J Vet Intern Med 2011;25:846–855

A s s o c i a t i o n o f Ce r e b r o s p i n a l F l u i d A n a l y s i s Fi n d i n g s wi t h C l i n i c a l
S i g n s a n d O u t c o m e i n Ac u t e N o n a m b u l a t o r y T h o r a c o l u m b a r D i s c
D i s e a s e in Do g s
I. Srugo, I. Aroch, M.M. Christopher, O. Chai, L. Goralnik, T. Bdolah-Abram, and M.H. Shamir

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 neu-
rologic 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 there-
fore 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 (45 cells/mL) 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 5 .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.
Key words: Canine; Intervertebral disc disease; Pleocytosis; Prognosis.

horacolumbar intervertebral disc disease (IVDD) is

T a common cause of hind limb paresis and paralysis
in dogs.1 Treatment protocols vary from conservative
Abbreviations:
AUC area under the curve
treatment to decompressive surgery.1,2 Laminectomy fol- CSF cerebrospinal fluid
lowed by meticulous removal of the herniated disc is the DPS deep pain sensation
treatment of choice for nonambulatory dogs.2 The over- IVDD intervertebral disc disease
all success rates of decompressive surgery range from 60 MF:M macrophage to monocyte ratio
to 95%.1–3 Several prognostic factors for thoracolumbar OR odds ratios
IVDD were previously studied, including severity and ROC receiver operator characteristics
duration of clinical signs, site of disc extrusion, severity SE standard error
of spinal cord compression and inflammation, intra- TCC total cell count
medullary T2-weighted hyperintensity observed in TNCC total nucleated cell count
magnetic resonance imaging (MRI), number of myelo-
graphic injections, use of corticosteroids, and duration of
anesthesia.3–8 However, the most reliable prognostic in-
dicator remains the presence or absence of deep pain tus is the most reliable prognostic indicator for recovery
sensation (DPS) at presentation.1,3,4,6 Although DPS sta- after spinal cord injury, the recovery rates of dogs pre-
sented with absence of DPS in cases of thoracolumbar
From the Department of Neurology (Srugo, Chai, Shamir), the disc herniation still vary between 0 and 76%.1,2,6,9 There-
Department of Small Animal Internal Medicine (Aroch), Veterinary fore, there is still a need for a more accurate prognostic
Teaching Hospital, Koret School of Veterinary Medicine, Hebrew tool to be used before surgical intervention in these dogs.
University of Jerusalem; the Department of Pathology, Microbiology Cerebrospinal fluid (CSF) characteristics in dogs with
and Immunology, School of Veterinary Medicine, University of Davis IVDD were described previously as normal or mildly in-
California, Davis, CA (Christopher); Teaching Services Unit, flammatory.2,10 It has been suggested that CSF analysis
Faculty of Medicine, Hebrew University of Jerusalem, Israel
(Bdolah-Abram); and Koret School of Veterinary Medicine, Hebrew
is unnecessary in those cases in which MRI findings sug-
University of Jerusalem, Israel (Goralnik). This work was performed gesting IVDD are obvious.11 However, in a recent study,
at Hebrew University Veterinary Teaching Hospital, Koret School of moderate to marked CSF pleocytosis and high CSF pro-
Veterinary Medicine, Hebrew University of Jerusalem. tein concentration were more commonly observed than
Corresponding author: Itay Srugo, Koret School of Veterinary was previously reported in dogs with type I disc disease,
Medicine, Hebrew University of Jerusalem, P.O. Box 12, Rehovot and paraplegic dogs with absent DPS had significantly
76100, Israel; e-mail: srugo@agri.huji.ac.il.
higher CSF TNCC than those with DPS.12 This associa-
Submitted December 26, 2010; Revised April 3, 2011; Accepted
April 25, 2011.
tion between the severity of neurologic abnormality and
Copyright r 2011 by the American College of Veterinary Internal the total nucleated cell count (TNCC) in dogs with
Medicine IVDD might be useful for assessing the prognosis in
10.1111/j.1939-1676.2011.0739.x more severe cases. When DPS is lost, the recovery rate
 CSF Analysis in Disc Disease
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 per-
centages 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 moder-
ate to severe CSF pleocytosis in many dogs with IVDD,
suggesting ongoing inflammation. The purpose of the pres-
ent study was to characterize the cerebellomedullary CSF
findings in dogs with acute, nonambulatory thoracolum-
bar 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 asso-
ciated 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, thoraco-
lumbar 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 47 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 conser-
vatively 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 4500 red blood cells
(RBC)/mL 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 adminis-
tration (with emphasis on glucocorticosteroids), location of the disc
herniation, and time period from decompressive surgery to regain-
ing 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.
847
CSF Collection and Analysis
Cerebellomedullary CSF samples were collected with hypoder-
mal 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 mL 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 pre-
pared 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 ra-
tio 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, mono-
cyte, and macrophage) was determined by manually counting 100
nucleated cells in both of the stained, cytocentrifuged CSF slides.
When o100 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 o40 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 mm), 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). Macro-
phages were defined as large mononuclear cells (450 mm) 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 mm), 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 concen-
tration is o25 mg/dL.
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 Dachs-
hunds, Pekingese, French Bulldog, Cocker Spaniel, Shih Tzu,
Maltese, Miniature Poodle, and Toy Poodle) and nonchondrodystro-
phic 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 de-
scribed 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, 4100
days, and failure to regain ambulation. For the purpose of describing
pleocytosis, dogs were divided into 3 groups based on TNCC: normal
(5 cells/mL), mild pleocytosis (45–20 cells/mL), and moderate to
severe pleocytosis (420 cells/mL).
The w2 test was used to compare neurologic grade at presentation
with categorical CSF morphologic characteristics such as erythro-
848
Fig 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).
phagia 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
[MF : M]). The Kruskal-Wallis test was used to check for associa-
tion of the time lag from onset of clinical signs to presentation,
breed, and presence of prior steroid treatment with all CSF charac-
teristics. 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
MF : M. Fisher’s exact test was used to check for association of the
outcome with presence of erythrophagia and neutrophil vacuolat-
ion. 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 MF : 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 di-
agnosed in 61 dogs that presented within 7 days from the
onset of clinical signs, in which CSF analysis and myelo-
graphy were performed. All dogs were treated by
decompressive surgery by hemilaminectomy as described
previously2 and acute disc herniation was confirmed dur-
ing surgery. CSF samples from 7 dogs had iatrogenic
blood contamination and these dogs thus were excluded
Srugo et al
from the study. Fifty-four dogs fulfilled all inclusion cri-
teria. 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 com-
mon (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 par-
enterally (IM or IV). Nonsteroidal anti-inflammatory
drugs (NSAIDs) were administered to 5 dogs (9%) be-
fore 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 signifi-
cant 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
 CSF Analysis in Disc Disease 849

