• Open Access

Association of Cerebrospinal Fluid Analysis Findings with Clinical Signs and Outcome in Acute Nonambulatory Thoracolumbar Disc Disease in Dogs

Authors


  • This work was performed at Hebrew University Veterinary Teaching Hospital, Koret School of Veterinary Medicine, Hebrew University of Jerusalem.

Corresponding author: Itay Srugo, Koret School of Veterinary Medicine, Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel; e-mail: srugo@agri.huji.ac.il.

Abstract

Background: Cerebrospinal fluid (CSF) pleocytosis recently was associated with the severity of neurologic signs in dogs with intervertebral disc disease (IVDD).

Hypothesis/Objectives: To look for an association among CSF cell counts, total protein concentration, and severity of neurologic signs at presentation with outcome in dogs with acute thoracolumbar IVDD. Our hypothesis was that CSF total nucleated cell count (TNCC) and percentage cell types would be associated with the severity of spinal cord damage and therefore with both the presenting clinical signs and the prognosis of affected dogs.

Animals: Fifty-four dogs with acute nonambulatory thoracolumbar IVDD were evaluated.

Methods: Retrospective study. Signalment, neurologic grade, CSF TNCC, protein concentration, red blood cells count and differential cell percentages, and short- and long-term outcomes were evaluated.

Results: CSF pleocytosis (>5 cells/μL) was present in 54% of dogs and was positively associated with neurologic grade at presentation and with postoperative time to regaining ambulation. Neutrophils were observed most frequently. The percentage of CSF macrophages and macrophage to monocyte ratio were higher (P= .001, for both) in dogs presented without deep pain sensation (DPS) that did not regain ambulation. Receiver operator characteristics curve analysis yielded a cut-off point of 13% macrophages with a sensitivity and specificity of 100 and 83%, respectively, for prediction of a negative outcome.

Conclusions and Clinical Importance: CSF pleocytosis is positively associated with the severity of spinal cord damage in dogs with thoracolumbar IVDD. The percentage of CSF macrophages can be used as a prognostic indicator for regaining ambulation in dogs that have lost DPS.

Abbreviations:
AUC

area under the curve

CSF

cerebrospinal fluid

DPS

deep pain sensation

IVDD

intervertebral disc disease

MΦ:M

macrophage to monocyte ratio

OR

odds ratios

ROC

receiver operator characteristics

SE

standard error

TCC

total cell count

TNCC

total nucleated cell count

Thoracolumbar intervertebral disc disease (IVDD) is a common cause of hind limb paresis and paralysis in dogs.1 Treatment protocols vary from conservative treatment to decompressive surgery.1,2 Laminectomy followed by meticulous removal of the herniated disc is the treatment of choice for nonambulatory dogs.2 The overall success rates of decompressive surgery range from 60 to 95%.1–3 Several prognostic factors for thoracolumbar IVDD were previously studied, including severity and duration of clinical signs, site of disc extrusion, severity of spinal cord compression and inflammation, intramedullary T2-weighted hyperintensity observed in magnetic resonance imaging (MRI), number of myelographic injections, use of corticosteroids, and duration of anesthesia.3–8 However, the most reliable prognostic indicator remains the presence or absence of deep pain sensation (DPS) at presentation.1,3,4,6 Although DPS status is the most reliable prognostic indicator for recovery after spinal cord injury, the recovery rates of dogs presented with absence of DPS in cases of thoracolumbar disc herniation still vary between 0 and 76%.1,2,6,9 Therefore, there is still a need for a more accurate prognostic tool to be used before surgical intervention in these dogs.

Cerebrospinal fluid (CSF) characteristics in dogs with IVDD were described previously as normal or mildly inflammatory.2,10 It has been suggested that CSF analysis is unnecessary in those cases in which MRI findings suggesting IVDD are obvious.11 However, in a recent study, moderate to marked CSF pleocytosis and high CSF protein concentration were more commonly observed than was previously reported in dogs with type I disc disease, and paraplegic dogs with absent DPS had significantly higher CSF TNCC than those with DPS.12 This association between the severity of neurologic abnormality and the total nucleated cell count (TNCC) in dogs with IVDD might be useful for assessing the prognosis in more severe cases. When DPS is lost, the recovery rate decreases to approximately 50%13; however, it varies between 0 and 76%.1,2,6,9 It is therefore important to find a prognostic indicator that will help differentiate between dogs that have a better chance to recover and those that have a poor prognosis. Although CSF analysis, including TNCC, protein concentration, and differential cell percentages have been reported previously in dogs with IVDD,12,14 these parameters have yet to be tested as prognostic indicators in this condition.

