Acute abdominal disease (ie, colic) is reportedly among the most common causes of death in some horse populations.1,2 The annual number of colic cases has been reported as 4 to 10/100 horses,1,3 although up to 46 cases/100 horses/y have been recorded.4 Although most cases resolve spontaneously or respond to medical management, 2% to 10% of horses with intestinal disease require surgical intervention.5,6
Morphological and physiologic abnormalities detected in the intestines of horses in response to ischemia and reperfusion injury include cytokine production, alterations in endothelial cells, and neutrophil activation and migration.7,8 Increased myeloperoxidase activity and a decrease in the myeloperoxidase index (ie, myeloperoxidase content per leukocyte) are indicators of neutrophil activation and can be detected in horses with intestinal disease.9,10 The resulting intestinal inflammation has been associated with loss of motility, systemic shock, and development of intestinal adhesions.11,12 Reperfusion of ischemic tissue results in endothelial cell production of reactive oxygen metabolites and cytokines that attract neutrophils, causing microvascular and tissue inflammation.13 As a consequence, neutrophils become activated, adhere to the endothelium, and migrate into the intestinal mucosa, muscle, and serosa.14–17
Neutrophil apoptosis is one of the principal mechanisms that maintain neutrophil homeostasis. Neutrophils have the ability to release clinically relevant amounts of antiapoptotic chemokines, which can recruit additional neutrophils to the site of inflammation and promote survival of neighboring neutrophils through paracrine mechanisms.18 In vitro inhibition of apoptosis in peripheral blood neutrophils with various inflammatory mediators prolongs their function.19 A significant decrease in the proportion of apoptotic peripheral blood neutrophils was detected in human patients with burn injury, systemic inflammatory response syndrome, sepsis, moderate to severe blunt trauma injury, or inflammatory bowel disease as well as in patients who underwent elective surgery, compared with values for respective control groups.20–28 Although prolonged functionality of these cells may be beneficial to promote an inflammatory response, this may also contribute to excessive inflammation, increasing the risk of complications and death in critically ill patients.
Intensity of the neutrophil response to intestinal obstruction or strangulation appears to be related to the severity of disease as well as the development of postoperative ileus and adhesion formation. Although neutrophil apoptosis has an important role in regulation of the inflammatory response, it is not known whether alterations in apoptotic signaling events occur in association with acute abdominal disease in horses, and, to our knowledge, the effects of diminished or delayed neutrophil apoptosis in horses have not been investigated. The purpose of the study reported here was to quantify peripheral blood neutrophil apoptosis in equine patients with intestinal injury. We hypothesized that the percentage of circulating neutrophils undergoing apoptosis in horses immediately before and after emergency surgery for obstructive or strangulating intestinal disease would be decreased, compared with the percentage in horses undergoing elective orthopedic surgery.
Materials and Methods
Animals—Twenty adult horses (> 2 years of age) undergoing surgery at the Marion duPont Scott Equine Medical Center were enrolled in the study between July 22, 2010, and December 9, 2011. Ten horses had surgery for treatment of colic, and 10 underwent elective arthroscopy. Data collected for each horse included age, breed, and sex; lesion treated at surgery and surgical procedure performed; anesthesia time; pre- and postoperative WBC and neutrophil counts; and outcome (survival to discharge or death). The study was approved by the Virginia Tech Institutional Animal Care and Use Committee.
Blood sample collection and isolation of neutrophils—Seventeen milliliters of peripheral blood was collected via jugular venipuncture into evacuated tubes containing acid citrate dextrose. Samples were collected at admission and 24 hours after surgery. Complete blood counts were performed, and the total neutrophil numbers were determined by multiplying the concentration of neutrophils per microliter of blood by the total volume of blood collected.
Blood was centrifuged twice (500 × g for 8 minutes at 22°C) to ensure that as much of the buffy coat layer as possible would be collected. The buffy coat was removed via pipette and mixed with an equal volume of 1× Hanks’ solutiona without calcium or magnesium.
