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    Serum concentration of lidocaine (squares) and the active metabolites MEGX (triangles) and glycinexylidide (circles) in 7 anesthetized horses. Lidocaine (2%) was administered as a bolus (1.3 mg/kg, IV) over a 15-minute period after induction of anesthesia, which was followed by a CRI (0.05 mg/kg/min) throughout anesthesia. The target steady-state range of lidocaine concentrations, as reported in another study,15 is indicated (gray-shaded area).

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    Representative photomicrographs of colonic mucosa obtained from a horse infused with saline (0.9% NaCl) solution during anesthesia. A—Control segment obtained at 0 hours (0 hours was defined as the time immediately after the abdominal incision and before intestinal ischemia-reperfusion or manipulation). B—Colonic injury after ischemia for 1 hour is characterized by edema of epithelial cells, hemorrhage in the lamina propria, and denuded, detached, and degenerated or necrotic epithelial cells. C—Tissues obtained after reperfusion for 4 hours have evidence of repair of the mucosa by restitution with flattened epithelial cells. H&E stain; bar = 100 μm.

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    Box-and-whisker plots of neutrophil scores (scale, 1 to 3, as described elsewhere26) for histologic layers of the colon obtained from anesthetized horses (7/treatment group) infused with lidocaine (L) or saline solution (S), with 0 hours as the time immediately after the abdominal incision and before intestinal ischemia-reperfusion or manipulation. Samples were collected at 0 hours as control tissues (0C, immediately after the abdominal incision and before ischemia-reperfusion and manipulation), after 1 hour of ischemia (1I), after reperfusion for 1 hour (1R), after reperfusion for 4 hours (4R), and at 4 hours as control tissues (4C). Boxes represent the interquartile range, the horizontal line in each box is the median, and the whiskers represent minimum and maximum values. Light gray boxes represent data from first quartile to median, and dark gray boxes represent data from the median to third quartile. A–CWithin a layer, boxes with different uppercase letters differ significantly (P < 0.05) within the lidocaine treatment. a–cWithin a layer, boxes with different lowercase letters differ significantly (P < 0.05) within the saline solution treatment. *Within a time point, values differ significantly (P < 0.05) between the lidocaine- and saline solution–treated tissues.

  • View in gallery

    Representative photomicrographs of jejunal mucosa obtained from a horse infused with lidocaine (2%) during anesthesia. A—Control segment obtained at 0 hours (immediately after the abdominal incision and before ischemia-reperfusion and manipulation). B—Jejunal injury after ischemia for 1 hour is characterized by a reduction in mucosal height, edema of epithelial cells, hemorrhage in the lamina propria, and denuded, detached, and degenerated or necrotic epithelial cells. C—Tissues obtained after reperfusion for 4 hours have evidence of repair of the mucosa by restitution with flattened epithelial cells. H&E stain; bar = 100 μm.

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    Box-and-whisker plots of neutrophil scores (scale, 1 to 3) for histologic layers of the jejunum obtained from anesthetized horses (7/treatment group) infused with lidocaine or saline solution. Samples were as follows: control samples collected at 0 hours (0C, immediately after the abdominal incision and before ischemia-reperfusion and manipulation), samples collected after ischemia for 0.5 hours (0.5I), samples collected after reperfusion for 1 hour (1R), samples collected after reperfusion for 4 hours (4R), and control samples collected at 4 hours (4C). See Figure 3 for remainder of key.

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    Representative photomicrographs of jejunal serosa and muscle layers obtained from a horse infused with lidocaine (2%) during anesthesia and in which the jejunum was manipulated to mimic surgical interventions. A—Control tissues obtained at 0 hours (immediately after the abdominal incision and before ischemia-reperfusion and manipulation). B—Neutrophil infiltration is evident at 1 hour after manipulation. C—A progressive increase in neutrophil infiltration is evident at 4 hours after manipulation. H&E stain; bar = 100 μm.

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    Box-and-whisker plots of neutrophil scores (scale, 1 to 3) for histologic layers of the jejunum obtained from anesthetized horses (7/treatment group) infused with lidocaine or saline solution and in which the jejunum was manipulated to mimic surgical interventions. Samples were as follows: control samples collected at 0 hours (0C, immediately after the abdominal incision and before ischemia-reperfusion and manipulation), samples collected 1 hour after manipulation (1M), samples collected 4 hours after manipulation (4M), and control samples collected at 4 hours (4C). *Within a time point, value differs significantly (P < 0.05) from the value for the lidocaine-treated tissues. See Figure 3 for remainder of key.

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    Mean ± SD values of TER for jejunal mucosa obtained from anesthetized horses before intestinal manipulation (control sample at 0 hours; solid line), jejunal mucosa obtained from anesthetized horses 4 hours after intestinal manipulation (dashed line), and a control sample at 4 hours after intestinal manipulation (dotted line) and incubated in Ussing chambers with Krebs-Ringer bicarbonate buffer. Values reported are the mean value for samples (evaluated in duplicate) from 4 horses. *Within a time point, the value for the control sample at 0 hours differs significantly (P < 0.05) from the value for the control sample at 4 hours. Within a time point, the value for the control sample at 0 hours differs significantly (P < 0.05) from the value for the sample at 4 hours after intestinal manipulation.

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    Mean ± SD values of permeability to 3H-labeled mannitol (transmucosal flux) for jejunal mucosa obtained from anesthetized horses before intestinal manipulation (control sample at 0 hours; black bars) and 4 hours after intestinal manipulation (white bars) and control samples obtained at 4 hours (gray bars) and incubated in Ussing chambers with Krebs-Ringer bicarbonate buffer. Values reported are the mean value for samples (evaluated in duplicate) from 4 horses. a–cWithin a time point, values with different letters differ significantly (P < 0.05).

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Effect of lidocaine on inflammation in equine jejunum subjected to manipulation only and remote to intestinal segments subjected to ischemia

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  • 1 Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32608.
  • | 2 Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32608.
  • | 3 Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32608.
  • | 4 Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32608.
  • | 5 Department of Physiological Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32608.
  • | 6 Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32608.

Abstract

OBJECTIVE To examine effects of continuous rate infusion of lidocaine on transmural neutrophil infiltration in equine intestine subjected to manipulation only and remote to ischemic intestine.

ANIMALS 14 healthy horses.

PROCEDURES Ventral midline celiotomy was performed (time 0). Mild ischemia was induced in segments of jejunum and large colon. A 1-m segment of jejunum was manipulated by massaging the jejunal wall 10 times. Horses received lidocaine (n = 7) or saline (0.9% NaCl) solution (7) throughout anesthesia. Biopsy specimens were collected and used to assess tissue injury, neutrophil influx, cyclooxygenase expression, and hypoxia-inducible factor 1α (HIF-1α) expression at 0, 1, and 4 hours after manipulation and ischemia. Transepithelial resistance (TER) and mannitol flux were measured by use of Ussing chambers.

RESULTS Lidocaine did not consistently decrease neutrophil infiltration in ischemic, manipulated, or control tissues at 4 hours. Lidocaine significantly reduced circular muscle and overall scores for cyclooxygenase-2 expression in manipulated tissues. Manipulated tissues had significantly less HIF-1α expression at 4 hours than did control tissues. Mucosa from manipulated and control segments obtained at 4 hours had lower TER and greater mannitol flux than did control tissues at 0 hours. Lidocaine did not significantly decrease calprotectin expression. Severity of neutrophil infiltration was similar in control, ischemic, and manipulated tissues at 4 hours.