Cerebellomedullary CSF analysis is routinely performed before myelography at the Hebrew University Veterinary Teaching Hospital (HUVTH). We have observed moderate to severe CSF pleocytosis in many dogs with IVDD, suggesting ongoing inflammation. The purpose of the present study was to characterize the cerebellomedullary CSF findings in dogs with acute, nonambulatory thoracolumbar IVDD, and to evaluate their association with the presenting neurologic abnormalities and final outcome after surgical intervention. We hypothesized that CSF TNCC and the differential cell percentage would be associated with the severity of spinal cord damage and therefore with both the presenting clinical signs and the prognosis of affected dogs.

Materials and Methods

Study Design and Animals

The HUVTH medical records (2005–2009) were retrospectively reviewed for dogs diagnosed with acute, nonambulatory, thoracolumbar IVDD. The inclusion criteria were performance of CSF analysis, myelography, surgical confirmation of acute intervertebral disc herniation, and neurologic grades of 3–6 at presentation (see below for details). Dogs were excluded if (1) clinical signs were present for >7 days before surgery in order to minimize the impact the time lag on CSF characteristics and final outcome,12 (2) if signs of another, additional neurologic disorder were present, (3) if the episode of clinical signs began with pain that was treated conservatively before occurrence of the nonambulatory state, in order to avoid cases that could be considered “acute on chronic”,12 and (4) if the CSF samples were assessed to be iatrogenically contaminated with blood, based on the combination of >500 red blood cells (RBC)/μL with the absence of erythrophagia.15,16

The information retrieved from the medical records included signalment (sex, age, breed), time from onset of clinical signs to presentation, neurologic grade at presentation, prior drug administration (with emphasis on glucocorticosteroids), location of the disc herniation, and time period from decompressive surgery to regaining ambulation. Short- (30 days) and long-term (over 3 months) follow-up evaluations were obtained either from the medical records or from telephone interviews with the owners.

Neurologic Grade

All dogs underwent neurologic examinations at presentation and on days 1, 2, 10, and 30 postoperatively. They were assigned a grade with a 0–6 scale, based on severity of neurologic dysfunction (0, normal; 1, spinal hyperesthesia; 2, ambulatory paraparesis, ataxia or proprioceptive deficits; 3, nonambulatory paraparesis; 4, paraplegia; 5, paraplegia with urinary or fecal incontinence; and 6, paraplegia with absence of DPS). The outcome was deemed successful if the dog regained ambulation and complete control of micturition (if the latter had been absent at presentation), even if mild ataxia still was evident.

CSF Collection and Analysis

Cerebellomedullary CSF samples were collected with hypodermal 1.5 in., 21 G needles into 2 sterile glass red top tubes and were analyzed within 10 minutes of collection. Total cell count (TCC) was determined with a hemocytometer, by counting all cells within 10 large squares of the grid.17 Two 200 μL CSF aliquots (1 from each tube) were centrifuged in a cytospina (10 minutes at 800 rpm) in order to prepare each of the slides. Two glass slidesb were thus prepared for morphologic evaluation and differential counts. One slide was stained with modified Wright's stainc and the other with a quick Romanowsky stain.d The smears were re-evaluated blindly for the purpose of the present study for CSF differential counts and the ratio between nucleated cells and RBC. Because the laboratory provides only the TCC, the TNCC and RBC counts were calculated based on the ratio between nucleated cells and RBCs in the stained, cytocentrifuged cytologic smears, by the mean ratio of at least 20 high-power fields. The CSF RBC count was calculated as TCC × mean percentage of RBCs in the stained smear.

The percentage of each cell type (neutrophil, lymphocyte, monocyte, and macrophage) was determined by manually counting 100 nucleated cells in both of the stained, cytocentrifuged CSF slides. When <100 nucleated cells were present, the differential count was based on all nucleated cells present in the smear. Differential counts were not done in samples with <40 nucleated cells, but other CSF results were included in the analyses.

Mononuclear CSF cells were classified as monocytes if their size was small (20–50 μm), usually with a high nucleus to cytoplasm (N : C) ratio, and with none to mild or moderate discrete cytoplasmic vacuolation, compared with macrophages (Fig 1A–C). Macrophages were defined as large mononuclear cells (>50 μm) usually with a lower N : C ratio than monocytes, lacy nuclear chromatin, and cytoplasm that was highly vacuolated or foamy, and sometimes contained phagocytized debris or erythrophagia (Fig 1D–F).18 Mononuclear CSF cells were classified as lymphocytes if smaller than monocytes (usually 10–15 μm), and in most cases their N : C ratio was higher compared with monocytes. Their nucleus usually showed some heterochromasia compared with the lacy nuclear chromatin pattern of monocytes, and their cytoplasm was smooth and not as granular as that of monocytes. CSF total protein was measured by a turbidimetric method with an autoanalyzer.e The HUVTH laboratory reference interval for CSF protein concentration is <25 mg/dL.

Figure 1.