A bilayer gradient of colloidal silica particles (diameter, 15 to 30 nm; 23% [wt/wt] in water) coated with polyvinylpyrrolidoneb (59% and 75% suspensions) was used to separate neutrophils from other WBCs. An isosmotic suspension of the colloidal silica particles was created by mixing 40 mL of the stock suspensionb with 3.63 mL of 10× Hanks’ solutionc without calcium or magnesium. The 59% suspension was then made by mixing 23.6 mL of the isosmotic suspensionb with 16.4 mL of 1× Hanks’ solution. The 75% suspension was made by mixing 30 mL of the isosmotic suspension with 10 mL of 1X Hanks’ solution. Suspension density was adjusted as needed by adding 1× Hanks’ solution or the isosmotic colloidal silica particle suspension until refraction (refractive index difference × 104) measured by use of a refractometerd was 115 × 104 to 120 × 104 for the 59% suspension and 140 × 104 to 145 × 104 for the 75% suspension. If the 75% suspension had a refractive index difference slightly > 145 × 104, no adjustments were made so as to prevent neutrophils from passing through the bottom layer of the gradient. The 59% and 75% suspensions were sometimes made up to 1 month in advance and stored in a refrigerator until used. Suspensions were warmed to room temperature (approx 22°C), and density was rechecked before bilayer gradients were made.
Each bilayer colloidal silica particle gradient was constructed in a 15-mL sterile conical tube. Five milliliters of the 59% suspension was loaded, taking care not to spill the suspension on the sides of the tube. Five milliliters of the 75% suspension was then slowly infused beneath the 59% suspension with an IV catheter,e taking care not to disturb the interface between layers. The mixture of buffy coat and 1× Hanks’ solution was then slowly applied to the prepared bilayer gradient, and the tube was centrifuged at 930 × g for 40 minutes at 22°C. Prior to centrifugation, distribution of tubes was carefully balanced. Minimal rates of acceleration and braking were used to avoid vibrations and mixing of the gradient contents. Neutrophils were collected from the interface between the 59% and 75% suspensions and washed 3 times in 30 mL of Dulbecco PBS solution.f Following each wash, cells were centrifuged at 600 × g for 8 minutes at 22°C, the supernatant was removed via gentle suction, and the pellet was resuspended in fresh wash solution. After the final wash, neutrophils were suspended in culture medium.
Cell culture—The isolated neutrophils were suspended in 5 mL of RPMI complete mediumg containing 0.5 mL of fetal bovine serum,h 0.018 μL of 2-mercaptoethanol (0.5 μmol),i 0.1 mL of penicillin (100.0 U) streptomycin (0.10 mg) solution,j 0.1 mL of l-glutamine (2.0 mmol),k 0.05 mL of sodium pyruvate solution (1.0 mmol),l and 0.1 mL of gentamicin (0.10 mg).m The neutrophil concentration per milliliter was determined via hemacytometer and was multiplied by 5 to determine the total numbers of neutrophils retrieved. This number was then divided by the total number of neutrophils in the initial blood sample (as indicated in the CBC results) to determine percentage recovery. Slides of the enriched neutrophil population were prepared, stained with 0.4% trypan blue solution,n and evaluated via light microscopy to assess purity of the population. Neutrophils were identified as having toxic changes if they contained Döhle bodies, basophilic cytoplasm, and intracytoplasmic vacuoles. Cells were suspended at 1 × 106 cells/mL of medium for culture and were incubated under conditions of 5% CO2 at 37°C for approximately 24 hours.
Flow cytometry—Following incubation, neutrophils and media were collected from the culture plate and centrifuged (600 × g for 8 minutes at 22°C). The supernatant was removed by gentle suction, and the neutrophils were washed twice in cold PBS solution (30 mL at 600 × g for 8 minutes at 22°C). The neutrophils were suspended in 1× binding buffer (provided with the apoptosis detection kito) at a concentration of 1 × 106 neutrophils/mL in 4 aliquots. Each aliquot of cells was prepared separately for flow cytometry as follows: unstained cells, cells incubated with only phycoerythrin-conjugated annexin Vo (a phosphatidyl serine marker), cells incubated with only 7-AADo (a vital DNA marker), and cells incubated with both of these labeling solutions. Five microliters of phycoerythrin-conjugated annexin V, 7-AAD, or both was added to 100 μL (1 × 105 cells) of the cell and buffer solution. The tubes were gently rotated and incubated for 15 minutes at room temperature in the dark. After 400 μL of binding buffer was added to each tube, all tubes were analyzed via flow cytometryp within 1 hour. Flow cytometric evaluation of all samples was completed between 21 and 27 hours after collection, with the exception of 1 sample tested at 48 hours.
Forward and side scatter dot plots were examined to set voltage parameters to move neutrophils into a gated region. Unstained cells, cells stained with only phycoerythrin-conjugated annexin V, and cells stained only with 7-AAD were used to set voltages. A second dot plot with fluorescence intensity channels for phycoerythrin (FL2) on the x-axis and 7-AAD (FL3) on the y-axis was used to assess results for the stained neutrophils. Ten thousand gated events were counted for each sample. Fluorescent compensation was adjusted by examination of cells stained only with phycoerythrin-conjugated annexin V and cells stained only with 7-AAD. After instrument settings were adjusted, the aliquot containing cells incubated with both stains was used to obtain the number of apoptotic (annexin V-positive) neutrophils for each horse. All steps for collection, isolation, culture, and flow cytometry were performed in the same manner and were completed by 1 investigator (KMK).