Table 1. Neurological grade at presentation and cerebrospinal fluid total nucleated cells in 54 dogs with nonambu-
latory thoracolumbar intervertebral disc disease.
Cerebrospinal Fluid TNCC Level

Number of Dogs (%)

Neurologic Signs at Presentation Neurologic Grade o5 cells/mL 45 and 20 cells/mL 420 cell/mL Number of Dogs
Nonambulatory paraparesis 3 5 (55.6%) 3 (33.3%) 1 (11.1%) 9
Paraplegia 4 12 (52.2%) 9 (39.1%) 2 (8.7%) 23
Paraplegia with urinary incontinence 5 3 (50%) 2 (33.3%) 1 (16.7%) 6
Paraplegia with absence of DPS 6 5 (31.3%) 1 (6.3%) 10 (62.5%) 16
Total 25 (46.3%) 15 (27.8%) 14 (25.9%) 54

DPC, deep pain sensation; TNCC, total nucleated cell count.

TNCC of all dogs was 6 cells/mL (range, 0–104). The me-
dian RBC count of all dogs was 0 cells/mL (range, 0–
2,522). Sixteen of 54 dogs (30%) had o40 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 MF : 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 pre-
sentation (n 5 32) had a significantly (P 5 .05) higher
percentage of neutrophils in CSF compared with gluco-
corticoid treated dogs (n 5 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 sig-
nificantly (P 5 .003) higher TNCC (median, 35 cells/mL;
range, 0–104 cells/mL) compared with dogs with
neurologic grades of 3–5 (median, 4.5 cells/mL; range, 0–
29 cells/mL). The percentage of CSF macrophages
was significantly (P 5 .0002) higher in dogs presented
with neurologic grade 6 (median, 8%; range, 0–40%)