 Cerebrospinal fluid monocytes and macrophages from dogs with thoracolumbar intervertebral disc herniation. (A–C) Monocytes, with mild (B) to moderate (C) vacuolation. A small lymphocyte is also noted in (A). (D–F) Macrophages, with abundant vacuolation (D, E) and occasionally with erythrophagia (F) (modified Wright's stain).

Statistical Analysis

For the purpose of analyzing the association of signalment and history with CSF characteristics, the dogs were divided into 2 breed groups: chondrodystrophic (including miniature and standard Dachshunds, Pekingese, French Bulldog, Cocker Spaniel, Shih Tzu, Maltese, Miniature Poodle, and Toy Poodle) and nonchondrodystrophic breeds (all others). In addition, dogs were divided into 2 groups based on presence or absence of prior steroid treatment. Dogs also were divided into 4 neurologic grade groups at presentation (as described above, grades 3–6). For the analysis of CSF results, TNCC, CSF protein concentration, and the percentages of each cell type were considered as continuous variables. For analyses of the outcome, dogs were divided into 2 groups (successful and unsuccessful outcomes) at 30 days postoperatively and then again for long-term outcome (ie end of the study). They also were divided into 5 groups based on time lag from surgery to regaining ambulation: 1–5, 6–30, 31–100, >100 days, and failure to regain ambulation. For the purpose of describing pleocytosis, dogs were divided into 3 groups based on TNCC: normal (≤5 cells/μL), mild pleocytosis (>5–≤20 cells/μL), and moderate to severe pleocytosis (>20 cells/μL).

The χ2 test was used to compare neurologic grade at presentation with categorical CSF morphologic characteristics such as erythrophagia The nonparametric Mann-Whitney U- and Kruskal-Wallis tests were used to compare neurologic grade at presentation with quantitive CSF characteristics (ie protein concentration, TNCC, differential cell percentages, and macrophage to monocyte ratio [MΦ : M]). The Kruskal-Wallis test was used to check for association of the time lag from onset of clinical signs to presentation, breed, and presence of prior steroid treatment with all CSF characteristics. The Mann-Whitney U-test was used to check for association of the outcome (30 days and long term) with CSF pleocytosis, CSF protein, TNCC, differential cell percentages, and MΦ : M. Fisher's exact test was used to check for association of the outcome with presence of erythrophagia and neutrophil vacuolation. Receiver operator characteristics (ROC) curve analysis including area under the curve (AUC) and its standard error (SE) was performed for several measures, and to identify optimal cut-off points, with their sensitivity and specificity, for predicting outcome. Selected optimal cut-off points were those with the least number of misclassifications. These cut-off points were used to divide the dogs into 2 categorical groups for each measure (ie TNCC, percentage of macrophages and MΦ : M), those below, equal to, or above the cut-off point. The odds ratio (OR) for the outcome then was calculated by Fisher's exact test. All statistical tests were 2-tailed, and a P value ≤.05 was considered statistically significant. Statistical analyses were performed by statistical software.f

Results

Signalment and Medical History

Acute nonambulatory thoracolumbar IVDD was diagnosed in 61 dogs that presented within 7 days from the onset of clinical signs, in which CSF analysis and myelography were performed. All dogs were treated by decompressive surgery by hemilaminectomy as described previously2 and acute disc herniation was confirmed during surgery. CSF samples from 7 dogs had iatrogenic blood contamination and these dogs thus were excluded from the study. Fifty-four dogs fulfilled all inclusion criteria. They had a median age of 4 years (range, 1–11 years), a median body weight of 9.8 kg (range, 3–34 kg), and included 29 males (6 castrated) and 25 females (16 spayed). Chondrodystrophic dogs were most common (37/54, 68%), including Dachshunds (13/54, 24%), Pekingese (12/54, 22%), French Bulldog (8/54, 15%), Cocker Spaniel, Shih Tzu, Maltese, and Poodle (1 each). There were 12 mixed breed dogs (22%). Glucocorticoids were administered to 22 dogs (40%) before presentation, of which 10 were PO medicated and 12 were treated parenterally (IM or IV). Nonsteroidal anti-inflammatory drugs (NSAIDs) were administered to 5 dogs (9%) before presentation. Because of this low number, no statistical analyses of the association of NSAIDs with CSF characteristics were made. The median time lag from onset of clinical signs to presentation in all dogs was 18 hours (range, 2–168 hours) and 12 hours in dogs without DPS (range, 2–48 hours). There was no significant difference in time lag between dogs presented with and without DPS.

Neurologic Grades at Presentation

The distribution of dogs based on neurologic grade at presentation was as follows: grade 3, 9 dogs (17%); grade 4, 23 dogs (42%); grade 5, 6 dogs (11%), and grade 6, 16 dogs (30%).