Statistical analysis—Data were analyzed with commercially available statistical software.q The Mann-Whitney U test was used to compare the percentage of apoptotic neutrophils between treatment groups (ie, horses that underwent arthroscopy and those that had surgery for colic); within the colic group, the same test was used to compare the percentage of apoptotic neutrophils after surgery in horses with strangulating versus nonstrangulating intestinal obstructions. Exact Mann-Whitney test methods were used for comparisons between groups of ≤ 10 animals. Other comparisons between treatment groups, including anesthesia time, percentage of apoptotic neutrophils before and after surgery, and percentage recovery of neutrophils, were also analyzed via the Mann-Whitney U test. Comparison of pre- and posttreatment neutrophil numbers, percentage apoptosis, and percentage recovery was performed with the Wilcoxon signed rank test for paired data. Correlations between anesthesia time or WBC count and percentages of apoptotic neutrophils were evaluated via the Spearman rank correlation coefficient (ρ). Values of P < 0.05 were accepted as significant.
Results
Horses in the arthroscopy group ranged from 2 to 17 years of age, and the horses in the colic group ranged from 5 to 26 years of age. Breeds in the colic group included Warmblood (n = 4), Thoroughbred (3), Percheron (2), and pony cross (1). Breeds in the arthroscopy group included Warmblood (n = 5), Warmblood cross (1), Thoroughbred (2), and Thoroughbred cross (2). The colic group included horses with strangulating (n = 5) and nonstrangulating (5) lesions. One horse with strangulating adhesions had previous abdominal surgery approximately 18 months prior to surgery in the present study; none of the other horses that underwent surgical treatment for colic had undergone prior surgery. All horses in the arthroscopy and colic groups survived to discharge from the hospital.
A significantly (P = 0.037) smaller percentage of neutrophils was recovered in preoperative blood samples from the colic group (median, 32.5%), compared with the arthroscopy group (median, 66.5%). However, the percentage of neutrophils recovered in postoperative samples did not differ significantly (P = 0.173) between these groups, and overall comparison revealed no significant (P = 0.184) differences between preoperative and postoperative samples for both groups combined (Table 1). Three of 10 preoperative samples and 4 of 10 postoperative samples from the colic group contained neutrophils with toxic changes.
Percentage recovery of neutrophils from peripheral blood samples collected from adult horses immediately prior to and 24 hours after surgery for treatment of colic (n = 10) or elective arthroscopic surgery (10).
| Sample and group | 25th percentile | Median value | 75th percentile |
|---|---|---|---|
| Preoperative and postoperative samples | |||
| Arthroscopy | 46 | 58.0* | 70 |
| Colic | 25.5 | 35.5* | 45 |
| Preoperative samples | |||
| Arthroscopy | 46 | 66.5* | 86 |
| Colic | 26 | 32.5* | 40 |
| Postoperative samples | |||
| Arthroscopy | 48 | 57.5 | 61 |
| Colic | 25 | 39.0 | 76 |
| All preoperative samples | 26.5 | 40.5 | 66.8 |
| All postoperative samples | 38 | 49.5 | 64.5 |
Neutrophils were isolated by use of a bilayer gradient of colloidal silica particles coated with polyvinylpyrrolidone and cultured overnight in a modified complete RPMI media before counting. Percentage recovery was calculated by comparison with results of a CBC.
Within a sample type, values are significantly (P < 0.05) lower in the colic group than in the arthroscopy group.
There were no significant differences between pre- and postoperative neutrophil counts for the colic group (P = 0.106) or the arthroscopy group (P = 0.125).
Apoptotic neutrophils were identified and compared among groups and sample types (Figure 1). Percentages of apoptotic neutrophils were not significantly different in any of the following comparisons: all preoperative versus all postoperative samples (P = 0.256), preoperative samples from the colic group versus those from the arthroscopy group (P = 0.436), preoperative versus postoperative samples from the arthroscopy group (P = 0.131), or preoperative versus postoperative samples from the colic group (P = 0.432). However, within the colic group, the median percentage of apoptotic neutrophils was significantly (P = 0.047) lower in postoperative samples from horses with strangulating intestinal lesions (18.0% [25th and 75th percentiles, 14.9% and 31.1%, respectively]) than in those of horses with nonstrangulating intestinal lesions (66.3% [25th and 75th percentiles, 65.1% and 68.7%, respectively]).
