Fetal bone engraftment reconstitutes the immune system in pigs with severe combined immunodeficiency.

Genetic modification of genes such as recombination activating gene 2 (RAG2) or interleukin-2 receptor-γ (IL2RG) results in pigs exhibiting severe combined immunodeficiency (SCID). Pigs presenting a SCID phenotype are important animal models that can be used to establish xenografts and to study immune system development and various immune-related pathologies. However, due to their immunocompromised nature, SCID pigs have shortened lifespans and are notoriously difficult to maintain. The failure-to-thrive phenotype makes the establishment of a breeding population of RAG2/IL2RG double-knockout pigs virtually impossible. Here, to overcome this limitation, we investigated whether reconstituting the immune system of SCID piglets with a fetal bone allograft would extend their lifespan. Following intramuscular transplantation, allografts gave rise to lymphocytes expressing T cell (CD3, CD4 and CD8), B cell (CD79α) and natural killer cell (CD335) lineage markers, which were detected in circulation as well in the spleen, liver, bone marrow and thymic tissues. The presence of lymphocytes indicates broad engraftment of donor cells in the recipient SCID pigs. Unlike unreconstituted SCID pigs, the engrafted animals thrived and reached puberty under standard housing conditions. This study demonstrates a novel method to extend the survival of SCID pigs, which may improve the availability and use of SCID pigs as a biomedical animal model.

Case report: Naturally occurring neurogenic stunned myocardium in a dog secondary to status epilepticus.

A 4-year-old male neutered Boston Terrier was presented with status epilepticus. He was diagnosed with idiopathic epilepsy and hospitalized with supportive care. During hospitalization, the patient developed both supraventricular and ventricular arrhythmias as well as focal left ventricular dyskinesis. Cardiac troponin I was significantly increased, which was supportive of myocardial damage. Neurogenic stunned myocardium was suspected, and the patient was treated and responded to esmolol. Follow-up echocardiography demonstrated the resolution of the ventricular dyskinesia. This report describes the clinical presentation, diagnostic findings, treatment, management, and outcome of the first reported case of naturally occurring neurogenic stunned myocardium in a dog. Electrocardiogram monitoring, cardiac troponin I, and echocardiography should be considered in patients presenting with seizure activity, especially when exhibiting cluster seizures or in status epilepticus.

Atlantoaxial joint stabilization using patient-specific 3-D-printed drill guides and 3-D-printed titanium plates or polymethyl methacrylate is effective in toy-breed dogs.

OBJECTIVE: To report the clinical outcomes in toy-breed dogs with atlantoaxial instability (AAI) stabilized with patient-specific 3-D-printed titanium plates or polymethyl methacrylate (PMMA), both with the assistance of 3-D-printed drill guides. ANIMALS: 15 client-owned dogs undergoing surgical treatment for AAI between January 1, 2020, and October 31, 2022. METHODS: The clinical characteristics, diagnostic images, and neurological outcomes of 15 dogs treated for AAI using 3-D-printing technology were reviewed. Postoperative CT images were examined to evaluate the screw placement accuracy in the atlas and axis. Clinical outcomes, including postoperative neurological improvement and screw loosening, were evaluated in dogs treated with a patient-specific titanium plate and those treated with PMMA. RESULTS: Patient-specific titanium plates (7 dogs) and PMMA (8 dogs) were used for AAI stabilization. The mean follow-up period was 15.2 months (range 7 to 22 months). A reduction of the axis without vertebral canal violation was confirmed on postoperative CT in 14 dogs. The mean deviation from the preoperative planning ranged from 0.30 to 1.27 mm at the insertion and exit points of 84 screws using this method. The neurological grade had improved in each dog postoperatively and at the final follow-up. Screw loosening was noted in 4 dogs in the titanium plates groups without neurological deterioration. CLINICAL RELEVANCE: Patient-specific 3-D-printed drill guides and titanium plates or PMMA are effective for AAI stabilization in toy-breed dogs, providing accurate guidance.

Neural stem/progenitor cells from adult canine cervical spinal cord have the potential to differentiate into neural lineage cells.

