Migration and differentiation of canine bone marrow stromal cells transplanted into the developing mouse brain.

To evaluate whether canine bone marrow stromal cells (BMSCs) can migrate and adopt neural phenotypes in the developing mouse brain we transplanted fluorescently labeled BMSCs into the lateral ventricle of immunocompromised neonatal mice. Most fibroblasts, used as a control, and BMSCs isolated from adult dogs remained around the injection site and exhibited a spindle-shaped appearance. A small number of BMSCs from young dogs were found in the subventricular zone, rostral migratory stream, and olfactory bulbs, and retained expression of neuron marker. Our findings suggest that BMSCs isolated from adult dogs have limited ability of migration and differentiation toward neural cells in the developing brain. Bone marrow of young dogs may contain a primitive stem cell population with neural differentiation capacity.

Nestin-positive spheres derived from canine bone marrow stromal cells generate cells with early neuronal and glial phenotypic characteristics.

Bone marrow stromal cells (BMSCs) isolated from humans and rodents have been shown to generate neural cells under specific culture conditions and after transplantation in the central nervous system. The apparent plasticity of BMSCs has therefore been a target of intensive research in attempt to develop a novel therapy for neurological diseases. Canines sustain neurological disorders (e.g., traumatic spinal cord injury) that closely mirror pathology of those in humans. Therefore, we evaluated neural differentiation properties of canine BMSCs to provide insights into basic characterization of these cells for future neurotransplantation trials in canine patients with neurological disorders. We demonstrate that canine BMSCs form spherical cellular aggregates on anti-adhesive culture substrate in serum-free culture media, which morphologically and phenotypically resemble spherical aggregates of neural progenitor cells, so-called neurospheres. Upon dissociation and subculture on adhesive substrate, canine BMSCs express neuronal (ss capital SHA, Cyrillic-tubulin) and glial (GFAP, A2B5, and CNPase) markers. Formation of spherical aggregates appears to be a critical preceding process for some of the glial marker expression (CNPase and A2B5). However, expression of more mature neuronal (MAP2) and glial (MBP) markers could not be induced with the protocol used in this study. We suggest that induction of canine BMSCs into cells with neural progenitor cell characteristics is possible and that these cells may have the potential for future cellular therapy for neurological disorders.

Detection of oligoclonal bands in cerebrospinal fluid from German Shepherd dogs with degenerative myelopathy by isoelectric focusing and immunofixation.

BACKGROUND: Detection of intrathecal IgG synthesis is important in evaluating inflammatory diseases in the central nervous system. Isoelectric focusing (IEF) is currently the most sensitive method to demonstrate intrathecal IgG synthesis and may have diagnostic value for German Shepherd degenerative myelopathy (GSDM). OBJECTIVE: The objective of this study was to adapt a modified IEF and immunofixation method for the detection of oligoclonal bands in cerebrospinal fluid (CSF) from dogs with GSDM. METHODS: Serum and lumbar CSF samples were collected from 6 German Shepherd dogs clinically diagnosed with GSDM. Samples were also collected from 6 clinically healthy dogs for comparison. The concentration of IgG was measured by quantitative ELISA and the concentration of total protein was measured by the Bradford protein assay. The presence of oligoclonal bands was evaluated by use of modified IEF followed by immunofixation. RESULTS: The concentrations of total protein and IgG, and the IgG/total protein ratio in CSF samples, were not significantly different between GSDM patients and control dogs. Four GSDM patients had oligoclonal bands in their CSF based on IEF-immunofixation. No oligoclonal bands were found in CSF from control dogs. CONCLUSION: The results suggest that the detection of oligoclonal bands by IEF-immunofixation may have diagnostic value for GSDM, and support the idea that humoral immune responses may play a role in the pathogenesis of GSDM.

Teratoma in the cervical spinal cord of a dog.

An 11-year-old, spayed female giant schnauzer was presented for evaluation of chronic, progressive tetraparesis. Diagnostic imaging was consistent with intervertebral disk protrusion, and surgical decompression and stabilization were performed. Postoperatively the dog did not improve, and further imaging suggested an intramedullary mass at the level of the sixth cervical vertebra. The dog was euthanized 7 days after surgery, and a teratoma was found postmortem.

Measurement of myelin basic protein in the cerebrospinal fluid of dogs with degenerative myelopathy.