CSF Analysis

Abnormally increased CSF TNCC was present in 29 of 54 dogs (54%), of which 15 (28%) had mild pleocytosis and 14 (26%) had marked pleocytosis. The median TNCC of all dogs was 6 cells/μL (range, 0–104). The median RBC count of all dogs was 0 cells/μL (range, 0–2,522). Sixteen of 54 dogs (30%) had <40 cells in CSF smears, and differential counts were obtained in 38/54 dogs (70%). Neutrophils were the most common cell type in 29/38 dogs (76%) (median, 60% neutrophils; range, 0–85%). Monocytes were the most common cell type in 7/38 dogs (19%) (median, 23.5% monocytes; range, 5–95%). Lymphocytes and macrophages were the most common cell types in 1/38 dogs (2.5%), each (medians 12 and 1.5%, respectively; ranges, 0–52 and 0–40, respectively). The median MΦ : M was 0 (range, 0–3). CSF protein was measured in 25/54 dogs (46%), with a median concentration of 4.2 mg/dL (range, 0–228.5 mg/dL). Increased CSF protein was present in 4/25 dogs (16%), with a median concentration of 38.8 mg/dL (range, 26–228.5 mg/dL).

Dogs that did not receive glucocorticoids before presentation (n = 32) had a significantly (P= .05) higher percentage of neutrophils in CSF compared with glucocorticoid treated dogs (n = 22) (medians, 60 and 42%, respectively). Other CSF results, and the outcome, were not influenced by glucocorticoid treatment. There was no association of route of glucocorticoid administration with any of the CSF characteristics.

CSF characteristics of dogs in each neurologic grade at presentation were tabulated (Tables 1 and 2). Dogs with absent DPS at presentation (neurologic grade 6) had significantly (P= .003) higher TNCC (median, 35 cells/μL; range, 0–104 cells/μL) compared with dogs with neurologic grades of 3–5 (median, 4.5 cells/μL; range, 0–29 cells/μL). The percentage of CSF macrophages was significantly (P= .0002) higher in dogs presented with neurologic grade 6 (median, 8%; range, 0–40%) compared with those presented with grades 3–5 (median, 0%; range, 0–9%). The MΦ : M ratio also was significantly (P= .0003) higher in dogs with neurologic grade 6 (median, 0.58; range, 0–3) compared with dogs with grades 3–5 (median, 0; range, 0–0.44). Erythrophagia was observed in CSF from 5 dogs (9%) and was significantly (P= .016) associated with high neurologic grade (all 5 dogs presented with grades 5 or 6).

Table 1.   Neurological grade at presentation and cerebrospinal fluid total nucleated cells in 54 dogs with nonambulatory thoracolumbar intervertebral disc disease.
Neurologic Signs at PresentationNeurologic GradeCerebrospinal Fluid TNCC LevelNumber of Dogs
Number of Dogs (%)
<5 cells/μL>5 and ≤20 cells/μL>20 cell/μL
  1. DPC, deep pain sensation; TNCC, total nucleated cell count.

Nonambulatory paraparesis35 (55.6%)3 (33.3%)1 (11.1%)9
Paraplegia412 (52.2%)9 (39.1%)2 (8.7%)23
Paraplegia with urinary incontinence53 (50%)2 (33.3%)1 (16.7%)6
Paraplegia with absence of DPS65 (31.3%)1 (6.3%)10 (62.5%)16
Total 25 (46.3%)15 (27.8%)14 (25.9%)54
Table 2.   Cerebrospinal fluid analysis findings and neurologic grade severity at presentation in 54 dogs with acute, nonambulatory thoracolumbar intervertebral disc disease.
Neurologic Signs at Presentation (neurological grade)CSF Protein (mg/dL)TNCC (cells/μL)RBC (cells/μL)Neu (%)Lym (%)Mon (%)MΦ (%)MΦ : M ratio
  • CSF, cerebrospinal fluid; DPS, deep pain sensation; Lym, lymphocytes; Mon, monocytes; MΦ, macrophages; Neu, neutrophils; RBC, red blood cells; TNCC, total nucleated cell count.

  • Statistically significant (P≤ .05) from all other groups.

Nonambulatory paraparesis (grade 3) (9 dogs)
 Mean36.877.7957.8868.3311.0020.500.170.01
 Median4.205.000.0067.0011.0022.000.000.00
 Range0–228.50–230–49059–823–186–300–10–0.05
 n79966666
Paraplegia (grade 4) (23 dogs)
 Mean11.506.329.5529.1315.7340.870.930.04
 Median6.504.000.0023.0017.0042.000.000.00
 Range0–33.50–28.80–148.80–623–305–950–40–0.44
 n723231515151515
Paraplegia with urinary incontinence (grade 5) (6 dogs)
 Mean1.267.0813.9257.6710.3326.335.670.16
 Median0.004.5015.8660.007.0027.006.000.15
 Range0–6.30–210–3253–604–2025–272–9.00
 n56633333
Paraplegia with absence of DPS (grade 6) (16 dogs)
 Mean17.0543.30186.8356.8613.0719.4311.000.84
 Median17.0535.0011.8063.509.0016.008.000.58
 Range0–440–1040–252214–850–525–600–400–3
 n616161414141414

There were no significant differences in CSF protein concentration among different neurologic grades at presentation, or between dogs with and without DPS at presentation.