CONCLUSIONS AND CLINICAL RELEVANCE Manipulated jejunum did not have a significantly greater increase in neutrophil infiltration, compared with 4-hour control (nonmanipulated) jejunum remote to sites of manipulation, ischemia, and reperfusion. Lidocaine did not consistently reduce neutrophil infiltration in jejunum.

Abstract

OBJECTIVE To examine effects of continuous rate infusion of lidocaine on transmural neutrophil infiltration in equine intestine subjected to manipulation only and remote to ischemic intestine.

ANIMALS 14 healthy horses.

PROCEDURES Ventral midline celiotomy was performed (time 0). Mild ischemia was induced in segments of jejunum and large colon. A 1-m segment of jejunum was manipulated by massaging the jejunal wall 10 times. Horses received lidocaine (n = 7) or saline (0.9% NaCl) solution (7) throughout anesthesia. Biopsy specimens were collected and used to assess tissue injury, neutrophil influx, cyclooxygenase expression, and hypoxia-inducible factor 1α (HIF-1α) expression at 0, 1, and 4 hours after manipulation and ischemia. Transepithelial resistance (TER) and mannitol flux were measured by use of Ussing chambers.

RESULTS Lidocaine did not consistently decrease neutrophil infiltration in ischemic, manipulated, or control tissues at 4 hours. Lidocaine significantly reduced circular muscle and overall scores for cyclooxygenase-2 expression in manipulated tissues. Manipulated tissues had significantly less HIF-1α expression at 4 hours than did control tissues. Mucosa from manipulated and control segments obtained at 4 hours had lower TER and greater mannitol flux than did control tissues at 0 hours. Lidocaine did not significantly decrease calprotectin expression. Severity of neutrophil infiltration was similar in control, ischemic, and manipulated tissues at 4 hours.

CONCLUSIONS AND CLINICAL RELEVANCE Manipulated jejunum did not have a significantly greater increase in neutrophil infiltration, compared with 4-hour control (nonmanipulated) jejunum remote to sites of manipulation, ischemia, and reperfusion. Lidocaine did not consistently reduce neutrophil infiltration in jejunum.

At least 50% of horses develop ileus after small intestinal surgery, and these horses are 1.4 times as likely to die as horses without this complication.1 The pathophysiologic mechanism for postoperative ileus currently is believed to include mural inflammation induced by preoperative distention, hypoperfusion, reperfusion injury, and intestinal manipulation.2–4 Inflammation induced by intestinal manipulation in laboratory animals can release proinflammatory cytokines and prostaglandins from activated macrophages.5 These mediators then trigger an influx of monocytes and neutrophils into the muscle layers, and these cells can release inflammatory products that reduce muscle contractility.5–7 Transmural intestinal inflammation could also contribute to other complications after colic surgery, including adhesions and endotoxemia–systemic inflammatory response syndrome.8

Manual decompression of the small intestine is a commonly performed procedure in horses with small intestinal obstruction9 and can cause mural inflammation in horses in research settings.2,4,10 Intestinal inflammation, evident as neutrophilic infiltration, has been identified in equine jejunum 18 hours after experimentally induced ischemia and intestinal manipulation, which coincides with the time at which postoperative ileus most commonly develops in clinically affected horses.4 This temporal relationship suggests that inflammatory pathways could play an important role in the development of postoperative ileus.4 In a studya of clinically affected horses, those that developed postoperative ileus had an increased number of neutrophils in the serosa of the distal resection margin and a slightly higher neutrophil count in the circular muscle of the proximal resection margin. These findings suggest that the inflammatory response to mechanical manipulation could contribute to postoperative ileus in horses.

Lidocaine has been used in horses after colic surgery11,12 on the basis that it can treat or prevent postoperative ileus.13–15 In a multicenter study14 of horses with nasogastric reflux attributable to postoperative ileus or enteritis, 65% of horses treated with a lidocaine infusion ceased to have reflux within 30 hours after infusion, compared with 27% of horses treated with infusion of saline (0.9% NaCl) solution. Lidocaine also reduced the hourly volume of reflux during and after the infusion, which decreased the duration of hospitalization.14 In another study,15 lidocaine reduced the prevalence of postoperative ileus in horses after small intestinal surgery and improved the short-term survival rate. Because lidocaine does not appear to act as a prokinetic drug in clinically normal horses,16–18 a potential benefit for use of lidocaine to prevent or treat postoperative ileus could be mediated through an anti-inflammatory effect19 or an ability to enhance mucosal repair.20

The study reported here was designed to measure inflammatory cell infiltration and expression of COX-1, COX-2, and HIF-1α in intestine subjected to manipulation and remote to segments of jejunum and colon subjected to ischemia. Although both COX enzymes appear to be constitutively expressed in equine jejunum, ischemia increases COX-2 expression, and that response is reduced by lidocaine.19 Blood flow to tissues can affect expression of HIF-1α, a transcription factor composed of an inducible α unit and a constitutive β unit.21 During periods of tissue hypoxia, the α and β units form a complex that initiates transcription of oxygen-regulated genes that improve survival of hypoxic cells.22

The hypothesis for the study reported here was that CRI of lidocaine would reduce neutrophil infiltration inflicted by intestinal manipulation in a segment of jejunum remote to intestinal segments subjected to ischemia. Another objective was to examine, as assessed in vitro, effects of inflammation on mucosal barrier permeability in manipulated jejunum. A 4-hour period was allowed after ischemia and manipulation to develop a neutrophil infiltration response.

Materials and Methods

Horses

Fourteen adult horses without signs of gastrointestinal tract disorders were included in the study. Horses were of various breeds. Median age of horses was 6 years (range, 3 to 25 years), and median body weight was 522 kg (range, 400 to 575 kg). Horses had not received NSAIDs during the 3 weeks preceding the study and did not have laminitis or a history of gastrointestinal tract disease. For the week before the start of the study, horses were housed on pasture, were fed grass hay (2% of body weight/d), and had unlimited access to water. The study was approved by the Institutional Animal Care and Use Committee of the University of Florida.

Anesthesia

Horses were randomly assigned by a random number generator to 2 groups (7 horses/group). Horses in 1 group received a CRI of lidocaine throughout anesthesia, and horses in the other group received a CRI of an equivalent volume of a physiologic saline solution; for both groups, the CRI was in addition to the polyionic fluids horses received as part of the anesthesia regimen. Time 0 was defined as the time immediately after the abdominal incision and before intestinal ischemia-reperfusion or manipulation.

Horses were sedated with xylazine hydrochloride (0.5 mg/kg, IV) and butorphanol tartrate (0.02 mg/kg, IV). Anesthesia was induced with diazepam (0.1 mg/kg, IV) and ketamine hydrochloride (2.2 mg/kg, IV). The trachea of each horse was intubated. Horses were positioned in dorsal recumbency, and anesthesia was maintained with isoflurane (1% to 3%) in oxygen. Mean arterial blood pressure, heart rate, respiratory rate, fraction of inspired oxygen, and inspiratory isoflurane concentrations were monitored continuously. Isotonic polyionic fluids were infused continuously (5 mL/kg/h, IV), and mean arterial blood pressure was maintained at ≥ 70 mm Hg with a CRI of dobutamine and adjustments to the flow of isoflurane. Horses were euthanized (100 mL of pentobarbital sodium with phenytoin,b IV) at the end of anesthesia (approx 6 hours after the induction of anesthesia).

