Case Description—A 7-year-old castrated male Great Dane was evaluated because of a 2-month history of fecal incontinence.
Clinical Findings—On the basis of the presence of paraparesis and apparently normal spinal reflexes, the neurologic signs were localized in the region of the third thoracic to the third lumbar spinal cord segments. On the basis of the findings of magnetic resonance imaging, a presumptive diagnosis of a compressive intervertebral disk extrusion with secondary hemorrhage and epidural hematoma formation was made.
Treatment and Outcome—A right-sided hemil-aminectomy was performed (centered at the T13-L1 intervertebral space) to further characterize the lesion and decompress the spinal cord. The histopathologic diagnosis was extruded intervertebral disk material with chronic hemorrhage and inflammation. Three weeks after surgery, there was complete resolution of the dog's fecal incontinence and moderate improvements in its hind limb function.
Clinical Relevance—Thoracolumbar spinal cord injuries can result in upper motor neuron fecal incontinence in ambulatory dogs. Epidural spinal hematomas may develop secondary to intervertebral disk herniations and cause spinal cord compression resulting in neurologic deficits.
OBJECTIVE To develop a spasticity scale for dogs with chronic deficits following severe spinal cord injury (SCI) for use in clinical assessment and outcome measurement in clinical trials.
ANIMALS 20 chronically paralyzed dogs with a persistent lack of hind limb pain perception caused by an acute SCI at least 3 months previously.
PROCEDURES Spasticity was assessed in both hind limbs via tests of muscle tone, clonus, and flexor and extensor spasms adapted from human scales. Measurement of patellar clonus duration and flexor spasm duration and degree was feasible. These components were used to create a canine spasticity scale (CSS; overall score range, 0 to 18). Temporal variation for individual dogs and interrater reliability were evaluated. Gait was quantified with published gait scales, and CSS scores were compared with gait scores and clinical variables. Owners were questioned regarding spasticity observed at home.
RESULTS 20 dogs were enrolled: 18 with no apparent hind limb pain perception and 2 with blunted responses; 5 were ambulatory. Testing was well tolerated, and scores were repeatable between raters. Median overall CSS score was 7 (range, 3 to 11), and flexor spasms were the most prominent finding. Overall CSS score was not associated with age, SCI duration, lesion location, or owner-reported spasticity. Overall CSS score and flexor spasm duration were associated with gait scores.
CONCLUSIONS AND CLINICAL RELEVANCE The CSS could be used to quantify hind limb spasticity in dogs with chronic thoracolumbar SCI and might be a useful outcome measure. Flexor spasms may represent an integral part of stepping in dogs with severe SCI.
OBJECTIVE To identify an optimal technique for isolation, purification, and amplification of Schwann cells (SCs) from biopsy specimens of the dorsal cutaneous branches of the cervical nerves of dogs.
SAMPLE Biopsy specimens of dorsal cervical cutaneous nerves from the cadavers of three 1- to 2-year-old dogs.
PROCEDURES Nerve specimens were dissected, predegenerated, and dissociated to isolate single cells. After culture to enhance SC growth, cells were immunopurified by use of magnetic beads. Cell purity was evaluated by assessing expression of cell surface antigens p75 (to detect SCs) and CD90 (to detect fibroblasts). Effects of various concentrations of recombinant human glial growth factor 2 (rhGGF2) on SC proliferation were tested. Cell doubling time was assessed in SC cultures with selected concentrations of rhGGF2.
RESULTS Mean ± SD wet weight of nerve fascicles obtained from the biopsy specimens was 16.8 ± 2.8 mg. A mean predegeneration period of 8.6 days yielded approximately 6,000 cells/mg of nerve tissue, and primary culture yielded 43,000 cells/mg of nerve tissue in a mean of 11 days, of which 39.9 ± 9.1% expressed p75. Immunopurification with magnetic beads yielded a mean of 85.4 ± 1.9% p75-positive cells. Two passages of subculture with 10μM cytosine arabinoside further enhanced SC purity to a mean of 97.8 ± 1.2% p75-positive cells. Finally, rhGGF2 supplementation at a range of 40 to 100 ng/mL increased the SC proliferation rate up to 3-fold.
CONCLUSIONS AND CLINICAL RELEVANCE SCs could be cultured from biopsy specimens of dorsal cervical cutaneous nerves and purified and expanded to generate adequate numbers for autologous transplants to treat dogs with spinal cord and peripheral nerve injuries.
OBJECTIVE To evaluate gene expression and DNA copy number in adipose tissue-derived stromal cells (ADSCs) and in ADSC-derived neurosphere-like cell clusters (ADSC-NSCs) generated from tissues of chronically paraplegic dogs.
ANIMALS 14 client-owned paraplegic dogs.