Outcome

Dogs were followed until they regained ambulation, or for at least 3 months (range, 3–12 months) when the outcome was less favorable. Successful outcome was documented in 47/54 dogs (87%). Ambulation was regained within 5 days postoperatively in 21 dogs (39%), within 6–30 days in 19 dogs (35%), within 31–100 days in 5 dogs (9%), and after >100 days in 2 dogs (4%). All dogs with neurologic grades 3–5 at presentation recovered. The outcome was negative in 7 dogs (13%), all of which had neurologic grade 6 at presentation. Five of these 7 dogs were euthanized at their owners' request owing to lack of, or insufficient, improvement (30–120 days postoperatively).

CSF Results and Outcome

CSF characteristics and their association with outcome are presented in Tables 3 and 4. Dogs that regained ambulation within 30 days postoperatively had significantly (P= .004) lower TNCC (median, 4.5 cells/μL; range, 0–30 cells/μL) compared with dogs that failed to regain ambulation within 30 days postoperatively (median, 45 cells/μL; range, 0–104 cells/μL). The percentage of neutrophils and macrophages were significantly (P= .016 and P < .0001, respectively) lower in dogs that regained ambulation within 30 days postoperatively (median 48%; range, 0–82; median, 0%; range, 0–6%, respectively) compared with dogs that failed to regain ambulation within 30 days postoperatively (median, 67%; range, 28–85; median, 9%; range, 0–40%, respectively). The MΦ : M ratio was significantly (P < .0001) lower in dogs that regained ambulation within 30 days postoperatively (median, 0; range, 0–0.4) compared with dogs that failed to regain ambulation within 30 days (median, 0.7; range, 0–3). CSF protein concentration was significantly (P= .04) lower in dogs that regained ambulation within 30 days postoperatively (median, 2.4 mg/dL; range, 0–228 mg/dL) compared with dogs that failed to regain ambulation within 30 days postoperatively (median, 19.5 mg/dL; range, 2.6–44 mg/dL). When testing the outcome at the end of the study period, dogs that have regained ambulation had significantly (P= .004) lower TNCC (median, 5 cells/μL; range, 0–96 cells/μL) compared with those having a negative outcome (median, 50 cells/μL; range, 0–104 cells/μL). The percentage of macrophages and the MΦ : M ratio were significantly (P < .0001, for both) lower in dogs that had regained ambulation at the end of the study period (median 0%; range, 0–12%; median, 0; range, 0–0.7, respectively) compared with those that had a negative outcome (median, 15.5%; range, 9–40%; median, 1.7; range, 0–3, respectively). CSF protein concentration also was significantly (P= .01) lower in dogs that regained ambulation (median, 2.6 mg/dL; range, 0–228 mg/dL) compared with dogs that failed to regain ambulation (median, 20.6 mg/dL; range, 14.6–44 mg/dL). A significant trend of increase in time lag from surgery to regaining ambulation was observed with higher TNCC (P < .001), higher percentage of macrophages (P < .001), and higher MΦ : M ratio (P < .001) (χ2 trend test; Table 5).

Table 3.   Cerebrospinal fluid analysis findings and outcome of 54 dogs with acute, nonambulatory thoracolumbar intervertebral disc disease.
OutcomeCSF Protein (mg/dL)TNCC (cells/μL)RBC (cells/μL)Neu (%)Lym (%)Mon (%)MΦ (%)MΦ : M ratio
  • CSF, cerebrospinal fluid; Lym, lymphocytes; Mon, monocytes; MΦ, macrophages; Neu, neutrophils; RBC, red blood cells; TNCC, total nucleated cell count.

  • Statistically significant (P≤ .05) from the other subgroup within the outcome group.