BACKGROUND: Analysis of cerebrospinal fluid (CSF) is part of a routine clinical workup in veterinary patients when neurologic disease is suspected. However, knowledge of particular protein markers of disease in CSF is limited. The concentration of myelin basic protein (MBP) in CSF is used as a biochemical marker in humans to evaluate demyelinating lesions in the central nervous system (CNS). OBJECTIVE: The purpose of this study was to evaluate an ELISA for determination of MBP concentration in the CSF of German shepherd dogs with degenerative myelopathy (GSDM). METHODS: Cross-reactivity of the anti-human polyclonal antibody used in a commercial ELISA (Active MBP ELISA, Diagnostic Systems Laboratories Inc, Webster, TX, USA) was tested with canine MBP by immunoblotting. CSF samples were collected from both the cisterna magna and the lumbar cistern of 8 clinically healthy control dogs and 8 German shepherd dogs clinically diagnosed with GSDM. MBP concentrations were measured in all CSF samples using the ELISA. RESULTS: The mean MBP concentration in CSF from the lumbar cistern of dogs with GSDM (3.13 -/+ 0.46 ng/mL) was significantly higher than that in the cisterna magna (0.70 -/+ 0.06 ng/mL) and from both cisternal (0.47 -/+ 0.07 ng/mL) and lumbar (0.94 -/+ 0.37 ng/mL) samples from control dogs. CONCLUSION: The MBP ELISA has potential as a supplemental test of CSF to diagnose demyelinating disorders in dogs.

Three-dimensional culture of feline articular chondrocytes in alginate microspheres.

Chondrocytes isolated from proximal femoral articular cartilage from 3 adult cat cadavers were expanded in monolayer culture and subsequently cultured in alginate microspheres for 24 days. Cell proliferation and production of proteoglycans in alginate microspheres were observed during day 18 and 24. Quantification of chondroitin sulfates (CS) by capillary electrophoresis revealed that cultured chondrocytes synthesized CS6 but not CS4. Three-dimensional culture using alginate microspheres is a useful in vitro technique to study proliferation and metabolism of chondrocytes; however, further modifications are needed to apply the technique to feline articular chondrocytes.

High field (4.7 T) magnetic resonance imaging of feline hip joints.

Magnetic resonance imaging (MRI) is a non-invasive technique widely used to investigate degenerative joint disease (DJD). In this study, we obtained magnetic resonance images of feline hip joints, using a high magnetic field MRI unit (4.7 tesla) with proton density (PD)-weighted and T2-weighted fast spin-echo (FSE). PD-weighted FSE provided detailed anatomical images of feline hip joints with superb depiction of subchondral bones of the femoral head and acetabulum. Articular cartilage (AC) was also visualized with PD-weighted and T2-weighted FSE; however, mild AC lesions noted on gross examination were not detectable with these sequences.

Expression of neural markers on bone marrow-derived canine mesenchymal stem cells.

OBJECTIVE: To evaluate cell surface markers of bone marrow-derived canine mesenchymal stem cells (MSCs) by use of flow cytometric analysis and determine whether canine MSCs express proteins specific to neuronal and glial cells. SAMPLE POPULATION: Bone marrow aspirates collected from iliac crests of 5 cadavers of young adult dogs. PROCEDURES: Flow cytometric analysis was performed to evaluate cell surface markers and homogeneity of third-passage MSCs. Neural differentiation of canine MSCs was induced by use of dibutyryl cAMP and methyl-isobutylxanthine. Expressions of neuronal (beta III-tubulin) and glial (glial fibrillary acidic protein [GFAP] and myelin basic protein) proteins were evaluated by use of immunocytochemical and western blot analyses before and after neural differentiation. RESULTS: Third-passage canine MSCs appeared morphologically homogeneous and shared phenotypic characteristics with human and rodent MSCs. Immunocytochemical and western blot analyses revealed that canine MSCs constitutively expressed beta III-tubulin and GFAP. After induction of neural differentiation, increased expression of GFAP was found in all samples, whereas such change was inconsistent in beta III-tubulin expression. Myelin basic protein remained undetectable on canine MSCs for these culture conditions. CONCLUSIONS AND CLINICAL RELEVANCE: Canine bone marrow-derived mononuclear cells yielded an apparently homogeneous population of MSCs after expansion in culture. Expanded canine MSCs constitutively expressed neuron or astrocyte specific proteins. Furthermore, increases of intracellular cAMP concentrations induced increased expression of GFAP on canine MSCs, which suggests that these cells may have the capacity to respond to external signals. Canine MSCs may hold therapeutic potential for treatment of dogs with neurologic disorders.

Temporal dynamic changes in synthesis of chondroitin sulfate isomers in canine articular chondrocyte culture.

To investigate temporal dynamic changes in the synthesis of chondroitin 6-sulfate (CS6) and chondroitin 4-sulfate (CS4) in vitro, normal articular cartilage of femoral heads was harvested from three dogs. Chondrocytes were isolated and cultured in alginate microspheres for 21 days. On days 7, 14 and 21, DNA content was quantified by fluorometric assay using Hoechst 33258. On days 14 and 21, proteoglycans were extracted, and the amounts of CS6 and CS4 were quantified after chondroitinase ABC digestion using capillary electrophoresis. The DNA content and amounts of CS6 and CS4 increased during the culture period. The amounts of CS6 and CS4 divided by DNA content revealed that the synthesis of CS6 was more up-regulated than CS4.