CRI

Lidocaine (2%) was administered to horses as a bolus (1.3 mg/kg, IV) over a 15-minute period after induction of anesthesia, which was followed by a CRI (0.05 mg/kg/min) throughout anesthesia. The control group received physiologic saline solutionc (equivalent volumes on a body weight basis) as a bolus and CRI. Surgeons responsible for inducing ischemia and manipulating the jejunum were not aware of which of these treatments was administered to each horse.

Ischemia and reperfusion

Standard aseptic preparation and surgical draping of the abdomen of each horse were performed. A 25-cm ventral midline incision was used to enter the abdominal cavity. The jejunum, beginning at least 4 arcades proximal to the ileum, was assigned to 3 equal portions. A 40-cm segment in the middle of each of the 2 most distal portions of jejunum was rendered ischemic by venous, arterial, and mural occlusion for 30 minutes, as described elsewhere.23,24 At the same time, two 20-cm-long segments of colon at the pelvic flexure were rendered ischemic by venous, arterial, and mural occlusion for 1 hour.23 At the end of the ischemic period, intestine was reperfused by removal of ligatures and clamps used to induce ischemia, as previously described.23,24

Jejunal manipulation

Intestinal manipulation similar to that applied to distended jejunum in a clinical setting was simulated. Each end of a 1-m segment of the most proximal portion of the jejunum was ligated with umbilical tape. Sufficient intestinal contents were allowed to remain in this segment, as determined by subjective assessment, to fill 30 cm of the intestine to approximately 7 cm in diameter. This fluid column was massaged proximally and distally between the umbilical tape ligatures (5 times in each direction; total, 10 massages). The umbilical tape ligatures were removed, a suture was placed on the antimesenteric side to mark each site of occlusion, and the segment was returned to the abdomen. All ischemic and manipulated segments had a loose umbilical tape tie that extended outside the abdomen and had a coding system that allowed each segment to be exteriorized individually for biopsy. This allowed exteriorization of the segment of interest from the abdomen without manipulation of other segments.

Collection of intestinal biopsy specimens

Immediately after the abdomen was incised, a full-thickness intestinal biopsy specimen (2 × 2 cm) was obtained from the midjejunum (control sample at time 0). A full-thickness biopsy specimen (2 × 2 cm) was obtained from the middle third of the manipulated segment at 1 hour after the end of manipulation and from ischemic segments at the end of ischemia and 1 hour later. At 4 hours after the end of ischemia and manipulation (all of which ended within a 15-minute period), ischemic and manipulated segments were exteriorized, and terminal biopsy specimens were collected. In addition, a control biopsy specimen was obtained from proximal or distal to the manipulated segment at that time. Biopsy sites were closed with suture in a continuous Lembert pattern.

Histologic examinations

All tissue samples were fixed in neutral-buffered 10% formalin for light microscopy. Tissues were subsequently embedded in paraffin, cut (4-μm-thick sections), and mounted on slides. Slides were stained with H&E stain in a routine manner.23,25 For histomorphometric assessment of mucosal damage, images obtained by use of light microscopy were analyzed with a computer-based imaging program,d and tissues were examined as previously described.23,25 The pathologist (AG) responsible for interpreting histologic and immunohistochemical changes and making other tissue measurements was not aware of the source of the tissues.

Neutrophil and eosinophil infiltration

Neutrophil infiltration in the serosa, muscle layers, submucosa, and mucosa was scored from 0 to 3 for presence of neutrophils by use of a 40X objective, as described elsewhere.25 Luna stain was used to detect eosinophils, and the absolute number of eosinophils was counted for each tissue sample, as previously described.26 Calprotectin was used as an additional marker of neutrophil infiltration in paraffin-embedded sections from manipulated jejunum and from a control segment obtained at 0 hours; calprotectin was detected by use of a mouse anti-human macrophage monoclonal antibody (MAC387) and biotin-free detection kit.25 The number of cells with positive staining results for calprotectin within the serosa and muscle layers was determined, and the mean number of calprotectin-containing cells per square millimeter was calculated for each layer.25 The number of calprotectin-positive cells per square millimeter of tissue was counted in 3 areas for each layer (length of each area, 217 μm, which was equal to the length of 1 image by use of a 40X objective).25

Immunohistochemical staining for COX-1, COX-2 and HIF-1α

Tissues only from manipulated jejunum were fixed in neutral-buffered 10% formalin, embedded in paraffin, and cut (5-μm-thick sections). Tissue sections were placed on silane-coated slides, deparaffinized, and rehydrated. Heat-antigen retrieval was performed, and slides were subsequently incubated in 1% H2O2. Slides were washed in PBS solution and incubated with normal goat serum for 1 hour. Slides then were incubated for 30 minutes with rabbit anti-human COX-1, COX-2, or HIF-1α polyclonal antibody.e Slides were washed 3 times in PBS solution and then incubated for 30 minutes with biotinylated donkey anti-goat antibody and streptavidin-labeled peroxidase. Sections were counterstained by immersion in Mayer hematoxylin stain for 3 minutes, and color was developed by use of 0.1% ammonium hydroxide (2.9mM). Slides then were washed in distilled water, dehydrated, and mounted in an aqueous mounting medium, and cover slips were applied. Serosa, muscularis, surface epithelial cells, upper and lower lamina propria cells, and crypts were scored (scale, 0 to 3) for intracellular expression of COX-1, COX-2, and HIF-1α by use of a previously described scoring system.27

Ussing chamber analysis

The jejunal mucosa sample collected at 0 hours and mucosa samples collected 4 hours after end of manipulation from the manipulated segment and the control segments were mounted in Ussing chambers.28 Krebs-Ringer bicarbonate buffer solution was used to bathe the tissues; the solution was maintained at a pH of 7.4 by constant perfusion with 95% O2 and 5% CO2 at 37°C by circulating with a gas lift through water-jacketed reservoirs.28 Each tissue was assayed in duplicate. At 15-minute intervals throughout the experimental period, the transepithelial potential difference was recorded with a potentiometer.f At other times, the transepithelial potential difference was continuously nullified by the passage of an external current across the tissue by use of an automatic voltage-current clamp amplifier.f The TER was calculated from Ohm's law as the potential difference divided by the external current across the tissue and used as a measure of integrity of the jejunal mucosa and permeability of the paracellular pathway.28 After the tissues were incubated for 30 minutes, 1 μCi of 3H mannitol (70 pmol) was added to the mucosal surface of each tissue to determine effects of various conditions on permeability for this low-molecular-weight marker of tissue leakiness.28 Mucosal-to-serosal flux of mannitol was determined by scintillation counting of 250-μL samples collected from both sides of tissues at 30-minute intervals, and the mannitol flux then was calculated.28