PROCEDURES Dorsal subcutaneous adipose tissue (< 1 cm3) was collected under general anesthesia; ADSCs were isolated and cultured. Third-passage ADSCs were cultured in neural cell induction medium to generate ADSC-NSCs. Relative gene expression of mesenchymal cell surface marker CD90 and neural progenitor marker nestin was assessed in ADSCs and ADSC-NSCs from 3 dogs by quantitative real-time PCR assay; expression of these and various neural lineage genes was evaluated for the same dogs by reverse transcription PCR assay. Percentages of cells expressing CD90, nestin, glial fibrillary acidic protein (GFAP), and tubulin β 3 class III (TUJ1) proteins were determined by flow cytometry for all dogs. The DNA copy number stability (in samples from 6 dogs) and neural cell differentiation (14 dogs) were assessed with array-comparative genomic hybridization analysis and immunocytochemical evaluation, respectively.
RESULTS ADSCs and ADSC-NSCs expressed neural cell progenitor and differentiation markers; GFAP and microtubule-associated protein 2 were expressed by ADSC-NSCs but not ADSCs. Relative gene expression of CD90 and nestin was subjectively higher in ADSC-NSCs than in ADSCs. Percentages of ADSC-NSCs expressing nestin, GFAP, and TUJ1 proteins were substantially higher than those of ADSCs. Cells expressing neuronal and glial markers were generated from ADSC-NSCs and had no DNA copy number instability detectable by the methods used.
CONCLUSIONS AND CLINICAL RELEVANCE Results suggested ADSCs can potentially be a safe and clinically relevant autologous source for canine neural progenitor cells. Further research is needed to verify these findings.
Objective—To develop a multiplex polymerase chain
reaction (PCR) assay for the detection of Toxoplasma
gondii and Neospora caninum DNA in canine and
feline biological samples.
Sample Population—Biological samples from 7 cats
with systemic (n = 4) or CNS (3) toxoplasmosis, 6 dogs
with neospora- or toxoplasma-associated encephalitis,
and 11 animals with nonprotozoal disease.
Procedure—Primers for T gondii, N caninum, and the
canine ferritin gene (dogs) or feline histone 3.3 gene
(cats) were combined in a single PCR assay. The DNA
was extracted from paraffin-embedded brain tissue,
CSF, or skeletal muscle. The PCR products with positive
results were cloned, and sequence identity was
Results—Of 7 cats and 4 dogs with immunohistochemical
or serologic evidence of toxoplasmosis, PCR
results were positive for all cats and 3 dogs for T gondii,
and positive for T gondii and N caninum for 1 dog.
Another dog had negative PCR results for both parasites.
Of 2 dogs with immunohistochemical or serologic
evidence of neosporosis, PCR results were positive
for 1 for N caninum and positive for the other for
T gondii. All negative-control samples yielded negative
results for T gondii and N caninum on the PCR assay.
Conclusions and Clinical Relevance—Standard tests
for toxoplasmosis or neosporosis associated with the
CNS rely on serologic, histologic, or immunohistochemical
analysis and can be difficult to interpret. The
multiplex PCR assay with built-in control reactions
could be a complementary clinical tool for the antemortem
diagnosis of toxoplasmosis or neosporosis
associated with the CNS. (Am J Vet Res 2003;64:1507–1513)
Objective—To assess the feasibility of measuring cord dorsum potentials (CDPs) in anesthetized clinically normal dogs after caudal nerve stimulation, determine the intervertebral site of maximum amplitude and best waveform of the CDP, and evaluate the effects of neuromuscular blockade.
Animals—8 male and 4 female dogs (age, 1 to 5 years).
Procedures—Dogs were anesthetized, and CDPs were recorded via needles placed on the dorsal lamina at intervertebral spaces L1–2 through L7–S1. Caudal nerves were stimulated with monopolar electrodes inserted laterally to the level of the caudal vertebrae. Dogs were tested without and during neuromuscular blockade induced with atracurium besylate. The CDP latency and amplitude were determined from the largest amplitude tracings.
Results—CDPs were recorded in 11 of 12 dogs without neuromuscular blockade and in all dogs during neuromuscular blockade. The CDP was largest and most isolated at the L4–5 intervertebral space (3 dogs) or the L5–6 intervertebral space (9 dogs); this site corresponded to the segment of insertion of the first caudal nerve. Onset latencies ranged from 2.0 to 4.7 milliseconds, and there was no effect of neuromuscular blockade on latencies. Amplitudes of the CDPs were highly variable for both experimental conditions.
Conclusions and Clinical Relevance—CDPs were recorded from all dogs tested in the study; neuromuscular blockade was not critical for successful CDP recording but reduced muscle artifact. This technique may be useful as a tool to assess the caudal nerve roots in dogs suspected of having compressive lumbosacral disease or myelopathies affecting the lumbar intumescence.
Objective—To isolate and characterize neural stem and progenitor cell populations in the brain of adult dogs.
Animals—7 healthy adult dogs.