Clinical and necropsy findings associated with increased mortality among American alligators of Lake Griffin, Florida.

From December, 1997, through November, 2000, 306 deaths were documented among adult and subadult American alligators (Alligator mississippiensis) of Lake Griffin, Florida (USA). Some live alligators were lethargic and unresponsive to approach. To determine the cause, we examined ten alligators captured from Lake Griffin between December 1997 and June 1999. Initially, four alligators, three of which were clinically unresponsive, were sacrificed for routine diagnostic necropsy. The other six Lake Griffin alligators, and five control alligators captured from Lake Woodruff National Wildlife Refuge, Florida, where mortality was negligible, were studied extensively by clinical neurologic examination, electromyography, hematology, serum chemical analyses, and blood culture, then sacrificed and necropsied. Samples of brain, spinal cord, peripheral nerves, skeletal muscle, and major internal organs were examined by light microscopy for abnormalities. Samples of nervous tissue also were examined by electron microscopy, and samples of various tissues were collected for toxicologic analyses. Clinical signs included swimming in circles, inability to submerge, lethargy, weakness, unresponsiveness, slow reflexes, dragging the dorsal surfaces of the hind feet, head tilt, and anisocoria. Lake Griffin alligators had significantly lower distal sciatic nerve conduction velocities than Lake Woodruff alligators, and the most severely affected alligators had the lowest velocities; but morphologic abnormalities in peripheral nerves were not evident in most cases. Three severely affected alligators had acute focal necrosis of the torus semicircularis in the midbrain, two had skeletal myofiber atrophy, another had diffuse nonsuppurative encephalomyelitis, and one mildly affected alligator had skeletal myodegeneration. The cause or causes have not yet been identified.

The effects of canine bone marrow stromal cells on neuritogenesis from dorsal root ganglion neurons in vitro.

The present in vitro study was designed to evaluate whether canine bone marrow stromal cells (BMSCs) promote neurite outgrowth from dorsal root ganglion (DRG) neurons. Bone marrow aspirates were collected from iliac crests of three young adult dogs. DRG neurons were cultured on BMSCs, fibroblasts, or laminin substrates. DRG neurons were also cultured in BMSC- or fibroblast-conditioned media. DRG neurons grown on BMSCs extended longer neurites and developed a much more elaborate conformation of branching neurites compared to those on fibroblasts or laminin. Quantitative analysis revealed that these effects were associated with the emergence of increased numbers of primary and branching neurites. The effect appears to be dependent upon cell-cell interactions rather than by elaboration of diffusible molecules. With more extensive investigations into the basic biology of canine BMSCs, their ability for promoting neurite outgrowth may be translated into a novel therapeutic strategy for dogs with a variety of neurological disorders.

The frequency, growth kinetics, and osteogenic/adipogenic differentiation properties of canine bone marrow stromal cells.

Bone marrow stromal cells (BMSCs) have gained considerable attention as a potential source for cell transplantation therapies for a variety of diseases due to their accessibility, proliferative capacity, and multilineage differentiation properties. Canine BMSCs have been shown to contribute to regeneration of osseous tissues, but knowledge about their biology is currently limited. In the present study, we investigated the frequency of adult canine BMSCs in bone marrow, morphological features, growth kinetics, and osteogenic as well as adipogenic differentiation properties in vitro. Our data suggest that adult canine bone marrow contains approximately one BMSC in every 2.38 x 10(4) bone marrow mononucleated cells (0.0042 +/- 0.0019%, n = 5). Primary BMSC cultures consisted of morphologically heterogeneous adherent cell populations from which spindle-shaped cells grew and became the predominant cell type. Growth kinetics patterns were dependent on the initial cell seeding densities, resulting in the highest fold increase at lower cell density. In the presence of osteogenic and adipogenic inducers, primary BMSCs underwent morphological and phenotypic changes characteristic of osteogenic and adipogenic differentiation, respectively. This study provides insights into basic characterization of adult canine BMSCs.

Cerebral phaeohyphomycosis caused by Cladosporium spp. in two domestic shorthair cats.

Two domestic shorthair cats presented for clinical signs related to multifocal central nervous system dysfunction. Both cats had signs of vestibular system involvement and anisocoria, and one had generalized seizure activity. Cerebrospinal fluid analysis revealed a neutrophilic pleocytosis with protein elevation in one cat and pyogranulomatous inflammation in the second. Electroencephalography and brain-stem auditory-evoked potentials in the first cat confirmed cerebral cortical and brain-stem involvement. Euthanasia was performed in both cats, and postmortem diagnoses of phaeohyphomycosis secondary to Cladosporium spp. were made based on histopathology and fungal culture in both cats.