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 CSF Protein TNCC RBC Neu Lym Mon MF MF :
(neurological grade) (mg/dL) (cells/mL) (cells/mL) (%) (%) (%) (%) M ratio
Nonambulatory paraparesis (grade 3) (9 dogs)
Mean 36.87 7.79 57.88 68.33 11.00 20.50 0.17 0.01
Median 4.20 5.00 0.00 67.00 11.00 22.00 0.00 0.00
Range 0–228.5 0–23 0–490 59–82 3–18 6–30 0–1 0–0.05
n 7 9 9 6 6 6 6 6
Paraplegia (grade 4) (23 dogs)
Mean 11.50 6.32 9.55 29.13 15.73 40.87 0.93 0.04
Median 6.50 4.00 0.00 23.00 17.00 42.00 0.00 0.00
Range 0–33.5 0–28.8 0–148.8 0–62 3–30 5–95 0–4 0–0.44
n 7 23 23 15 15 15 15 15
Paraplegia with urinary incontinence (grade 5) (6 dogs)
Mean 1.26 7.08 13.92 57.67 10.33 26.33 5.67 0.16
Median 0.00 4.50 15.86 60.00 7.00 27.00 6.00 0.15
Range 0–6.3 0–21 0–32 53–60 4–20 25–27 2–9 .00
n 5 6 6 3 3 3 3 3
Paraplegia with absence of DPS (grade 6) (16 dogs)
Mean 17.05 43.30 186.83 56.86 13.07 19.43 11.00 0.84
Median 17.05 35.00w 11.80 63.50 9.00 16.00 8.00w 0.58w
Range 0–44 0–104 0–2522 14–85 0–52 5–60 0–40 0–3
n 6 16 16 14 14 14 14 14

CSF, cerebrospinal fluid; DPS, deep pain sensation; Lym, lymphocytes; Mon, monocytes; MF, macrophages; Neu, neutrophils; RBC, red
blood cells; TNCC, total nucleated cell count.
w
Statistically significant (P  .05) from all other groups.
850 Srugo et al

compared with those presented with grades 3–5 (median,
0%; range, 0–9%). The MF : M ratio also was signifi-
cantly (P 5 .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 signifi-
cantly (P 5 .016) associated with high neurologic grade
(all 5 dogs presented with grades 5 or 6).
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 out-
come was less favorable. Successful outcome was
documented in 47/54 dogs (87%). Ambulation was re-
gained 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 4100 days in 2 dogs (4%). All
dogs with neurologic grades 3–5 at presentation recov-
ered. 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 out-
come are presented in Tables 3 and 4. Dogs that
regained ambulation within 30 days postoperatively had
significantly (P 5 .004) lower TNCC (median, 4.5 cells/
mL; range, 0–30 cells/mL) compared with dogs that failed
to regain ambulation within 30 days postoperatively (me-
dian, 45 cells/mL; range, 0–104 cells/mL). The percentage
of neutrophils and macrophages were significantly (P 5
.016 and P o .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%, respec-
tively). The MF : M ratio was significantly (P o .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 (me-
dian, 0.7; range, 0–3). CSF protein concentration was
significantly (P 5 .04) lower in dogs that regained ambu-
lation 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 (me-
dian, 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 5 .004)
lower TNCC (median, 5 cells/mL; range, 0–96 cells/mL)
compared with those having a negative outcome
(median, 50 cells/mL; range, 0–104 cells/mL). The percent-
age of macrophages and the MF : M ratio were
significantly (P o .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, re-
spectively) 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 5 .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 signifi-
cant trend of increase in time lag from surgery

Table 3. Cerebrospinal fluid analysis findings and outcome of 54 dogs with acute, nonambulatory thoracolumbar
intervertebral disc disease.
Outcome CSF Protein (mg/dL) TNCC (cells/mL) RBC (cells/mL) Neu (%) Lym (%) Mon (%) MF (%) MF : M ratio
At 30 days postoperative
Negative (14 dogs)
Median 19.5w 45.0w 20.0w 67.0w 7.0 15.0 9.0w 0.7w
Range 2.6–44 0–104 0–2522 28–85 0–20 5–60 0–40 0–3
n 5 14 14 13 13 13 13 13
Positive (40 dogs)
Median 2.4 4.5 0.0 48.0 15.0 28.0 0.0 0.0
Range 0–228.5 0–30 0–490 0–82 0–52 0–95 0–6 0–0.4
n 20 40 40 25 25 25 25 25
At the end of study
Negative (7 dogs)
Median 20.6w 50.0w 10.9 63.5 5.5 11.5 15.5w 1.7w
Range 14.6–44 0–104 0–182 30–85 0–20 5–20 9–40 0–3
n 4 7 7 6 6 6 6 6
Positive (47 dogs)
Median 2.6 5.0 0.0 59.5 13.0 26.5 0.0 0.0
Range 0–228.5 0–96 0–2522 0–82 0–52 0–95 0 12 0–0.7
n 21 47 47 32 32 32 32 32