Serum lidocaine concentrations

Serum samples were obtained from all horses at time 0 (immediately after the abdominal incision, before intestinal ischemia-reperfusion or manipulation, and before start of a lidocaine or saline CRI) and then at 15, 75, 145, and 255 minutes after the CRI started. Serum concentrations of lidocaineg and 2 lidocaine metabolites (MEGXg and glycinexylidideg) were determined by use of high-performance liquid chromatography with UV absorbance detection. Duplicate 1-mL samples of serum from saline solution– or lidocaine-treated horses were alkalinized with 1N NaOH (100 μL) and then extracted by mixing with dichloromethane (4 mL) followed by centrifugation (3,000 × g) for 20 minutes. The organic layer was removed, and the extraction process was repeated 3 times; all portions of the dichloromethane extracts were combined and dried under a stream of nitrogen gas at 60°C. Control serum samples were spiked with standard solutions of lidocaine, MEGX, and glycinexylidide and then extracted for determination of recovery. All dried extracts were reconstituted in mobile phase (30mM phosphate buffer [pH, 3.7] and acetonitrile in a 92:8 volumetric mixture) and filtered through 0.45-μm syringe filters prior to analysis. Analyte separation and quantification was performed with a high-performance liquid chromatography systemh by use of a C18 analytic column (150 × 4.6 mm; particle size, 5 μm)i with a mobile phase flow rate of 1 mL/min and UV detector wavelength of 205 nm. Peak areas for lidocaine, MEGX, and glycinexylidide in samples from lidocaine-treated horses were compared with standard curves (0.05 to 5 μg/mL) that were constructed for each analyte by fortification of equine serum with known amounts of reference standards. Coefficient of determination was ≥ 0.99 for each standard curve, and extracted spiked samples were within ± 15% of predicted concentrations across the range of each standard curve.

Data analysis

Statistical tests were performed with a commercially available program.j Power analysis was performed with online power analysis software.k On the basis of significant results of histomorphometric changes and tissue neutrophil scores obtained from previous ischemia-reperfusion studies,24,28,29 the calculated required sample size was 6 (α = 0.05 and power = 0.8), which supported the chosen sample size of 7 horses/group.

The Kruskal-Wallis test was used to compare histologic and histochemical data at 0, 1, and 4 hours. When a significant P value was identified, a post hoc Bonferroni test was used for pairwise comparisons. The Shapiro-Wilk test for normality was used for Ussing chamber data (TER and mannitol flux), and an ANOVA was performed to compare data. Whenever a significant P value was identified, a post hoc Bonferroni test was used for pairwise comparisons. The Mann-Whitney U test was used to compare values between the 2 treatment groups (saline solution vs lidocaine) at each time point but not between time points. Values of P < 0.05 were considered significant for all analyses.

Results

Serum lidocaine concentrations

Serum lidocaine concentrations of all horses were within the reported target steady-state rangel of 1 to 2 μg/mL within 15 minutes after start of the infusion and remained within that range throughout anesthesia and the CRI (Figure 1). The active metabolites MEGX and glycinexylidide continued to accumulate throughout the experimental period.

Figure 1—
Figure 1—

Serum concentration of lidocaine (squares) and the active metabolites MEGX (triangles) and glycinexylidide (circles) in 7 anesthetized horses. Lidocaine (2%) was administered as a bolus (1.3 mg/kg, IV) over a 15-minute period after induction of anesthesia, which was followed by a CRI (0.05 mg/kg/min) throughout anesthesia. The target steady-state range of lidocaine concentrations, as reported in another study,15 is indicated (gray-shaded area).

Citation: American Journal of Veterinary Research 78, 8; 10.2460/ajvr.78.8.977

Colonic ischemia

Ischemia for 1 hour caused tissue edema, and the serosa was purple and had evidence of petechia. By 10 to 15 minutes after removal of the clamps and ligatures, the reperfused segment had an appearance similar to that of adjacent control segments. Histologic evidence of mucosal injury after ischemia for 1 hour was mild and included hemorrhage in the lamina propria, detachment of surface epithelial cells, and epithelial degeneration and swollen necrotic cells (Figure 2).

Figure 2—
Figure 2—

Representative photomicrographs of colonic mucosa obtained from a horse infused with saline (0.9% NaCl) solution during anesthesia. A—Control segment obtained at 0 hours (0 hours was defined as the time immediately after the abdominal incision and before intestinal ischemia-reperfusion or manipulation). B—Colonic injury after ischemia for 1 hour is characterized by edema of epithelial cells, hemorrhage in the lamina propria, and denuded, detached, and degenerated or necrotic epithelial cells. C—Tissues obtained after reperfusion for 4 hours have evidence of repair of the mucosa by restitution with flattened epithelial cells. H&E stain; bar = 100 μm.

Citation: American Journal of Veterinary Research 78, 8; 10.2460/ajvr.78.8.977

The epithelial surface of segments obtained after reperfusion for 1 and 4 hours were almost completely covered with cells that were flatter than normal epithelial cells. Histomorphometric changes were the same in the lidocaine- and saline solution–treated groups, except that there were significantly more degenerated epithelium and swollen necrotic cells for the lidocaine group than for the saline solution group.

In all layers, ischemia alone did not affect neutrophil infiltration, but neutrophil scores were highest in the colon obtained after reperfusion for 4 hours (Figure 3). In the longitudinal and circular muscle layers, and especially in the serosa, neutrophil scores were significantly increased in the control tissues obtained at 4 hours, compared with the control tissues obtained at 0 hours. In the longitudinal muscle and serosal layers, control tissues obtained at 4 hours were not significantly different from the tissues obtained after reperfusion for 4 hours. Compared with results for infusion of saline solution, lidocaine significantly reduced neutrophil scores in the colonic mucosa after ischemia for 1 hour and in the longitudinal muscle after reperfusion for 1 hour.

Figure 3—
Figure 3—

Box-and-whisker plots of neutrophil scores (scale, 1 to 3, as described elsewhere26) for histologic layers of the colon obtained from anesthetized horses (7/treatment group) infused with lidocaine (L) or saline solution (S), with 0 hours as the time immediately after the abdominal incision and before intestinal ischemia-reperfusion or manipulation. Samples were collected at 0 hours as control tissues (0C, immediately after the abdominal incision and before ischemia-reperfusion and manipulation), after 1 hour of ischemia (1I), after reperfusion for 1 hour (1R), after reperfusion for 4 hours (4R), and at 4 hours as control tissues (4C). Boxes represent the interquartile range, the horizontal line in each box is the median, and the whiskers represent minimum and maximum values. Light gray boxes represent data from first quartile to median, and dark gray boxes represent data from the median to third quartile. A–CWithin a layer, boxes with different uppercase letters differ significantly (P < 0.05) within the lidocaine treatment. a–cWithin a layer, boxes with different lowercase letters differ significantly (P < 0.05) within the saline solution treatment. *Within a time point, values differ significantly (P < 0.05) between the lidocaine- and saline solution–treated tissues.

Citation: American Journal of Veterinary Research 78, 8; 10.2460/ajvr.78.8.977

Jejunal ischemia

Ischemia for 0.5 hours caused tissue edema, and the serosa was purple and had evidence of petechia. The tissues returned to a more normal appearance after reperfusion for 10 to 15 minutes. Histologic evidence of mucosal injury after ischemia for 0.5 hours was mild and included hemorrhage and detachment of surface epithelial cells (Figure 4). Restitution of the epithelial lining was evident in samples obtained after reperfusion for 4 hours, with some fluid accumulation in the villi. Ischemia significantly reduced villus and epithelial height but did not change villus and epithelial width. The epithelial surfaces of segments obtained after reperfusion for 1 and 4 hours were almost completely covered with cells that were flatter than normal epithelial cells. Lidocaine significantly reduced mucosal height and the number of swollen necrotic cells in samples obtained after reperfusion for 4 hours, compared with results for saline solution–treated tissues.

