Circulating neutrophil activation in dogs with naturally occurring spinal cord injury secondary to intervertebral disk herniation

Rae L. Van Sandt Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX
Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX

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C. Jane Welsh Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX

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Nick D. Jeffery Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX

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Colin R. Young Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX

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Dylan A. McCreedy Department of Biology, College of Science, Texas A&M University, College Station, TX

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Gus A. Wright Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX
Flow Cytometry Facility, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX

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C. Elizabeth Boudreau Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX

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Gwendolyn J. Levine Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX

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Jonathan M. Levine Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX

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Abstract

OBJECTIVE

To investigate the time course of circulating neutrophil priming and activity in dogs with spinal cord injury secondary to intervertebral disk herniation that undergo decompressive surgery.

ANIMALS

9 dogs with spinal cord injury and 9 healthy dogs (controls).

PROCEDURES

For dogs with spinal cord injury, blood samples were collected on the day of hospital admission and 3, 7, 30, and 90 days after injury and decompressive surgery. A single blood sample was collected from the control dogs. Flow cytometry analysis was performed on isolated neutrophils incubated with antibody against CD11b and nonfluorescent dihydrorhodamine 123, which was converted to fluorescent rhodamine 123 to measure oxidative burst activity.

RESULTS

Expression of CD11b was increased in dogs with spinal cord injury 3 days after injury and decompressive surgery, relative to day 7 expression. Neutrophils expressed high oxidative burst activity both 3 and 7 days after injury and decompressive surgery, compared with activity in healthy dogs.

CLINICAL RELEVANCE

For dogs with spinal cord injury, high CD11b expression 3 days after injury and decompressive surgery was consistent with findings for rodents with experimentally induced spinal cord injury. However, the high oxidative burst activity 3 and 7 days after injury and decompressive surgery was not consistent with data from other species, and additional studies on inflammatory events in dogs with naturally occurring spinal cord injury are needed.

Abstract

OBJECTIVE

To investigate the time course of circulating neutrophil priming and activity in dogs with spinal cord injury secondary to intervertebral disk herniation that undergo decompressive surgery.

ANIMALS

9 dogs with spinal cord injury and 9 healthy dogs (controls).

PROCEDURES

For dogs with spinal cord injury, blood samples were collected on the day of hospital admission and 3, 7, 30, and 90 days after injury and decompressive surgery. A single blood sample was collected from the control dogs. Flow cytometry analysis was performed on isolated neutrophils incubated with antibody against CD11b and nonfluorescent dihydrorhodamine 123, which was converted to fluorescent rhodamine 123 to measure oxidative burst activity.

RESULTS

Expression of CD11b was increased in dogs with spinal cord injury 3 days after injury and decompressive surgery, relative to day 7 expression. Neutrophils expressed high oxidative burst activity both 3 and 7 days after injury and decompressive surgery, compared with activity in healthy dogs.

CLINICAL RELEVANCE

For dogs with spinal cord injury, high CD11b expression 3 days after injury and decompressive surgery was consistent with findings for rodents with experimentally induced spinal cord injury. However, the high oxidative burst activity 3 and 7 days after injury and decompressive surgery was not consistent with data from other species, and additional studies on inflammatory events in dogs with naturally occurring spinal cord injury are needed.

Introduction

Intervertebral disk herniation (IVDH) in dogs is a naturally occurring form of spinal cord injury (SCI) that bears critical similarities to human SCI with respect to pathomechanisms and treatment.14 SCIs resulting from IVDH in dogs consist of various components of compression and contusion and are typically treated with a combination of decompressive surgery and physical rehabilitation.57 Histologic characteristics of SCIs in dogs parallel those detected in humans with naturally occurring SCIs and in animals with experimentally induced SCIs, including axon destruction, demyelination, and centrally oriented necrosis and cavitation.8 Spinal cord lesions in affected dogs contain activated microglia9; have aberrantly increased expression of interleukin 6, interleukin 8,3,10 and matrix metalloproteinase 911; and contain a population of peripherally derived leukocytes.9,12 These inflammatory events result in loss of integrity of the blood–spinal cord barrier and increased oxidative stress.13 Several independent groups have used dogs with IVDH to evaluate potential neuroprotective and neuroregenerative strategies for use in human clinical trials.5,1416