CSF, cerebrospinal fluid; Lym, lymphocytes; Mon, monocytes; MF, macrophages; Neu, neutrophils; RBC, red blood cells; TNCC, total
nucleated cell count.
w
Statistically significant (P  .05) from the other subgroup within the outcome group.
 CSF Analysis in Disc Disease 851

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.
Cerebrospinal Fluid Total Nucleated Cell Count Level

Normal Mildly Increased Moderately to Markedly

Outcome (o5 cells/mL) n (%) (45 and 20 cells/mL) n (%) Increased (420 cell/mL) n (%)
At 30 days postoperative
Negative 3 (12.0%) 1 (6.7%) 10 (71.4%)
Positive 22 (88.0%) 14 (93.3%) 4 (28.6%)
At the end of study
Negative 1 (4.0%) 0 (0%) 6 (42.9%)
Positive 24 (96.0%) 15 (100%) 8 (57.1%)

to regaining ambulation was observed with higher
TNCC (P o .001), higher percentage of macrophages
(P o .001), and higher MF : M ratio (P o .001) (w2 trend
test; Table 5).
ROC analysis of TNCC yielded an AUC of 0.821 (SE,
0.121; Fig 2A) with an optimal cut-off point for predic-
tion of a negative long-term outcome of 23.3 cells/mL,
corresponding to a sensitivity and specificity of 89 and
85%, respectively. Dogs with TNCC  23.3 cells/mL were
50-fold more likely to regain ambulation compared with
those with TNCC 4 23.3 cells/mL (CI 95%, 5–500; P o
.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 specific-
ity of 100 and 83%, respectively. ROC analysis of
MF : 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 MF : M ratio 0.64 were 7-fold more
likely to have a successful long-term outcome com-
pared with those with an MF : M ratio 4 0.64 (CI 95%,
2–43; P o.01).
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 5 .001) and MF : M ratio (P 5
.001) compared with those presented with absent DPS
that failed to regain ambulation.
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 am-
bulation and TNCC, percentage of CSF macrophages,
and MF : M ratio might potentially aid clinicians in
estimating the postoperative recovery period and the
value of by other therapeutic modalities such as physio-
therapy. The present findings are particularly relevant

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 CSF Protein TNCC RBC Neu Lym Mon MF MF :
Ambulation (mg/dL) (cells/mL) (cells/mL) (%) (%) (%) (%) M ratio
1–5 days (21 dogs)
Median 5.25 3.00 0 59.00 15.00 25.00 0.00 0.00
Range 0–228 0–11 0–490 0–82 0–30 0–70 0–4 0–0.4
n 12 21 21 11 11 11 11 11
6–30 days (19 dogs)
Median 0 7.00 0 45.00 17.00 29.00 0.00 0.00
Range 0–33.5 1–30 0–149 4–62 0–52 0–95 0–6 0–0.2
n 8 19 19 14 14 14 14 14
31–100 days (5 dogs)
Median 2.6 15.40 31.8 60.00 8.00 20.00 4.00 0.29
Range 2.6–2.6 2–96 13–64 28–77 4–20 14–60 0–9 0–0.5
n 1 5 5 5 5 5 5 5
Above 100 days (2 dogs)
Median — 79.00 1261 69.00 7.00 14.50 9.50 0.64
Range — 78, 80 0, 2522 67, 71 4, 10 12, 17 7, 12 0.6–0.7
n 0 2 2 2 2 2 2 2

CSF, cerebrospinal fluid; Lym, lymphocytes; Mon, monocytes; MF, macrophages; Neu, neutrophils; RBC, red blood cells; TNCC, total
nucleated cell count.
852 Srugo et al

Fig 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.

for dogs with thoracolumbar IVDD that present with
absence of DPS, in which the percentage of CSF macro-
phages and the MF : M had prognostic value. To the best
of our knowledge, this is the first description of a poten-
tially 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 post-
injury, 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 in-
flammation may evolve to a more lymphocytic
inflammation later in the course of disease.12 The patho-
physiology 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 dis-
ease.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 dam-
age 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 im-
portant role in the pathophysiology of acute spinal cord
injury: their percentage was higher with increasing sever-
ity of spinal cord injury and in dogs with a negative
outcome, and macrophages were more frequently ob-
served 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 initia-
tion 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