Figure 4—
Figure 4—

Representative photomicrographs of jejunal mucosa obtained from a horse infused with lidocaine (2%) during anesthesia. A—Control segment obtained at 0 hours (immediately after the abdominal incision and before ischemia-reperfusion and manipulation). B—Jejunal injury after ischemia for 1 hour is characterized by a reduction in mucosal height, edema of epithelial cells, hemorrhage in the lamina propria, and denuded, detached, and degenerated or necrotic epithelial cells. C—Tissues obtained after reperfusion for 4 hours have evidence of repair of the mucosa by restitution with flattened epithelial cells. H&E stain; bar = 100 μm.

Citation: American Journal of Veterinary Research 78, 8; 10.2460/ajvr.78.8.977

No significant differences were found in neutrophil scores over time in the mucosal layer or villi (Figure 5). In the circular muscle, longitudinal muscle, and serosa, neutrophil scores did not differ significantly between samples obtained after reperfusion for 4 hours and control tissues obtained at 4 hours, but these layers had significantly higher neutrophil scores than corresponding layers for control samples obtained at 0 hours, samples obtained after ischemia for 0.5 hours, and samples obtained after reperfusion for 1 hour for both the lidocaine and saline solution groups. There were no significant differences in neutrophil scores between the saline solution– or lidocaine-treated tissues in any layer at any time point.

Figure 5—
Figure 5—

Box-and-whisker plots of neutrophil scores (scale, 1 to 3) for histologic layers of the jejunum obtained from anesthetized horses (7/treatment group) infused with lidocaine or saline solution. Samples were as follows: control samples collected at 0 hours (0C, immediately after the abdominal incision and before ischemia-reperfusion and manipulation), samples collected after ischemia for 0.5 hours (0.5I), samples collected after reperfusion for 1 hour (1R), samples collected after reperfusion for 4 hours (4R), and control samples collected at 4 hours (4C). See Figure 3 for remainder of key.

Citation: American Journal of Veterinary Research 78, 8; 10.2460/ajvr.78.8.977

Jejunal manipulation

Manipulated segments of jejunum subjectively appeared more hyperemic than adjacent unmanipulated segments at 4 hours after manipulation. Manipulation of the jejunum did not cause histologic evidence of mucosal edema, hemorrhage, or epithelial injury in the saline solution and lidocaine groups at 1 or 4 hours after manipulation or in the control tissues at 4 hours. However, marked neutrophil infiltration was evident in the serosal layer at 1 and 4 hours after manipulation (Figure 6).

Figure 6—
Figure 6—

Representative photomicrographs of jejunal serosa and muscle layers obtained from a horse infused with lidocaine (2%) during anesthesia and in which the jejunum was manipulated to mimic surgical interventions. A—Control tissues obtained at 0 hours (immediately after the abdominal incision and before ischemia-reperfusion and manipulation). B—Neutrophil infiltration is evident at 1 hour after manipulation. C—A progressive increase in neutrophil infiltration is evident at 4 hours after manipulation. H&E stain; bar = 100 μm.

Citation: American Journal of Veterinary Research 78, 8; 10.2460/ajvr.78.8.977

For both saline solution– and lidocaine-treated horses, neutrophil scores were significantly increased in all layers, except the villi, at 4 hours after manipulation, compared with neutrophil scores in control tissues at 0 hours and tissues obtained 1 hour after manipulation (Figure 7). The neutrophil score was significantly lower in the villi 1 hour after manipulation in the lidocaine group, compared with the score for villi in the saline solution group, but there was no significant difference between the neutrophil scores in the lidocaine-treated versus the saline solution–treated tissues at other time points. There was no significant difference in the neutrophil scores in any layers between manipulated and control tissues at 4 hours after manipulation in the saline solution or lidocaine groups.

Figure 7—
Figure 7—

Box-and-whisker plots of neutrophil scores (scale, 1 to 3) for histologic layers of the jejunum obtained from anesthetized horses (7/treatment group) infused with lidocaine or saline solution and in which the jejunum was manipulated to mimic surgical interventions. Samples were as follows: control samples collected at 0 hours (0C, immediately after the abdominal incision and before ischemia-reperfusion and manipulation), samples collected 1 hour after manipulation (1M), samples collected 4 hours after manipulation (4M), and control samples collected at 4 hours (4C). *Within a time point, value differs significantly (P < 0.05) from the value for the lidocaine-treated tissues. See Figure 3 for remainder of key.

Citation: American Journal of Veterinary Research 78, 8; 10.2460/ajvr.78.8.977

Calprotectin results were similar to the neutrophil findings, except that calprotectin was not detected in the mucosa (Table 1). The serosal layer had a significantly higher number of calprotectin-positive cells per square millimeter in the manipulated and control tissues at 4 hours after manipulation, compared with the number in control tissues obtained at 0 hours and tissues obtained 1 hour after manipulation in both the lidocaine and saline solution groups. Calprotectin results were similar for the saline solution– and lidocaine-treated tissues in all layers at all times.

Table 1—

Median (range) number of cells with positive staining results for calprotectin per square millimeter for various histologic layers of the jejunum obtained from horses (7 horses/treatment) after infusion with lidocaine or saline (0.9% NaCl) solution during anesthesia and in which the jejunum was manipulated to mimic surgical interventions.

Time pointTreatmentCircular muscleLongitudinal muscleSerosa
0CLidocaine0 (0–9.43)0 (0–9.43)0 (0–9.43)A
 Saline solution0 (0–18.86)a0 (0–28.30)a9.43 (0–9.43)a
1MLidocaine0 (0–282.97)0 (0–697.98)28.30 (9.43–1,433.69)A
 Saline solution9.43 (0–56.59)a9.43 (0–37.73)a235.80 (37.73–688.55)a
4MLidocaine18.86 (0–301.83)169.78 (47.16–1,169.59)1,584.61 (962.08–3,320.13)B
 Saline solution113.19 (18.86–499.91)b396.15 (245.24–754.57)b1,952.46 (1,490.28–3,084.32)b
4CLidocaine20.54 (0–135.92)92.30 (23.08–530.77)2,187.00 (164.08–3,133.31)B
 Saline solution71.77 (7.69–325.62)a,b69.23 (20.54–451.31)a1,410.23 (679.46–2,302.54)b

Samples were as follows: control samples at 0 (0C), samples at 1 hour after manipulation (1M), samples at 4 hours after manipulation (4M), and control samples at 4 hours (4C).

Within the saline solution treatment within a column, values with different superscript letters differ significantly (P < 0.05).

Within the lidocaine treatment within a column, values with different superscript letters differ significantly (P < 0.05).

In both jejunum and colon, Luna staining indicated that there was no change in eosinophil numbers in any layer following ischemia or manipulation, in both the saline solution– and lidocaine-treated tissues.