BACKGROUND: • Neural stem/progenitor cells (NSPCs) are multipotent self-renewing cells that can be isolated from the brain or spinal cord. As they need to be isolated from neural tissues, it is difficult to study human NSPCs. To facilitate NSPC research, we attempted to isolate NSPCs from dogs, as dogs share the environment and having many similar diseases with humans. We collected and established primary cultures of ependymal and subependymal cells from the central canal of the cervical spinal cord of adult dogs. To isolate pure NSPCs, we employed the monolayer culture and selective medium culture methods. We further tested the ability of the NSPCs to form neurospheres (using the suspension culture method) and evaluated their differentiation potential. RESULTS: • The cells had the ability to grow as cultures for up to 10 passages; the growth curves of the cells at the 3rd, 6th, and 9th passages showed similar patterns. The NSPCs were able to grow as neurospheres as well as monolayers, and immunostaining at the 3rd, 6th, and 9th passages showed that these cells expressed NSPC markers such as nestin and SOX2 (immunofluorescent staining). Monolayer cultures of NSPCs at the 3rd, 6th, and 9th passages were cultured for approximately 14 days using a differentiation medium and were observed to successfully differentiate into neural lineage and glial cells (astrocytes, neurons, and oligodendrocytes) at all the three passages tested. CONCLUSION: • It is feasible to isolate and propagate (up to at least 10 passages) canine cervical spinal cord-derived NSPCs with the capacity to differentiate into neuronal and glial cells. To the best of our knowledge this is the first study to successfully isolate, propagate, and differentiate canine NSPCs derived from cervical spinal cord in the adult canine, and we believe that these cells will contribute to the field of spinal cord regeneration in veterinary and comparative medicine.

Time course and prognostic value of serum GFAP, pNFH, and S100ß concentrations in dogs with complete spinal cord injury because of intervertebral disc extrusion.

BACKGROUND: A noninvasive biomarker is needed to predict recovery from severe spinal cord injury (SCI) because of thoracolumbar intervertebral disc extrusion (TL-IVDE). Proteins released from neural and glial cells can be detected in the blood and show promise as prognostic tools, but their concentration is influenced by time after injury. HYPOTHESIS/OBJECTIVES: Serum concentrations of glial fibrillary acidic protein (GFAP), phosphorylated neurofilament heavy chain (pNFH), and S100ß will follow different time courses; measurement of combinations of these proteins will predict outcome. ANIMALS: Thirty-one dogs with TL-IVDE causing paralysis with no pain perception. METHODS: Prospective study. Serum samples were taken at presentation and intervals over 56 days and banked at -80°C. Glial fibrillary acidic protein, pNFH, and S100ß concentrations were measured using ELISA tests and plotted against time from onset of nonambulatory status. Outcome was established at 6 months. The association between biomarker concentration and outcome was examined using logistic regression, receiver operator characteristics curve analysis, and model development. RESULTS: Thirty-one dogs participated, 3/31 (10%) developed progressive myelomalacia and 19/31 (62%) recovered ambulation. Glial fibrillary acidic protein and S100ß concentrations rose for the first 1 to 3 days, and were undetectable by 14 and 28 days, respectively. Phosphorylated neurofilament heavy chain concentrations peaked at 14 days and were detectable at 56 days. Glial fibrillary acidic protein concentrations in the first 72 hours after onset of nonambulatory status predicted recovery with an accuracy of 76.7%-89% depending on sample timing. CONCLUSIONS AND CLINICAL IMPORTANCE: Serum GFAP concentrations can be used to predict outcome in clinically complete SCI. A rapid inexpensive bedside test is needed.

Evaluation of gene expression and DNA copy number profiles of adipose tissue-derived stromal cells and consecutive neurosphere-like cells generated from dogs with naturally occurring spinal cord injury.

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 ß 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.

Generation of pure cultures of autologous Schwann cells by use of biopsy specimens of the dorsal cutaneous branches of the cervical nerves of young adult dogs.

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.

Gait scoring in dogs with thoracolumbar spinal cord injuries when walking on a treadmill.