The role of neutrophils in early acute secondary SCIs has been characterized in animals with experimentally induced injuries.1719 Neutrophils are believed to migrate from the periphery into injury sites in response to cytokines released by endothelium and resident microglia.2023 Arriving neutrophils can nonspecifically exacerbate injury through the release of multiple substances that degrade extracellular matrix and cause cytotoxicosis.2428 In the subacute phase of injury, neutrophils have been shown to improve axon growth and recovery.2931 Histologic studies in dogs with IVDH have shown neutrophils within the spinal cord soon after injury12,32,33 and have likewise identified cytokines that can induce circulating leukocyte priming, transmigration, and activation.810,13,34,35 Neutrophils are set apart from other circulating leukocytes because they can be primed or partially activated while still in the circulation, whereas other peripherally derived cells enter the damaged spinal cord prior to activation.3639

The objective of the study reported here was to evaluate the time course of circulating neutrophil priming and activity in dogs with an SCI secondary to IVDH by measuring CD11b expression and oxidative burst activity (OBA). We hypothesized that circulating neutrophils would become active following naturally occurring SCI and decompressive surgery and that there would be evidence of increased activity of circulating neutrophils, compared with activity in healthy control dogs.

Materials and Methods

Animals

All animal procedures were conducted with approval from the Texas A&M University Animal Care and Use Committee (Animal Use Protocols No. 2015-0129 and No. 2018-0146). Procedures in dogs with naturally occurring SCIs were performed with owner consent, and dogs were included in the study if they met the following criteria: 1) the dog was nonambulatory for < 48 hours at the time of hospital admission, 2) the dog had no history of back pain, 3) the dog underwent no administration of NSAIDs or glucocorticoids within the previous 30 days, 4) the dog underwent no vaccine administration within the previous 14 days, 5) the dog had an SCI involving the T3-L3 spinal cord segments, and 6) the dog was scheduled to receive decompressive surgery on the day of hospital admission. Dogs were excluded from further participation if they had any additional neurologic disease unrelated to the SCI, any illness or disease involving abnormal function of the immune system (eg, cancer or inflammatory bowel disease), or any inflammation unrelated to the SCI (eg, skin infection or urinary tract infection) or if they were uncooperative or pregnant or weighed < 4 kg.

Healthy dogs were recruited through 2 pathways. First, a cohort of healthy dogs matched with the SCI dogs on the basis of age (within 20%), breed, and sex was developed with owner consent. Second, a cohort of healthy reference control dogs representing chondrodystrophic breeds was accessed.40 Healthy matched and reference control dogs met the same inclusion and exclusion criteria as the SCI dogs, except they did not have features associated with SCI.

A power calculation was performed on the basis of neutrophil OBA measurements in humans41 to detect a 1.5-fold change in OBA between healthy dogs and SCI dogs 3 days after injury, which resulted in an estimate of 19 SCI dogs.42

Decompressive surgery

Dogs with SCI underwent complete physical and neurologic examinations, a CBC, and serum biochemical testing prior to anesthesia. Dogs were premedicated with glycopyrrolate and oxymorphone or hydromorphone. Anesthesia was induced with propofol, dogs were intubated, and anesthesia was maintained with sevoflurane. Magnetic resonance imaging or CT of the thoracolumbar vertebral column was performed to identify the site of IVDH, and a hemilaminectomy was performed to remove herniated intervertebral disk material and associated hemorrhage from the epidural space. The diagnosis of IVDH was confirmed on the basis of gross or histologic examination of the removed material.

Following anesthetic recovery, dogs were given fentanyl IV for pain management, and manual bladder expression was performed as necessary. Twenty-four hours after surgery, physical rehabilitation consisting of supported walking, passive range-of-motion exercises, and standing strength exercises was initiated. Dogs were released to their owner’s care after pain could be controlled with orally administered tramadol and urine was voluntarily voided or the bladder could be manually expressed. Owners were instructed to continue the physical rehabilitation exercises for 6 weeks after surgery.