Scatterplots showing results of 2-color flow cytometry analysis for apoptotic neutrophils in representative samples from equine patients. A lower percentage of apoptotic neutrophils is present in panel A, and a higher percentage of these cells is present in panel B. Neutrophils were isolated by use of a bilayer colloidal silica particle gradient, evaluated for purity via light microscopy, and cultured for approximately 24 hours in modified RPMI medium. Cells were subsequently washed and stained with phycoerythrin-conjugated annexin V and 7-AAD. Neutrophils with positive results for annexin V (ie, apoptotic neutrophils) are shown in the lower right quadrant. Numbers in the plot represent the percentage of cells in each quadrant; numbers on the x- and y-axes indicate fluorescence (arbitrary units). Comp-FL2-H = Fluorescence intensity channel 2. FL3-H = Fluorescence intensity channel 3.
Citation: American Journal of Veterinary Research 74, 7; 10.2460/ajvr.74.7.999
No significant (P = 0.80) difference in median anesthesia time was detected between horses of the arthroscopy (112.5 minutes [25th and 75th percentiles, 90 and 135 minutes, respectively]) and colic (135 minutes [25th and 75th percentiles, 90 and 180 minutes, respectively]) groups. There was also no significant correlation between anesthesia times and percentages of apoptotic neutrophils for the arthroscopy group (P = 0.234; ρ = 0.41), the colic group (P = 0.590; ρ = −0.19), or the colic and arthroscopy groups combined (P = 0.801; ρ = 0.06). In addition, there was no significant (P = 0.221) correlation (ρ = 0.28) between postoperative WBC counts and percentages of apoptotic neutrophils.
Discussion
Few studies have been performed to evaluate changes in peripheral blood neutrophils in horses with clinical disease. Neutrophil activation has been associated with strangulating intestinal obstruction and inflammatory bowel disease in humans and strangulating intestinal disease in horses, with no evidence of activation in horses with nonstrangulating obstructions.9,10 Other studies evaluating neutrophil changes in horses have been limited to the responses to respiratory disease29 and endometritis.30 Neutrophil activation may decrease the normal rate of neutrophil apoptosis as has been reported for horses with strangulating intestinal disease, thereby increasing neutrophil availability in inflammatory processes.9
In humans, decreases in the proportion of apoptotic neutrophils have been associated with burns, cardiac bypass surgery, other surgical trauma, endotoxemia, inflammatory bowel disease, and systemic inflammatory response syndrome.19–25,31 Results of these studies19–25,31 suggest that increased neutrophil survival time is potentially responsible for worsening clinical signs, and inducing neutrophil apoptosis may result in improved outcome.23 Horses with ischemic intestine are at risk for endotoxemia, increased production of TNF-α, and clinical signs of shock, all of which have been associated with decreased neutrophil apoptosis.23,28,31,32 Decreased neutrophil apoptosis in horses with strangulating intestinal lesions may be linked to systemic inflammatory reactions that occur in horses with shock resulting from the response to the injured tissue.9,10,33,34
Suppression of apoptosis in human neutrophils occurs in response to various cytokines, including IL-1β,35 IL-2,36 IL-6 (both alone and via a mechanism involving platelet activating factor),35,37,38 IL-8 (both spontaneously and mediated by TNF-α),18,28,39,40 and transforming growth factor-β.41 Systemic inflammatory mediators such as TNF-α and IL-1β, associated with delayed apoptosis in human neutrophils,25,28 have a similar systemic effect in horses.32,42
In the present study, the lower percentage of apoptotic neutrophils in postoperative blood samples from horses with strangulating intestinal lesions, compared to that in samples from horses with nonstrangulating lesions (18.0% and 66.3%, respectively), suggests that delayed neutrophil apoptosis may be associated with the severity of intestinal inflammation and the systemic effects observed in horses with ischemia and reperfusion of intestine in clinical or experimental settings.8,9,15–17 Activated neutrophils marginate and migrate into the intestinal interstitium during periods of low-flow ischemia and reperfusion, and a decrease in the proportion of circulating apoptotic neutrophils may result from activation or from apoptosis occurring in neutrophils that are no longer circulating.12,15 This finding has potential implications for development of novel treatments that may reduce postoperative complications through manipulation of neutrophil apoptosis to mitigate systemic and local activity. The question remains whether intervening in key signaling pathways, such as those involving nuclear factor κ light polypeptide gene enhancer in B cells, mitogen-activated protein kinase, phosphoinositide-3-kinase, B-cell leukemia-lymphoma 2, some lipid mediators, and cyclin-dependent kinase,43 to increase neutrophil apoptosis could help prevent postoperative complications in horses. Hypothetically, drugs such as inhibitors of nuclear factor κ light polypeptide gene enhancer in B cells and prostaglandin D2 metabolites43 could be used to regulate apoptosis in horses with signs of strangulating intestinal disease, thereby decreasing intestinal inflammation and improving outcome.