At 30 days postoperative
 Negative (14 dogs)
  Median19.545.020.067.07.015.09.00.7
  Range2.6–440–1040–252228–850–205–600–400–3
  n514141313131313
 Positive (40 dogs)
  Median2.44.50.048.015.028.00.00.0
  Range0–228.50–300–4900–820–520–950–60–0.4
  n2040402525252525
At the end of study
 Negative (7 dogs)
  Median20.650.010.963.55.511.515.51.7
  Range14.6–440–1040–18230–850–205–209–400–3
  n47766666
 Positive (47 dogs)
  Median2.65.00.059.513.026.50.00.0
  Range0–228.50–960–25220–820–520–950 120–0.7
  n2147473232323232
Table 4.   Number of cerebrospinal total nucleated cells and outcome at 30 days postsurgery and at the end of the study in dogs with acute, nonambulatory thoracolumbar intervertebral disc disease.
OutcomeCerebrospinal Fluid Total Nucleated Cell Count Level
Normal (<5 cells/μL) n (%)Mildly Increased (>5 and ≤20 cells/μL) n (%)Moderately to Markedly Increased (>20 cell/μL) n (%)
At 30 days postoperative
 Negative3 (12.0%)1 (6.7%)10 (71.4%)
 Positive22 (88.0%)14 (93.3%)4 (28.6%)
At the end of study
 Negative1 (4.0%)0 (0%)6 (42.9%)
 Positive24 (96.0%)15 (100%)8 (57.1%)
Table 5.   Cerebrospinal fluid analysis findings and time to regain of ambulation of 47 dogs with acute, nonambulatory thoracolumbar intervertebral disc disease and positive outcomes.
Time to Regain of AmbulationCSF Protein (mg/dL)TNCC (cells/μL)RBC (cells/μL)Neu (%)Lym (%)Mon (%)MΦ (%)MΦ : M ratio
  1. CSF, cerebrospinal fluid; Lym, lymphocytes; Mon, monocytes; MΦ, macrophages; Neu, neutrophils; RBC, red blood cells; TNCC, total nucleated cell count.

1–5 days (21 dogs)
 Median5.253.00059.0015.0025.000.000.00
 Range0–2280–110–4900–820–300–700–40–0.4
 n1221211111111111
6–30 days (19 dogs)
 Median07.00045.0017.0029.000.000.00
 Range0–33.51–300–1494–620–520–950–60–0.2
 n819191414141414
31–100 days (5 dogs)
 Median2.615.4031.860.008.0020.004.000.29
 Range2.6–2.62–9613–6428–774–2014–600–90–0.5
 n15555555
Above 100 days (2 dogs)
 Median79.00126169.007.0014.509.500.64
 Range78, 800, 252267, 714, 1012, 177, 120.6–0.7
 n02222222

ROC analysis of TNCC yielded an AUC of 0.821 (SE, 0.121; Fig 2A) with an optimal cut-off point for prediction of a negative long-term outcome of 23.3 cells/μL, corresponding to a sensitivity and specificity of 89 and 85%, respectively. Dogs with TNCC ≤ 23.3 cells/μL were 50-fold more likely to regain ambulation compared with those with TNCC > 23.3 cells/μL (CI 95%, 5–500; P < .01). An ROC curve for percentage of macrophages as a predictive value of a negative long-term outcome had an AUC of 0.92 (SE, 0.011; Fig 2B) with an optimal cut-off point of 13%, corresponding to a sensitivity and specificity of 100 and 83%, respectively. ROC analysis of MΦ : M ratio as a predictive value of a negative long-term outcome had an AUC of 0.95 (SE, 0.08; Fig 2C) with an optimal cut-off point of 0.64, corresponding to a sensitivity and specificity of 97 and 100%, respectively. Dogs with a MΦ : M ratio ≤0.64 were 7-fold more likely to have a successful long-term outcome compared with those with an MΦ : M ratio > 0.64 (CI 95%, 2–43; P <.01).

Figure 2.

 Receiver operator characteristic curves of several cerebrospinal fluid characteristics as predictors of a successful outcome in dogs with acute nonambulatory thoracolumbar disc herniation. (A) Total nucleated cell count; (B) percentage of macrophages; (C) macrophage to monocyte ratio. AUC, area under the curve; SE, standard error.

The CSF characteristics of dogs without DPS and their outcomes were tabulated (Table 6). Such dogs that later regained ambulation had a significantly lower percentage of CSF macrophages (P= .001) and MΦ : M ratio (P= .001) compared with those presented with absent DPS that failed to regain ambulation.

Table 6.   Cerebrospinal fluid analysis findings and the outcome in 16 dogs with acute thoracolumbar intervertebral disc disease with absence of deep pain sensation at presentation.
OutcomeCSF Protein (mg/dL)TNCC (cells/μL)RBC (cells/μL)Neu (%)Lym (%)Mon (%)MΦ (%)MΦ : M ratio
  • CSF, cerebrospinal fluid; Lym, lymphocytes; Mon, monocytes; MΦ, macrophages; Neu, neutrophils; RBC, red blood cells; TNCC, total nucleated cell count.

  • 1

    P value of comparison of dogs with positive and negative outcomes.