Neurologic scoring

Neurologic function was scored with the modified Frankel scoring system at the time of hospital admission (day 0) and 3, 7, 30, and 90 days after surgery. The modified Frankel scoring system has been validated previously in dogs with IVDH-associated SCI and correlates well with MRI-based measures of SCI and post-SCI motor outcome.43 Scores were assigned on a scale from 0 to 5, where 0 = paraplegic with absent pelvic limb pain sensation, 1 = paraplegic with pain sensation in response to severe stimuli, 2 = paraplegic with pain sensation in response to mild stimuli, 3 = nonambulatory paraparesis, 4 = ambulatory paraparesis, and 5 = signs consistent with spinal pain only. Dogs were considered ambulatory if they could rise unassisted and take ≥ 10 steps without falling. In dogs that were not ambulatory, pelvic limb movements were evaluated by providing tail support. Conscious proprioception was evaluated by supporting the dog in a standing position and placing the dorsum of the paw in contact with the ground. Conscious perception of mild and severe stimuli was evaluated by pinching the interdigital webbing and clamping the nail bed with hemostats, respectively. Pain sensation was considered intact if there was a behavioral (eg, orienting to the stimulus or vocalization) or physiologic (eg, tachycardia or tachypnea) response to stimulation.

Neutrophil isolation

For dogs with SCI, blood samples were collected on the day of hospital admission (day 0) and 3, 7, 30, and 90 days after surgery. Blood samples were obtained on the day of hospital admission prior to anesthetic induction. For healthy dogs, a single blood sample was collected.

At each sampling time, 8 to 10 mL of blood was collected by means of external jugular venous puncture and split evenly between 2 EDTA-coated 6-mL plastic tubes. Samples were cooled to 4°C immediately after collection and centrifuged at 506 × g in a swinging bucket rotor for 10 minutes at 4°C with the brake on. The supernatant was removed, and the remaining blood was gently mixed in a 1:1 ratio with sterile PBS solution layered over a room-temperature Histopaque-1077 and -1119 (Sigma-Aldrich) gradient. This mixture was then centrifuged at 400 × g in a swinging bucket rotor for 30 minutes at room temperature with the brake off. The layer of cells between the Histopaque-1119 and -1077 layers represented neutrophils and was collected. Isolated neutrophils were washed 3 times with 3 mL of PBS solution. Red blood cells mixed with the neutrophils were lysed by gently freeing the cell pellet with 80 μL of PBS solution and incubating the cells with 1 mL of ice-cold sterile water for exactly 30 seconds; isotonicity was restored with double-strength PBS solution. Cells were washed 3 times with PBS solution, and total cell counts were obtained with 0.2% trypan blue exclusion dye and a Cellometer (Nexcelom) in accordance with the manufacturer’s protocol. Neutrophils were brought to a concentration of 1 × 106 cells/mL with RPMI 1640 medium (Thermo Fisher Scientific) and set aside on ice for further experiments.

CD11b expression

A count of 300,000 neutrophils was aliquoted and centrifuged at 172 × g for 4 minutes at 4 °C. Cells were resuspended in 100 µL of ice-cold flow cytometry staining buffer (Thermo Fisher Scientific) and Human BD Fc Block (2.5 µg/1 million cells; BD Biosciences) in accordance with the manufacturers’ protocols and incubated on ice for 10 minutes. Cells were then brought to a volume of 350 μL with flow cytometry staining buffer and incubated on ice in the dark for 30 minutes with FITC antimouse CD11b (Clone M1/70; Biolegend) and 7-aminoactinomycin D staining solution for cell viability (1:80 dilution) in accordance with the manufacturers’ protocols. Neutrophils were then centrifuged at 172 × g for 4 minutes at 4 °C and resuspended in 350 μL of fresh fluorescence-activated cell sorting buffer. Anti-rat and anti-mouse CompBeads (BD Biosciences) incubated with 1 μL of antibody were run on the flow cytometer (FACSCalibur; BD Biosciences) prior to all neutrophil samples and used for compensation. Neutrophil suspensions with and without antibodies (unstained controls) were then run, and fluorescent intensities were measured. At least 10,000 events from the middle of the run were recorded. Flow cytometry standard files were exported and analyzed with standard software (FlowJo version 10.4.2; Becton, Dickinson and Co). The ellipse tool was used to gate neutrophils on the basis of forward- and side-scatter values. Next, dead cells were excluded with a rectangular gate on cells that were negative for 7-aminoactinomycin D. Viable neutrophils were gated with the rectangle tool to isolate only those cells that were CD11b+, and their median fluorescent intensity (MFI) was obtained (Figure 1). A small percentage of counted cells (approx 5%) were lymphocytes rather than neutrophils.