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.
CSF Protein TNCC RBC Neu Lym Mon MF MF :
Outcome (mg/dL) (cells/mL) (cells/mL) (%) (%) (%) (%) M ratio
Negative (7 dogs)
Mean 24.93 54.88 44.41 61.17 7.33 12.33 20.00 1.73
Median 20.55 50.00 10.85 63.50 5.50 11.50 15.50 1.65
Range 14.6–44 0–104 0–182 30–85 0–20 5–20 9–40 0.69–3
n 4.00 7.00 7.00 6.00 6.00 6.00 6.00 6.00
Positive (9 dogs)
Mean 1.30 34.29 297.59 53.63 17.38 24.75 4.25 0.24
Median 1.30 15.40 12.75 63.50 11.00 18.50 3.00 0.07
Range 0–2.6 0–96 0–2522 14–77 4–52 12–60 0–12 0–0.71
n 2.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00
P value1 0.133 0.351 1.000 0.491 0.108 0.059 0.001 0.001

CSF, cerebrospinal fluid; Lym, lymphocytes; Mon, monocytes; MF, macrophages; Neu, neutrophils; RBC, red blood cells; TNCC, total
nucleated cell count.
1
P value of comparison of dogs with positive and negative outcomes.
 CSF Analysis in Disc Disease
protective cytokines that promote neuronal regenera-
tion, wound healing, and tissue repair.27,28 Macrophages
also promote axonal loss through release of pro-inflam-
matory cytokines and proteases, reactive oxygen species,
and nitric oxide formation.25,26,29 Invasion of macro-
phages into the lesion site is promoted by disruption of
the blood-spinal cord barrier and by proinflammatory
cytokines released from the injured spinal cord pa-
renchyma.22,26,30 We assume that the number of macro-
phages at the lesion site in the spinal cord is reflected by
their number in the CSF. It is thus that the high percent-
age of CSF macrophages in of dogs presented with
neurologic grade 6 is positively associated with the
severity of the damage.
The MF : 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 how-
ever, to our knowledge, a ratio has not been determined
previously. A CSF MF : M ratio would be expected to be
a more sensitive indicator of spinal cord injury than the
percentage of macrophages or monocytes alone if macro-
phages are primarily derived from resident CSF
monocytes, whose percentage then would decrease concur-
rent with the increase in macrophages. Although there is
some inherent subjectivity involved in evaluating cell mor-
phology (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
MF : 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 homo-
genous 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 o7
days because time from onset of neurologic signs to sur-
gical decompression is known to influence the prognosis
of dogs with IVDD that present without DPS.4,6 Fur-
thermore, 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 re-
ported 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 MF : M ratio were not
associated with breed type (ie chondrodystrophic or non-
chondrodystrophic).
There are several limitations in the sampling and anal-
ysis 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 re-
sults because of uneven cell distribution, variable cell
853
recovery, and the additional calculations involved. How-
ever, 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 exclu-
sion of several samples in this study. The cut-off of
500 RBCs/mL 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 evi-
dence for noniatrogenic hemorrhage. Furthermore,
presence of up to 13,200 RBC/mL does not appear to sig-
nificantly affect the CSF protein concentration or
TNCC, such that the inclusion of the samples with hem-
orrhage 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 thoraco-
lumbar area compared with cerebellomedullary cistern
samples because of the caudal flow of CSF.10,32 This
might explain the relatively low median TNCC and per-
centage 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 un-
derestimated as well. Finally, the number of CSF samples
available for protein measurement limited the conclu-
sions 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 indica-
tor 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/mL, TNCC was a sensi-
tive and specific predictor of outcome (ie regaining
ambulation) in the present study. However, unlike the
MF : M ratio, the TNCC appeared to provide little ad-
vantage over assessment of the prognosis based solely on
neurologic grade, because all dogs with grades 3–5 even-
tually 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 exam-
ine the association of TNCC and outcome in this
subgroup of dogs.
This study had 2 general limitations. First, its retro-
spective design limited the available data and thus
854 Srugo et al
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 sub-
category 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 in-
cluded 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, con-
clusions 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 MF : M ratio have strong potential as useful
preoperative prognostic indicators of outcome in dogs
with acute thoracolumbar IVDD, especially in paraple-
gic 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 371; 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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