Significantly smaller percentages of neutrophils were recovered overall and in preoperative blood samples from the colic group (median, 35.5% and 32.5%, respectively), compared with values for the arthroscopy group (median, 58% and 66.5%, respectively), but the lower recovery rates did not appear to be associated with the presence of neutrophils with toxic changes, as might have been expected if increased fragility or changes in cell density due to activation were present.44 It is possible that the neutrophils collected from the horses that underwent abdominal surgery were more likely to be activated, owing to some degree of increased systemic inflammation, even without detectable toxic changes. Activation of human peripheral blood neutrophils in response to a synthetic chemotactic peptide has been shown to decrease cellular density.45 Decreased density of equine neutrophils resulting from cellular activation could result in changes in the cell location within the colloidal silica particle gradient and decreased cell recovery.46 Previous evaluation of human neutrophils has shown that activation does not result from incubation with colloidal silica particles,47 suggesting that similar treatment of equine neutrophils would not have caused activation of the cells in our study.
Selecting a strangulating lesion type for all horses in the colic group would likely have resulted in significantly decreased neutrophil apoptosis, compared with horses in the arthroscopy group. The low yield of neutrophils in some samples, particularly from horses in the colic group, was a considerable limitation, given that evaluation of the missing cells could have affected the number of apoptotic neutrophils. Further evaluation of the recovery of neutrophils is needed to assess the effect on apoptotic cell counts in horses with colic.
Similar to studies of neutrophils from humans and laboratory animals, apoptosis in equine neutrophils can be detected by culture and staining in vitro with phycoerythrin-conjugated annexin V followed by flow cytometry, which was shown to have good correlation with changes detected via light microscopy.r In that study,r induced apoptosis in peripheral blood neutrophils from clinically normal horses was effectively detected with the same phycoerythrin-conjugated annexin V assayn as used in the present study. Further testing should be completed to assess the repeatability and accuracy of phycoerythrin-conjugated annexin V assays for use in detecting apoptosis of peripheral blood neutrophils from horses with naturally occurring diseases.
In the study reported here, we found that proportions of apoptotic circulating neutrophils from horses with strangulating intestinal lesions were significantly lower than those of horses with nonstrangulating lesions. Further investigations are needed to better understand neutrophil activation during intestinal disease and the relationship between neutrophil apoptosis and inflammation during intestinal injury.
ABBREVIATIONS
| 7-AAD | 7-aminoactinomycin D |
| IL | Interleukin |
| TNF | Tumor necrosis factor |
Hanks’ Balanced Salt Solution, Sigma-Aldrich Co, St Louis, Mo.
Percoll, Sigma-Aldrich Co, St Louis, Mo.
Hanks’ Balanced Salt Solution 10×, Sigma-Aldrich Co, St Louis, Mo.
TS Meter Hand-Held Refractometer, Reichert Technologies, Depew, NY.
Milacath, 16 gauge × 13 cm (5.25 in), Mila International Inc, Erlanger, Ky.
HyClone 1X Dulbecco PBS solution, Thermo Fisher Scientific Inc, Waltham, Mass.
Gibco RPMI 1640, Life Technologies Corp, Carlsbad, Calif.
Gibco Fetal Bovine Serum, qualified, heat inactivated, USDA-approved regions, Life Technologies Corp, Carlsbad, Calif.
2-mercaptoethanol, Sigma-Aldrich Co, St Louis, Mo.
Penicillin-streptomycin, Life Technologies Corp, Carlsbad, Calif.
L-glutamine, Life Technologies Corp, Carlsbad, Calif.
Sodium pyruvate, Sigma-Aldrich Co, St Louis, Mo.
Gentamicin, Life Technologies, Carlsbad, Calif.
Typan blue, Sigma-Aldrich Co, St Louis, Mo.
PE Annexin V Apoptosis Detection Kit I, BD Pharmingen, Franklin Lakes, NJ.
BD FACSCalibur, BD, Franklin Lakes, NJ.
SAS, version 9.3, SAS Institute, Cary, NC.
Wereszka MM. Methods to detect apoptosis in equine peripheral blood neutrophils from normal healthy horses. MS thesis, Virginia Polytechnic Institute and State University, Blacksburg, Va, 2007.
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