Figure 1
Figure 1

Illustration of the gating strategy used to measure CD11b expression in circulating neutrophils from dogs with a spinal cord injury (SCI) secondary to intervertebral disk herniation that underwent decompressive surgery. Flow cytometry files were exported, and the ellipse tool was used to gate neutrophils on the basis of forward- and side-scatter values (A). Next, dead cells were excluded with a rectangular gate on cells that were negative for 7-aminoactinomycin D (7AAD; B). Finally, viable neutrophils were gated with the rectangle tool to isolate only those cells that were CD11b+, and their median fluorescent intensity (MFI) was obtained (C).

Citation: American Journal of Veterinary Research 83, 4; 10.2460/ajvr.21.05.0073

Oxidative burst activity

A count of 400,000 neutrophils in RPMI medium was incubated in a 24-well culture plate at 37 °C and 5.0% CO2 for 90 minutes, after which 50μM dihydrorhodamine 123 (Millipore Sigma) was added. After 20 minutes of incubation with dihydrorhodamine 123, cells were transferred to round-bottom tubes and centrifuged at 172 × g for 4 minutes at 4 °C, and the supernatant was removed. Cells were resuspended in ice-cold PBS solution with 6% fetal bovine serum (fluorescence-activated cell sorting buffer) and underwent flow cytometry to measure conversion of nonfluorescent dihydrorhodamine 123 to rhodamine 123 in the fluorescein isothiocyanate channel. Flow cytometry standard files were exported and analyzed with standard software (FlowJo version 10.4.2; Becton, Dickinson and Co). The ellipse tool was used to gate neutrophils on the basis of forward- and side-scatter values, and MFIs were obtained. Standard software (Prism 7; GraphPad Software) was used to create scatterplots and box plots.

Statistical analysis

The primary objective of the study was to determine differences in CD11b expression and OBA of circulating neutrophils between healthy dogs and dogs with naturally occurring SCI secondary to IVDH. The 4 assumptions for the nonparametric Mann-Whitney U test were met: 1) neutrophil activity was measured as a continuous variable, 2) the SCI and healthy dogs consisted of 2 independent groups, 3) all observations were independent, and 4) the MFIs were not normally distributed. For CD11b expression MFIs and OBA MFIs, values obtained at each time point for the SCI dogs were compared with values obtained for the healthy dogs.

A secondary objective of the study was to explore the time course of circulating neutrophil activation after decompressive surgery. This analysis was conducted with the Friedman test to identify differences among the repeated measures after decompressive surgery (days 3, 7, 30, and 90) for the SCI dogs. This analysis could only be conducted for time points at which values for all dogs were obtained. Therefore, the Wilcoxon signed rank test was used to compare values between time points for SCI dogs from which at least 2 blood samples were obtained.

All statistical analyses were performed with standard software (SPSS Statistics version 27; IBM Corp). Values of P ≤ 0.05 were considered significant.

Results

Because of the stringent inclusion criteria and recruitment issues, only 11 dogs with an SCI secondary to IVDH were recruited for the study. One dog was excluded because of urinary tract infection detected on day 7, and another was excluded because of reinjury prior to day 30. The remaining 9 dogs were included in the study. In addition, 6 matched healthy control dogs and 3 reference control dogs were included in the study.