Procedures—Dogs (age, 10 to 60 months) were euthanized for reasons unrelated to the study. The subventricular zone (SVZ) adjacent to the lateral ventricles and subgranular zone (SGZ) of the hippocampus were isolated and used to generate single cell suspensions for nonadherent culture. The resulting primary neurospheres were serially passaged to assess self-renewal capacity. Neurospheres were differentiated by the withdrawal of growth factors and the addition of serum. Differentiated and undifferentiated neurospheres were analyzed via reverse transcriptase PCR assay or immunocytochemical staining for markers of pluripotency and neural lineage.
Results—Neurospheres were generated from the SVZ and SGZ in all dogs. The SVZ generated more primary neurospheres than did the SGZ. Serial passage was successful, although few neurospheres could be generated after the fifth passage. Undifferentiated neurospheres were positive for SOX2, nestin, and glial fibrillary acidic protein (GFAP) and negative for OCT4 and NANOG. After differentiation, GFAP, neuronal class III β-tubulin, and 2′, 3′-cyclic nucleotide 3′-phosphodiesterase–positive progeny were noted migrating out of the neurospheres.
Conclusions and Clinical Relevance—Results suggested the persistence of SOX2-positive, nestin-positive, GFAP-positive, OCT4-negative, and NANOG-negative neural progenitor cells in the SVZ and SGZ regions of mature canine brains, which are capable of producing multiple cell lineages. This study may serve as a basis for future studies investigating the role of these cells in various disease processes, such as neoplasia, or for regenerative purposes.
Objective—To determine long-term (> 6 months) outcome
of dogs with paraplegia and loss of hind limb
deep pain perception (DPP) resulting from intervertebral
disk herniation or trauma.
Procedure—Outcome was determined as successful
or unsuccessful. The association of neuroanatomic
localization, breed, age, weight, sex, and (for dogs
with intervertebral disk herniation) speed of onset of
signs and duration of paraplegia prior to surgery with
outcome was evaluated. Owners were contacted by
telephone to identify long-term health problems.
Results—Nine of 17 dogs with traumatic injuries
were treated, and 2 regained the ability to walk;
none of the 17 dogs regained DPP. Sixty-four of 70
dogs with intervertebral disk herniation underwent
surgery; 9 (14%) were euthanatized within 3 weeks
after surgery (7 because of ascending myelomalacia),
37 (58%) regained DPP and the ability to walk,
7 (11%) regained the ability to walk without regaining
DPP, and 11 (17%) remained paraplegic without
DPP. Outcome was not associated with any of the
factors evaluated, but speed of recovery of ambulation
was significantly associated with body weight
and age. Fifteen (41%) and 12 (32%) dogs that
regained DPP had intermittent fecal and urinary
Conclusions and Clinical Relevance—Results suggested
that the prognosis for paraplegic dogs without
DPP because of trauma was guarded, while dogs
with disk herniation had a better chance of recovering
motor function. A third of the dogs that recovered
motor function had intermittent incontinence.
Persistent loss of DPP did not preclude recovery of
motor function, but such dogs remained incontinent.
(J Am Vet Med Assoc 2003;222:762–769)
Objective—To determine historical, physical examination,
hematologic, and serologic findings in dogs
with Ehrlichia ewingii infection.
Procedure—In all dogs, infection with E ewingii was
confirmed with a polymerase chain reaction (PCR)
assay. Follow-up information and clarification of information
recorded in the medical records was obtained
by telephone interviews and facsimile correspondence
with referring veterinarians and owners.
Results—Fever and lameness were the most common
findings with each occurring in 8 dogs. Five dogs
had neurologic abnormalities including ataxia, paresis,
proprioceptive deficits, anisocoria, intention tremor,
and head tilt. Neutrophilic polyarthritis was identified
in 4 dogs. No clinical signs were reported in 3 dogs.
The predominant hematologic abnormality was
thrombocytopenia, which was identified in all 12 dogs
for which a platelet count was available. Reactive lymphocytes
were seen in 5 of 13 dogs. Concurrent
infection with another rickettsial organism was identified
in 4 dogs. Of the 13 dogs tested, 7 were seroreactive
to E canis antigens. Morulae consistent with E
ewingii infection were identified in neutrophils in 8
dogs. Treatment with doxycycline, with or without
prednisone, resulted in a rapid, favorable clinical
response in the 9 dogs for which follow-up information
Conclusions and Clinical Relevance—Results suggest
that PCR testing for E ewingii infection should
be considered in dogs with fever, neutrophilic polyarthritis,
unexplained ataxia or paresis, thrombocytopenia,
or unexplained reactive lymphocytes, and in
dogs with clinical signs suggestive of ehrlichiosis that
are seronegative for E canis. Following treatment
with doxycycline, the prognosis for recovery is good.
(J Am Vet Med Assoc 2003;222:1102–1107)