expressed as positive cells per mm 2 (median, minimum-maximum). Group C is the control group, and groups L and X are preconditioned with lidocaine and xylazine respectively.

F I G U R E 4
Box-plot diagram of the cleaved caspase-3-positive cells in the mucosa, expressed as percentage of the cell count in the pre-ischaemia sample. The horizontal bar displays the median, the interquartile range is represented by the box and the minimum and maximum by the whisker plots. Significant differences (P < .05) are marked with an asterisk. P, pre-ischaemia; I, ischaemia; R, reperfusion The MAC387 stain used in the current study to identify inflammatory cells is not specific for neutrophils, as macrophages and monocytes also express cytosolic calprotectin during inflammation. 22 However, a significant correlation between neutrophils identified histomorphologically and calprotectin-positive cells has been found in the equine colon. 23 Therefore, it is believed that this immunohistochemical stain provides a good estimate of the neutrophil count in the equine intestine. In the current study, a significant increase in calprotectin-positive cells was noted during reperfusion.
Neutrophilic inflammation, initiated by the presence of superoxide radicals during reperfusion, has been indicated as a cause of reperfusion injury. 1 On the contrary, the influx of neutrophils may be part of normal tissue repair, and one cannot assume a direct relationship between higher inflammatory cell counts and increased ischaemia/ reperfusion injury.
The occurrence of reperfusion injury in small intestinal strangulation in horses is under debate, and it has been suggested that this does not contribute significantly to injury in clinical cases. 24 The results of the caspase-3 immunohistochemistry could indicate an effect of reperfusion in the current model, although this did not appear to cause mucosal changes on histological examination. It has been suggested that reperfusion injury may be more likely to occur in models of low-flow ischaemia, where the blood flow is typically reduced to 20%. 25 In the current study, the flow was reduced to 10%, verified by In the current study, isoflurane could have ameliorated ischaemia/ reperfusion injury across the groups, which might explain the lack of progression of histomorphological damage from ischaemia to reperfusion. Considering all horses were anaesthetised with isoflurane, this effect would be present across all groups. There is evidence indicating that the protective effect of isoflurane is dose-dependent. 27 In the current study, the mean isoflurane concentration was higher in the control group; however, this group did not have better results for any of the tested variables.
The to be younger than those in groups L and X. Even though a statistically significant difference could not be detected, an effect of age on the test results cannot be excluded. Another limitation of the study design was that only the short-term effects of preconditioning could be examined, and thereby limiting this to the early phase of protection.
Furthermore, only one observer graded the histology, and the site of tissue sampling was not randomised.
The occurrence of equine strangulating colic is unpredictable, thereby precluding the application of preconditioning before the ischaemic insult has commenced. Nevertheless, there may still be blood flow to the tissue in the early stages of ischaemia, presenting the opportunity to precondition the tissue incorporated within the lesion.
For the type of strangulating obstructions where increasingly more intestine is incorporated over time, the surrounding intestinal segments may be preconditioned before their blood supply is affected.
Furthermore, several studies have found that prestenotic and remote intestinal segments also sustain injury. 11,30 Therefore, there are several situations where the concept of preconditioning could be a feasible therapeutic strategy to reduce intestinal ischaemia/reperfusion injury in colic horses.
In conclusion, the results of this study indicate a beneficial effect of xylazine on apoptosis rate and inflammation. A concurrent reduction in mucosal histomorphological injury could not be found; therefore, the clinical significance of these findings remains

E TH I C A L A N I M A L R E S E A RCH
The study was reviewed by the Ethics Committee for Animal Experiments of Lower Saxony, Germany, and approved according to §8 of the German Animal Welfare Act (LAVES 33.8-42502-04-17/2595).

OWN E R I N FO R M E D CO N S E NT
Not applicable.

DATA ACCE SS I B I LIT Y S TATE M E NT
The data that support the findings of this study are openly available under the following reference: Verhaar, Nicole (2019)

ACK N OWLED G EM ENTS
We are grateful to Doris Voigtländer for her expert support in tissue processing and immunohistochemistry. We thank all involved employees of the clinic for horses who contributed to the care of the horses or who gave their support in the execution of the study.
Furthermore, we thank the Institute for Pathology of the University of Veterinary Medicine Hannover for their technical support and helpful advice.

AUTH O R CO NTR I B UTI O N S
All authors contributed to the manuscript. N. Verhaar contributed to the study design and execution, and performed the data analysis and interpretation. C. Pfarrer and S. Kästner contributed to the study design as well as the data analysis and interpretation. N. Neudeck, L. Twele and K. König contributed to the study design and its execution. K. Rohn contributed to the data analysis.

CO N FLI C T O F I NTE R E S T
No competing interests have been declared.