Expression of COX-1 was detected in all layers, but COX-1 expression did not change between treatments or over time. At 4 hours after manipulation, COX-2 scores in all layers (overall score) and in the circular muscle were significantly higher in the saline solution–treated tissues, compared with COX-2 scores for the lidocaine-treated tissues (Table 2). For the saline solution–treated tissues, COX-2 scores were significantly lower in the epithelium, villi, lower lamina propria, and submucosa and for overall score in control tissues obtained at 4 hours than in the manipulated tissues at 1 and 4 hours after manipulation. There were also significantly lower COX-2 scores in the serosa of control tissues obtained at 4 hours, compared with COX-2 scores for the tissues obtained at 4 hours after manipulation. For the saline solution–treated tissues, control tissues obtained at 4 hours had significantly lower COX-2 scores in the submucosa, compared with COX-2 scores for tissues obtained 1 hour after manipulation. The villi and serosal layers also had significantly lower scores in control samples obtained at 0 hours, compared with scores for tissues obtained 1 and 4 hours after manipulation.

Table 2—

Median (range) COX-2 scores (scale, 0 to 3, as described elsewhere27) for jejunal tissues obtained from horses (7 horses/treatment) after infusion with lidocaine or saline solution during anesthesia and in which the jejunum was manipulated to mimic surgical interventions.

Time pointTreatmentOverall scoreEpitheliumVilliULPLLPCryptsSubmucosaCircular musclesLongitudinal musclesSerosa
0CLidocaine18.83 (16.00–24.50)2.33 (1.00–2.83)1.83 (1.50–2.50)A2.00 (1.50–2.83)1.67 (1.33–2.67)2.33 (1.67–3.00)2.00 (1.50–2.83)A,B2.67 (1.67–3.00)2.67 (2.50–3.00)1.83 (0.67–2.50)A
 Saline solution15.67 (7.83–22.50)a,d1.50 (0.33–3.00)a,d1.67 (1.33–2.50)a2.00 (1.33–2.50)a1.33 (1.00–2.17)a1.83 (0.83–3.00)1.67 (0.83–2.33)a2.67 (0.50–3.00)2.83 (0.50–3.00)1.50 (0.67–2.33)a,b
1MLidocaine22.83 (17.17–24.50)2.67 (2.50–3.00)2.67 (1.67–3.00)22.33 (1.50–2.67)2.50 (1.17–2.83)2.33 (1.17–2.83)2.50 (1.83–3.00)A2.33 (2.17–3.00)2.67 (2.33–2.83)2.17 (1.50–2.67)A,B
 Saline solution23.50 (21.33–25.00)b2.83 (2.50–2.83)b2.83 (2.50–3.00)b2.67 (1.83–3.00)b2.33 (2.00–2.83)b2.33 (1.83–2.83)2.67 (2.50–3.00)b2.83 (2.17–3.00)2.67 (1.67–2.83)2.50 (2.17–2.67)a,b
4MLidocaine18.33 (17.67–25.67)*2.67 (1.83–3.00)2.67 (1.83–3.00)A,B2.00 (1.33–2.83)1.83 (1.33–2.83)1.83 (1.17–3.00)2.50 (1.50–2.83)A,B2.00 (1.33–2.50)*2.33 (1.83–3.00)2.83 (1.67–3.00)B
 Saline solution24.67 (20.50–29.67)†c3.00 (2.17–3.00)c3.00 (2.50–3.00)c2.50 (2.00–2.83)b2.33 (2.00–2.83)b2.50 (1.67–3.00)2.50 (2.17–3.00)b2.67 (2.33–2.83)†2.67 (1.83–3.00)3.00 (2.67–10.33)a
4CLidocaine19.33 (16.83–26.17)1.83 (1.67–3.00)1.83 (1.67–3.00)A,B1.83 (1.67–2.83)1.83 (1.67–2.83)2.5 (1.67–3.00)1.83 (1.50–2.50)B2.83 (2.00–3.00)3.00 (1.83–3.00)2.17 (2.00–3.00)A,B
 Saline solution17.67 (12.67–20.50)b,d1.83 (0.83–2.50)b,d1.83 (1.5–2.50)a,d1.83 (1.50–2.33)a,c1.67 (1.00–1.83)a,c2.00 (1.17–2.50)1.67 (1.17–2.17)a,c2.33 (0.83–3.00)2.00 (1.50–3.00)2.17 (1.83–2.50)b

Overall score is the sum of the scores of all layers.

Within a time point, value differs significantly (P < 0.05) from the value for the lidocaine treatment.

LLP = Lower lamina propria. ULP = Upper lamina propria.

See Table 1 for remainder of key.

Intestinal manipulation decreased HIF-1α expression at 1 and 4 hours after manipulation, compared with HIF-1α scores for control tissues obtained at 0 and 4 hours; HIF-1α scores were similar for control tissues obtained at 0 and 4 hours (Table 3). There was no difference in HIF-1α scores between the lidocaine- or saline solution–treated tissues at any time point.

Table 3—

Median (range) scores (scale, 0 to 3, as described elsewhere27) for HIF-1α expression in jejunal tissues obtained from horses (7 horses/treatment) after infusion with lidocaine or saline solution during anesthesia and in which the jejunum was manipulated to mimic surgical interventions.

Time pointTreatmentOverall scoreEpitheliumVilliULPLLPCryptsSubmucosaCircular musclesLongitudinal musclesSerosa
0CLidocaine18.00 (15.50–20.83)A,C1.67 (1.50–2.83)A,C2.50 (2.17–2.67)A,C1.67 (1.17–2.00)A,C1.33 (1.00–1.83)A,C 2.33 (1.83–3.00)A,C2.17 (2.00–2.50)A,C2.33 (1.83–2.83)A,C2.17 (1.50–2.50)A,C1.83 (1.67–2.50)A,C 
 Saline solution17.17 (10.83–21.33)a,c2.00 (1.00–2.33)a,c2.33 (1.67–3.00)a,c1.50 (1.17–2.50)a,c1.17 (0.83–2.33)a,c 2.50 (2.17–2.67)a,c2.17 (0.83–3.00)2.17 (1.00–3.00)1.83 (0.83–3.00)1.67 (1.00–2.33)a,b 
1MLidocaine6.33 (3.50–12.00)B0.33 (0.0–1.33)B1.33 (0.33–1.67)B0.33 (0.17–1.50)B0.50 (0.0–1.33)B 1.83 (1.00–2.17)A0.67 (0.0–2.17)B0.33 (0.0–1.33)B0.17 (0.0–1.50)B0.17 (0.0–1.67)B 
 Saline solution7.00 (3.50–13.67)b0.67 (0.17–1.50)b1.33 (1.17–2.33)a0.33 (0.0–1.17)b0.50 (0.17–1.00)b 1.67 (1.33–2.33)a0.17 (0.0–1.67)0.67 (0.0–1.83)0.50 (0.0–2.17)0.67 (0.0–2.17)a 
4MLidocaine4.00 (0.67–15.33)B0.33 (0.00–1.67)B0.67 (0.17–2.33)B0.17 (0.0–0.67)B0.33 (0.0–0.83)B 1.33 (0.33–2.00)A,B0.17 (0.0–1.67)B0.17 (0.0–1.83)B1.00 (0.0–2.00)B1.00 (0.0–2.50)B 
 Saline solution8.17 (1.50–14.83)b1.00 (0.0–1.33)b1.50 (0.33–2.00)a,b0.33 (0.0–1.0)b0.67 (0.0–1.33)b 2.00 (0.33–2.33)a,b0.33 (0.0–1.17)0.17 (0.0–1.33)0.33 (0.0–2.17)0.67 (0.33–3.00)a 
4CLidocaine20.83 (16.50–25.33)C2.17 (1.67–3.00)C2.83 (2.00–3.00)C1.83 (1.17–2.67)C1.67 (1.17–2.67)C 2.33 (2.00–3.00)C2.83 (1.83–3.00)C2.50 (2.17–2.83)C2.33 (1.83–2.83)C3.00 (2.17–3.00)C 
 Saline solution19.83 (16.58–23.50)c2.33 (1.83–2.33)c2.67 (2.17–3.00)c1.67 (1.17–2.33)c1.83 (1.25–2.00)c 2.5 (2.17–3.00)c2.67 (1.50–3.00)2.17 (1.67–2.832.17 (1.83–2.67)2.67 (1.67–3.00)b 