Negative (7 dogs)
 Mean24.9354.8844.4161.177.3312.3320.001.73
 Median20.5550.0010.8563.505.5011.5015.501.65
 Range14.6–440–1040–18230–850–205–209–400.69–3
 n4.007.007.006.006.006.006.006.00
Positive (9 dogs)
 Mean1.3034.29297.5953.6317.3824.754.250.24
 Median1.3015.4012.7563.5011.0018.503.000.07
 Range0–2.60–960–252214–774–5212–600–120–0.71
 n2.009.009.009.009.009.009.009.00
P value10.1330.3511.0000.4910.1080.0590.0010.001

Discussion

Acute intervertebral disc herniation is considered the most common cause of spinal cord injury in dogs.19 The results of the present study demonstrate that routine CSF analysis yields useful prognostic findings in dogs with acute nonambulatory thoracolumbar disc herniation. Specifically, the positive association between time to ambulation and TNCC, percentage of CSF macrophages, and MΦ : M ratio might potentially aid clinicians in estimating the postoperative recovery period and the value of by other therapeutic modalities such as physiotherapy. The present findings are particularly relevant for dogs with thoracolumbar IVDD that present with absence of DPS, in which the percentage of CSF macrophages and the MΦ : M had prognostic value. To the best of our knowledge, this is the first description of a potentially useful prognostic indicator in dogs with acute thoracolumbar IVDD that are presented with neurologic Grade 6.

The frequency of occurrence of neutrophils in the CSF of most dogs in this study was in agreement with previous findings in dogs and human patients with acute spinal cord injury.14,20 In rodent models of spinal cord injury, neutrophils appear at the primary lesion 3–6 hours postinjury, peak at 12–24 hours, and decline within 5 days.21 The predominance of lymphocytes reported in the CSF of dogs in a previous study of chronic and acute-on-chronic IVDD suggests the initial neutrophilic inflammation may evolve to a more lymphocytic inflammation later in the course of disease.12 The pathophysiology of IVDD includes the initial trauma as well as secondary injury, which can result in further tissue damage and should always be considered part of the disease.2,22 Although the initial damage to the spinal cord is induced by contusion and compression, the severity of the final lesion often markedly exceeds the damage observed early in the disease course.22 This spread of damage through the spinal cord over time is considered secondary injury, and includes ischemia, excitatory amino acid toxicity, free radical formation, release of proteases, energy depletion, and inflammation.22 The inflammatory mechanisms and progression of inflammation over time have been well documented in animal models of SCI.21–24

Our results also suggest that macrophages have an important role in the pathophysiology of acute spinal cord injury: their percentage was higher with increasing severity of spinal cord injury and in dogs with a negative outcome, and macrophages were more frequently observed in dogs without DPS. Macrophages in injured spinal cord are derived from blood-borne monocytes and resident microglia.22,25 Microglial cells are activated and transform into macrophages within minutes after initiation of spinal cord microenvironmental disturbances.26 Blood-borne monocytes have been shown to infiltrate the lesion site 2 days after acute spinal cord injury in rats, peak at 5–7 days from the insult, and persist at the lesion site for several months.22,23,26 Macrophages participate in the removal of injured tissue debris, and release protective cytokines that promote neuronal regeneration, wound healing, and tissue repair.27,28 Macrophages also promote axonal loss through release of pro-inflammatory cytokines and proteases, reactive oxygen species, and nitric oxide formation.25,26,29 Invasion of macrophages into the lesion site is promoted by disruption of the blood-spinal cord barrier and by proinflammatory cytokines released from the injured spinal cord parenchyma.22,26,30 We assume that the number of macrophages at the lesion site in the spinal cord is reflected by their number in the CSF. It is thus that the high percentage of CSF macrophages in of dogs presented with neurologic grade 6 is positively associated with the severity of the damage.

The MΦ : M ratio also appeared to be a good predictor of outcome in dogs presenting without DPS, consistent with the transformation of monocytes into macrophages in response to tissue injury. Differentiation of monocytes and macrophages in CSF cytologic analysis is routine;18 however, to our knowledge, a ratio has not been determined previously. A CSF MΦ : M ratio would be expected to be a more sensitive indicator of spinal cord injury than the percentage of macrophages or monocytes alone if macrophages are primarily derived from resident CSF monocytes, whose percentage then would decrease concurrent with the increase in macrophages. Although there is some inherent subjectivity involved in evaluating cell morphology (ie in differentiating macrophages from monocytes), the use of defined criteria whereas the CSF smears were reassessed for the purpose of this study helped limit variation in this study. Our results suggest the MΦ : M ratio has prognostic value and therefore warrants further study in a larger number of dogs with acute IVDD.

Inclusion and exclusion criteria in the present study (eg restriction of IVDD location to the thoracolumbar area, inclusion of relatively acute cases, and exclusion of cases with CSF iatrogenic blood contamination) were designed to gain, as much as possible, a clinically homogenous population such that CSF findings were more likely to be related to the clinical disease findings and outcome. Dogs were included in the study only if the time lag from onset of clinical signs to presentation was <7 days because time from onset of neurologic signs to surgical decompression is known to influence the prognosis of dogs with IVDD that present without DPS.4,6 Furthermore, as noted above, a previous study found CSF lymphocytic pleocytosis from 7 days onward after the initial cord insult, suggesting chronic changes at this later stage.12 In contrast with a previous study that has reported that lymphocytes predominate in CSF samples of chondrodystrophic dogs with IVDD,12 the percentage of neutrophils in the present study was significantly higher in these breeds. The present results show that the TNCC, percentage of macrophages, and MΦ : M ratio were not associated with breed type (ie chondrodystrophic or nonchondrodystrophic).