Median age of the SCI dogs was 4 years (range, 2 to 9 years). Six of the 9 dogs were Dachshunds, 1 was a Dachshund-Chihuahua mix, 1 was a Havanese, and 1 was a Chihuahua. There were 5 neutered males, 3 spayed females, and 1 sexually intact female. Median time between the onset of neurologic signs and collection of the day 0 blood sample was 18 hours (range, 10 to 48 hours). Median modified Frankel score at the time of blood sample collection on day 0 was 2 (range, 0 to 3), indicating that all dogs were nonambulatory. Blood samples were collected from all 9 dogs on day 0, 8 dogs on day 3, 7 dogs on day 7, 6 dogs on day 30, and 7 dogs on day 90. Blood samples were collected at all 5 time points from only 5 dogs.

The healthy dogs ranged from 2 to 9 years old. There were 3 Dachshunds, 1 Dachshund-Chihuahua mix, 2 Beagles, 1 Cocker Spaniel, 1 Chihuahua, and 1 Shih Tzu. There were 4 neutered males, 4 spayed females, and 1 sexually intact male. All dogs were considered neurologically normal on the day blood samples were collected.

CD11b expression for all 5 time points for the SCI dogs did not differ significantly from expression for the healthy dogs (Figure 2). However, for the SCI dogs, CD11b expression on day 3 was significantly (P = 0.018) higher than expression on day 7.

Figure 2
Figure 2

Scatterplots of CD11b expression, measured as MFI, in circulating neutrophils from 9 dogs with an SCI secondary to intervertebral disk herniation that underwent decompressive surgery and in 9 healthy dogs. For dogs with SCI, blood samples were collected on the day of hospital admission (day 0) and 3, 7, 30, and 90 days after surgery. For healthy dogs, a single blood sample was collected (single). In each plot, the long horizontal line represents the median value, and the error bars represent 1 SD. Different symbols represent individual dogs. *Values were significantly (P = 0.018) different.

Citation: American Journal of Veterinary Research 83, 4; 10.2460/ajvr.21.05.0073

For the SCI dogs, OBA did not differ significantly among time points (Figure 3). However, OBA on days 3 and 7 for the SCI dogs was significantly (P = 0.021 and 0.014, respectively) higher than OBA for the healthy dogs.

Figure 3
Figure 3

Scatterplots of oxidative burst activity, measured as MFI of dihydrorhodamine 123 conversion to rhodamine 123 (R 123), for the dogs in Figure 2. *Values were significantly (P = 0.021 for day 3 vs healthy dogs and P = 0.014 for day 7 vs healthy dogs) different. See Figure 2 for remainder of key.

Citation: American Journal of Veterinary Research 83, 4; 10.2460/ajvr.21.05.0073

Discussion

Findings of the present study suggested that dogs with an SCI secondary to IVDH that undergo decompressive surgery exhibit features consistent with circulating neutrophil activation. In particular, for dogs with an SCI, CD11b expression was higher on day 3 than on day 7, and OBA was higher on days 3 and 7 in SCI dogs than in healthy dogs. The apparent lack of circulating neutrophil activation on day 0 in the SCI dogs may have reflected the relatively small number of dogs, a delay in response to cytokine signals following SCI, or the possibility that decompressive surgery was the major driver of neutrophil activation in our population of injured dogs.

Cell surface expression of CD11b, as a subunit of the Mac-1 complex, functions to allow firm adhesion during transendothelial migration and is required for neutrophils to leave the circulation.44,45 During neutrophil activation, Mac-1 is added to the cell surface as different granule populations fuse with the membrane. As such, neutrophil CD11b expression levels are partially indicative of neutrophil activation.46 For the SCI dogs in our study, CD11b expression was higher on day 3 than on day 7, which was consistent with results of studies37,47,48 involving rodents and humans that have shown circulating leukocytes have increased expression of both CD11b and the endothelial ligand I-CAM1 between 1 and 4 days after SCI. Injury severity did not appear to be associated with expression of CD11b in our study, which might have been a result of variability in age, sex, breed, and injury location in our population as well as the small sample size.