See Tables 1 and 2 for key.

Because there was limited availability of Ussing chambers, tissues from 4 horses were used in duplicate in the Ussing chambers. There was no difference in raw numbers for TER between the lidocaine- and saline solution–treated tissues at 4 hours; therefore, data from these tissues were pooled for analyses (n = 4 horses; 2 horses/treatment group) for tissues obtained at 4 hours after manipulation and control tissues obtained at 0 and 4 hours. The TER was significantly higher in the control tissues obtained at 0 hours, compared with the TER for control tissues obtained at 4 hours after incubation in the Ussing chambers for 30 to 135 minutes; the TER was significantly higher in control tissues at 0 hours, compared with the TER in tissues obtained at 4 hours after manipulation, after incubation for 60 to 135 minutes (Figure 8). The TER for tissues at 4 hours after manipulation and control tissues obtained at 4 hours was similar at all time points.

Figure 8—
Figure 8—

Mean ± SD values of TER for jejunal mucosa obtained from anesthetized horses before intestinal manipulation (control sample at 0 hours; solid line), jejunal mucosa obtained from anesthetized horses 4 hours after intestinal manipulation (dashed line), and a control sample at 4 hours after intestinal manipulation (dotted line) and incubated in Ussing chambers with Krebs-Ringer bicarbonate buffer. Values reported are the mean value for samples (evaluated in duplicate) from 4 horses. *Within a time point, the value for the control sample at 0 hours differs significantly (P < 0.05) from the value for the control sample at 4 hours. Within a time point, the value for the control sample at 0 hours differs significantly (P < 0.05) from the value for the sample at 4 hours after intestinal manipulation.

Citation: American Journal of Veterinary Research 78, 8; 10.2460/ajvr.78.8.977

The transmucosal flux of mannitol was lower in the control tissues obtained at 0 hours, compared with flux in control tissues obtained at 4 hours and tissues obtained at 4 hours after manipulation, after incubation for > 45 minutes (Figure 9). After incubation for 135 minutes, transmucosal flux of mannitol was significantly higher in the control samples obtained at 4 hours, compared with results for the tissues obtained at 4 hours after manipulation.

Figure 9—
Figure 9—

Mean ± SD values of permeability to 3H-labeled mannitol (transmucosal flux) for jejunal mucosa obtained from anesthetized horses before intestinal manipulation (control sample at 0 hours; black bars) and 4 hours after intestinal manipulation (white bars) and control samples obtained at 4 hours (gray bars) and incubated in Ussing chambers with Krebs-Ringer bicarbonate buffer. Values reported are the mean value for samples (evaluated in duplicate) from 4 horses. a–cWithin a time point, values with different letters differ significantly (P < 0.05).

Citation: American Journal of Veterinary Research 78, 8; 10.2460/ajvr.78.8.977

Discussion

A combination of ischemia in the colon and jejunum and manipulation of the jejunum caused a measurable inflammatory response in the present study, as indicated by a significant influx of neutrophils, especially in the seromuscular layers. This method was designed to simulate a typical intestinal strangulation, which involves intestinal ischemia and manual decompression. Intestinal distention was not induced in this study, although this change would develop in the manipulated segment in clinically affected horses and could add to the neutrophil response. Although jejunal and colonic ischemia would be unusual in a clinical setting, the purpose of the study was to maximize the yield of information from a small number of horses.

The finding that the response in time-matched control jejunal segments was similar to that in manipulated segments underscored the importance of the remote response in nonmanipulated jejunum when inflammation is induced in focal segments of intestine.6 Results of the present study also indicated that the degree of neutrophil infiltration in the serosal and muscle layers was similar between injured segments (manipulated or ischemia-reperfusion) and segments remote from those segments and not manipulated. Other studies4,10 on the inflammatory response to manipulation did not provide a comparison between manipulated tissues and nonmanipulated or control tissues at the same time points. Therefore, to our knowledge, the study reported here was the first in which it has been reported that the remote inflammatory response to intestinal injury could be similar to that in injured tissue, which might be an important factor in development of complications after colic surgery.

A 4-hour period was chosen to assess inflammation so that the entire study could be completed during anesthesia within a reasonable time frame (≤ 6 hours). However, longer intervals after manipulation were used in another study19 and might have yielded different results. We were concerned that recovery from anesthesia in horses with ischemic intestine would be an animal welfare issue, especially considering that high pain scores have been recorded in horses with ischemic colon that were allowed to recover from anesthesia.28 Also, the inflammation induced was sufficiently robust to reveal differences over time and among layers and should be relevant to clinically affected horses that would receive lidocaine during anesthesia to reduce the required amount of inhalation anesthetics.29 In a study30 of porcine ileum, 6 hours was sufficient to achieve peak neutrophil numbers in the mucosa after ischemic injury, and the time frame of the study reported here was similar.

Luna stain can be used in equine intestine as a marker of eosinophils,26 and calprotectin can be used to detect the pattern of neutrophil accumulation.25 Although calprotectin is also found in macrophages and monocytes, these would seem to contribute little to the inflammatory cell population in equine intestine.24 However, the mean number of calprotectin-positive cells typically underrepresents the number of H&E-stained neutrophils, presumably because neutrophil expression of calprotectin is variable or activated neutrophils lose cytoplasmic calprotectin.25 That this pattern was apparent in the lidocaine-treated horses raised the possibility that lidocaine played a role in activating the calprotectin-positive neutrophils. This would be consistent with in vitro evidence that lidocaine can increase adhesion and migration of equine neutrophils in response to various chemoattractants.31 However, concentrations of lidocaine required for this in vitro response are greater than the concentrations that can be achieved in vivo.30

The method used for jejunal manipulation in the present study should be relevant to the degree of trauma sustained by the small intestine during decompression, as used in our veterinary hospital.9 In another study,10 investigators used a different and more traumatic method of intestinal manipulation (eg, repeated manual decompression of intestine at 1 stroke/s for a total of 10 minutes). Details about the method of inducing manipulation trauma and effects of that trauma were not provided in another study4 in which investigators examined inflammation after intestinal manipulation. To our knowledge, no other studies have been conducted to examine the effects of lidocaine on manipulated equine small intestine.