There are several limitations in the sampling and analysis of CSF in this study. First, the estimation of TNCC and RBC count, based on evaluation of cytocentrifuged preparations, likely introduced some error into the results because of uneven cell distribution, variable cell recovery, and the additional calculations involved. However, the examination of a large number of fields in the smears helped reduce this error, and the TNCC results and their positive association with neurologic grade were in agreement with previous studies of dogs with IVDD.12 Second, CSF contamination with RBCs is a frequent problem when analyzing CSF, and has led to the exclusion of several samples in this study. The cut-off of 500 RBCs/μL is more restricted compared with previous similar studies12,14 and the additional requirement of observing erythrophagia helped ensure that when RBCs were present in high numbers, there was cytologic evidence for noniatrogenic hemorrhage. Furthermore, presence of up to 13,200 RBC/μL does not appear to significantly affect the CSF protein concentration or TNCC, such that the inclusion of the samples with hemorrhage did not likely affect these other CSF results.15,31 Third, all CSF samples in this study were collected from the cerebellomedullary cistern. Lumbar samples are more sensitive for detection of abnormalities at the thoracolumbar area compared with cerebellomedullary cistern samples because of the caudal flow of CSF.10,32 This might explain the relatively low median TNCC and percentage of cases showing abnormal TNCC observed in the present study (54%) compared with the those in a previous study (61%), in which lumbar samples were used.12 Thus, the present results might underestimate CSF changes in dogs with thoracolumbar IVDD, and the prognostic value of our findings might have been underestimated as well. Finally, the number of CSF samples available for protein measurement limited the conclusions that can be drawn from this parameter. The median CSF protein concentration in the severe cases of IVDD in our study was lower compared with a previous study,12 and although protein was increased in dogs without DPS, the difference was not significant. Nevertheless, significantly higher protein concentration was recorded in dogs with negative outcomes, suggesting that CSF protein might be a useful preoperative prognostic indicator of the outcome in dogs with acute thoracolumbar IVDD, and should thus be further studied.

The significantly higher TNCC in CSF from Grade 6 dogs compared with grades 3–5 and the insignificant difference among grades 3–5 was consistent with the more severe inflammation expected in severe IVDD cases and also was in agreement with a previous study in which the relationship between the severity of the neurologic signs in IVDD and the degree of CSF pleocytosis was not linear. Using a cutoff of 23 cells/μL, TNCC was a sensitive and specific predictor of outcome (ie regaining ambulation) in the present study. However, unlike the MΦ : M ratio, the TNCC appeared to provide little advantage over assessment of the prognosis based solely on neurologic grade, because all dogs with grades 3–5 eventually recovered. ROC analysis of only grade 6 dogs may have been warranted, but was precluded by the small number of dogs. Future large-scale studies should examine the association of TNCC and outcome in this subgroup of dogs.

This study had 2 general limitations. First, its retrospective design limited the available data and thus weakened some of our statistical analyses (eg, CSF total protein). Second, the number of dogs included in the study was low, and thus the number of cases in each subcategory is limited (eg the number of dogs in each neurologic grade) and this had a limiting impact on the statistical analyses. Nevertheless, the number of cases included in the present study was similar to that of previous studies that have assessed different prognostic markers in canine IVDD.8,14,33,34 Because of these limitations, conclusions cannot be drawn from all aspects of the study, but the findings related to outcome provide new insights into the value of CSF parameters in acute thoracolumbar IVDD and warrant additional research.

In conclusion, a high percentage of CSF macrophages and a high MΦ : M ratio have strong potential as useful preoperative prognostic indicators of outcome in dogs with acute thoracolumbar IVDD, especially in paraplegic dogs without DPS at presentation. Additional study is warranted to better elucidate the importance of these parameters in prognosis and in the pathogenesis of acute spinal cord injury.

Footnotes

a Shandon Cytospin 4, Thermo-Shandon Electron Corporation, Pittsburgh, PA

b Cytoslide, Thermo-Shandon Electron Corporation

c Hema-Tek 2000 Slide Stainer, model 4488B; Stain: Hematek stain pack; Modified Wright's stain; Bayer Corporation, Elkhart, IN

d Romanowsky-based quick stain, Jorgensen Laboratories Inc, Loveland, CO

e Cobas-Mira, Roche, Rottkreutz, Switzerland, at 37°; Reagent, Roche/Hitachi U/CSF total protein (TPUC3), Roche Diagnostics, Mannheim, Germany

f SPSS 17.0 for Windows, SPSS Inc, Chicago, IL

Acknowledgment

This work was not supported by any grant.

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