Activated neutrophils that have left the circulation amplify inflammation by releasing proinflammatory cytokines and reactive oxygen species, which drive acute secondary SCI,26,41 thus fulfilling their role as first responders. However, release of volatile reactive oxygen species and high OBA from circulating neutrophils can propagate inflammation far from the injury site and outside the CNS.49,50 In the present study, OBA of circulating neutrophils was higher 3 and 7 days after injury in dogs with an SCI than in healthy dogs. This increased circulating neutrophil OBA was similar to the increased circulating neutrophil OBA previously recognized up to 1 week after injury in humans with an SCI, relative to control individuals. This robust circulating neutrophil OBA in humans has been speculated to result from dysregulated CNS immune feedback.41 In dogs with naturally occurring SCIs, neutrophils have been identified in the CSF, and CSF cytokine profiles would suggest that neutrophil responses should be present after injury.11,34,35 Histologic data from dogs with naturally occurring injuries, however, have suggested macrophage activation with minimal neutrophil activity and infiltration early after severe SCI, which is contrary to data from animals and humans with SCI.3,9,10,21,5154

Our results showing that OBA was increased 3 and 7 days after SCI highlighted a potential prominent role for neutrophils following naturally occurring SCI in dogs and the need for further research. Further studies correlating circulating biomarkers, circulating neutrophil responses, and histologic data from dogs specifically between 3 and 7 days after injury could assist in clarifying inflammatory events, with the hope of informing therapeutic clinical trials. Interventions that block facets of the innate immune response following SCI, including neutrophil infiltration, have enhanced recovery in certain animal with experimentally induced injury.25,27 However, approaches that have selectively and rapidly blocked neutrophil infiltration into the injured spinal cord have resulted in more significant histologic injury, perhaps due to the key role that neutrophils play in healing.29 These previous studies suggest that the timing and specificity of strategies that block neutrophil infiltration following SCI will be critical to consider in future clinical trial design.

The primary objective of the present study was to evaluate neutrophil activity after SCI in dogs, and to our knowledge, this was the first time a longitudinal analysis of neutrophil activity has been conducted in dogs with naturally occurring SCI. For our study, inclusion of a day 0 time point was important. Enrolled dogs were clinical patients with spontaneous injury, and day 0 blood samples were obtained prior to skin and muscle incisions and laminectomy. Therefore, neutrophil activity on day 0 was in response to SCI and not any extraneous damage to the skin, muscle, or bone, as would be the case with experimentally induced SCI. However, there was no significant difference in either CD11b expression or OBA in SCI dogs on day 0, compared with healthy dogs. This could possibly have been because of the small sample size and the inclusion of both males and females, in that sexual dimorphisms following SCI have been identified in rodents, although they remain controversial.5558 Another factor that may have contributed to the variability in circulating neutrophil activity on day 0 could have been the variability in injury severity among SCI dogs. Another possibility for the lack of significant difference in day 0 neutrophil activity between SCI and healthy dogs was the inclusion of dogs whose initial clinical signs had first been noticed up to 48 hours earlier. Often, dogs with IVDH do not show obvious signs of acute SCI, such as a sudden inability to walk, but rather first show mild signs that progress over time. Dogs enrolled in the present study had a clear event in that the owners noticed their dogs were suddenly unable to walk at a specific time or during a small window of time (eg, the dog appeared fine when they left for work and could not walk when they arrived home). However, allowing up to 48 hours after injury for enrollment may have allowed activated circulating neutrophils to leave the circulation before the initial blood sample was obtained. This 48-hour enrollment period could have also accounted for the high variability on day 0 in both CD11b expression and OBA.

In conclusion, the present study was the first, to our knowledge, to characterize neutrophil activity in a longitudinal manner following naturally occurring SCI in dogs. Although the increased CD11b expression 3 days after injury was consistent with results of studies of SCI in other species, the prolonged OBA of circulating neutrophils was not. These initial experiments will be useful in powering future studies that target the inflammatory response in the first week following SCI in dogs. Our findings seem worthy of independent validation in a distinct, larger cohort of dogs.

Acknowledgments

Supported in part by the Texas A&M University Institute for Neuroscience.

The authors declare that there were no conflicts of interest.

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