Some significant differences in COX-2 scores were found among time points, and lidocaine reduced COX-2 expression in the circular muscle and in overall scores in the manipulated segments at 4 hours. Lidocaine also reduced neutrophil scores in the colonic mucosa after ischemia for 1 hour, in the jejunal villi after ischemia for 1 hour, and in colonic longitudinal muscles after reperfusion for 1 hour. These apparent inhibitory effects on neutrophil influx were consistent with evidence of reduced COX-2 expression in horses treated with lidocaine for ischemia-induced small intestinal injury, compared with results for horses treated with saline solution or flunixin meglumine alone.32 Also consistent with findings of that study,32 COX-2 appeared to be constitutively expressed in all intestinal layers of equine jejunum in the study reported here.

Lidocaine is one of the most widely used drugs for prevention of postoperative ileus and other complications of colic surgery.8,12 However, analysis of results of the present study did not reveal a consistent effect on neutrophil infiltration in response to ischemia and manipulation. The aforementioned benefits did not follow a consistent pattern with regard to tissue layer, type of injury, or time point to indicate a clinically relevant anti-inflammatory effect. This is consistent with results of an in vitro study31 for which investigators found that lidocaine at therapeutic concentrations did not inhibit migration or adhesion of equine neutrophils. Lidocaine does not inhibit inflammatory events in the laminae or skin of horses with laminitis experimentally induced by the use of black walnut extract.33 Despite the ability of lidocaine to reduce inflammation and improve lung function in human asthmatic patients, it appears to increase total numbers of inflammatory cells in bronchoalveolar lavage fluid obtained from horses with recurrent airway obstruction.34 Lidocaine was found to exert an anti-inflammatory effect in equine jejunum with experimentally induced ischemic injury,19 but this effect was evident only for horses treated with a combination of flunixin meglumine and lidocaine and was not evident after treatment with saline solution or lidocaine individually. Other properties of lidocaine, such as anesthetic-sparing effects,29,35,36 somatic analgesic properties,37,38 and the ability to ameliorate injury in equine lung tissue induced by intestinal ischemia and reperfusion,39 could be beneficial in horses with colic.

The neutrophil score was used as the predominant indicator of inflammation in the present study, although its role in inflammation is complicated and other indicators of inflammation could have yielded different results. Neutrophil influx is traditionally considered harmful to tissues, and neutrophil activation has been correlated with the mortality rate in horses with strangulating lesions.40 However, neutrophils can also be protective and support tissue repair, possibly by regulating the equilibrium between proinflammatory and anti-inflammatory conditions in injured tissues and by reducing concentrations of proinflammatory mediators and toxic radicals.41–44 Neutrophil accumulation within the equine large colon does not appear to have a negative effect on mucosal repair.23,25,28 Coincubation of ischemic-injured porcine ileal mucosa with activated neutrophils can augment the recovery of barrier function through an interleukin-1β– and COX-2–dependent mechanism.45

Inflammation in the seromuscular layer induced by manipulation did not appear to affect mucosal integrity, as determined by results of histologic examination. However, analysis of TER and mannitol flux revealed an increase in mucosal permeability in manipulated tissues and time-matched control samples, which presumably could be attributed to changes in tight junctions induced by inflammation.46 In porcine ileum, TER decreased at times when the number of mucosal neutrophils reached peak values,30 whereas results of the study reported here indicated that mucosal neutrophil infiltration was not required to induce this permeability response. Presumably, some inflammatory mediators released from neutrophils remote to the mucosa could be the reason for these findings. The TER in the control tissues at 0 hours was considerably higher than reported previously in equine jejunum,32 possibly because it was recorded before inflammation was induced and the tissues were obtained from anesthetized horses rather than from horses after euthanasia.32,47,48

Although the TER changes were similar to the changes in mannitol flux, the TER failed to confirm the difference between manipulated and control tissues obtained at 4 hours that was revealed by the mannitol flux. This could have reflected a difference between data obtained with 2 methods of measuring permeability or by a type II error caused by insufficient power to detect a difference in the TER measurements. A similar discrepancy has been reported in equine jejunum47 and was explained by differences in size and charge of pores in tight junctions as they responded to inflammatory mediators.46

Expression of HIF-1α has been examined in intestine obtained from horses with colic as a marker of intestinal ischemia.49 To our knowledge, oxygenation status of equine intestinal segments subjected to manipulation has not been reported previously. Although there was no significant difference in the expression of HIF-1α between saline solution and lidocaine treatments in the present study, HIF-1α scores were lower in manipulated tissues than in control tissues. It is possible that manipulation increased blood flow to those segments of small intestine, thereby reducing the expression of HIF-1α.49 Subjective evaluation of tissues obtained at 4 hours after manipulation revealed that the manipulated jejunum was diffusely and mildly hyperemic, compared with the appearance of the adjacent intestine, which supported the possibility that manipulation could have increased blood flow to the intestines.

Findings of the study reported here suggested that the remote inflammatory cell response to manipulation and ischemia in nonmanipulated (control) intestine could be of similar magnitude to that in intestinal segments subjected to injury resulting from manipulation and ischemia. This could explain the development of postoperative adhesions remote to the primary site of intestinal injury in horses with colic.50 Furthermore, evidence that manipulated small intestine might not have inflammation that is greater than inflammation in remote nonmanipulated intestine could support the use of manual decompression to relieve small intestinal distention.9 A consistent anti-inflammatory effect of lidocaine was not detected for the method of inducing intestinal injury and by measurement of the indicators of inflammation in the study reported here.

Acknowledgments

Supported by the Morris Animal Foundation and the University of Florida College of Veterinary Medicine Competitive Grants.

Presented as a poster at the 11th International Equine Colic Research Symposium, Dublin, July 2014, and as an abstract at the 2015 American College of Veterinary Surgeons Surgery Summit, Nashville, Tenn, October 2015.

ABBREVIATIONS

COX

Cyclooxygenase

CRI

Constant rate infusion

HIF-1α

Hypoxia-inducible factor 1α

MEGX

Monoethylglycinexylidide

TER

Transepithelial electrical resistance

Footnotes

a.

Rathmanner M, Staszyk C, Rötting AK, et al. Comparison of the inflammatory reaction in horses with and without development of post operative ileus (POI) (abstr), in Proceedings. 10th Int Equine Colic Res Symp 2011;57.

b.

Beuthanasia-D Special, Merck Animal Health Intervet Inc, Madison, NJ.

c.

Normosol-R, Abbott Laboratories, Irving, Tex.

d.

Image Pro Express, version 6.3, Media Cybernetics Inc, Rockville, Md.

e.

Alexis Co, San Diego, Calif.

f.

World Precision Instruments, Sarasota, Fla.

g.

AstraZeneca Pharmaceuticals UK, Macclesfield, Cheshire, England.

h.

Perkin Elmer Instruments, Norwalk, Conn.

i.

Phenomenex Co, Torrance, Calif.

j.

SPSS, version 22, IBM Corp, Armonk, NY.

k.

G*Power, version 3.1, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany.

l.

Malone ED, Turner TA, Wilson JH. Intravenous lidocaine for the treatment of equine ileus (abstr), in Proceedings. 6th Equine Colic Res Symp 1998;42.

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Contributor Notes

Address correspondences to Dr. Bauck (baucka@ufl.edu).