BACKGROUND: An inexpensive method of generating continuous data on hind limb function in dogs with spinal cord injury is needed to facilitate multicentre clinical trials. This study aimed to define normal fore limb, hind limb coordination in dogs walking on a treadmill and then to determine whether reliable data could be generated on the frequency of hind limb stepping and the frequency of coordinated stepping in dogs with a wide range of severities of thoracolumbar spinal cord injury. RESULTS: Sixty-nine neurologically normal dogs of different body sizes including seven lame dogs were videotaped walking on the treadmill without prior training and all used the lateral gait of right fore, left hind, left fore, right hind (RF-LH-LF-RH). Severely paraparetic dogs were able to walk on the treadmill for a minimum of 75 seconds, scoring of which generated data representative of function in animals with extremely variable gaits. Fifty consecutive stepping cycles were scored by three observers in 18 dogs with a wide range of disability due to acute thoracolumbar spinal cord injury using a stepping score (hind limb steps/fore limb steps ×100), and a coordination score (coordinated hind limb steps/total hind limb steps ×100). Dogs were also scored using a previously validated ordinal open field score (OFS). Inter- and intraobserver agreement was high as assessed with Cronbach's alpha test for internal reliability. The stepping and coordination scores were significantly correlated to each other and to the OFS. CONCLUSIONS: Dogs with naturally occurring spinal cord injury can walk on a treadmill without prior training and their hind limb function can be scored reliably using a stepping score and coordination score. The only requirements for data acquisition are a treadmill and appropriately positioned video camera and so the system can be used in multicentre clinical trials to generate continuous data on neurologic recovery in dogs.

Potassium channel antagonists 4-aminopyridine and the T-butyl carbamate derivative of 4-aminopyridine improve hind limb function in chronically non-ambulatory dogs; a blinded, placebo-controlled trial.

4-Aminopyridine (4-AP) blocks voltage gated potassium channels, restoring conduction to demyelinated axons and improving function in demyelinating conditions, but its use is associated with adverse effects and benefit in spinal cord injury is limited. Derivatives of 4-AP have been developed to improve clinical efficacy while reducing toxicity. We compared the therapeutic effects of orally administered 4-AP and its t-butyl carbamate derivative (t-butyl) with placebo in dogs that had suffered an acute spinal cord injury that left them chronically paralyzed. Nineteen dogs were entered into the trial, conducted in two-week treatment blocks starting with placebo, followed by random assignment to 4-AP or t-butyl, a washout and then the opposite medication followed by placebo. Investigators and owners were blinded to treatment group. Primary outcome measures included open field gait score (OFS), and treadmill based stepping score and regularity index, with additional secondary measures also considered. Thirteen of 19 dogs completed the protocol. Two were euthanized due to unrelated heath problems, two developed side effects and two were unable to complete for unrelated reasons. Dogs showed significant improvement in supported stepping score (from 17.39 to 37.24% with 4-AP; 16.85 to 29.18% with t-butyl p<0.0001) and OFS (from 3.63 to 4.73 with 4-AP; 3.78 to 4.45 with t-butyl, p = 0.005). Response was individually variable and most dramatic in three dogs that were able to walk without support with treatment. No significant difference was found between 4-AP and t-butyl. No adverse effects were reported with t-butyl but gastrointestinal upset and seizures were observed in two dogs with 4-AP. In conclusion, both 4-AP and t-butyl significantly improved supported stepping ability in dogs with chronic spinal cord injury with no adverse effects noted with t-butyl. Drug response varied widely between individuals, highlighting the need to understand the factors that influence canine and human patients' response to therapy.

Alternating current electric fields of varying frequencies: effects on proliferation and differentiation of porcine neural progenitor cells.

Application of sinusoidal electric fields (EFs) has been observed to affect cellular processes, including alignment, proliferation, and differentiation. In the present study, we applied low-frequency alternating current (AC) EFs to porcine neural progenitor cells (pNPCs) and investigated the effects on cell patterning, proliferation, and differentiation. pNPCs were grown directly on interdigitated electrodes (IDEs) localizing the EFs to a region accessible visually for fluorescence-based assays. Cultures of pNPCs were exposed to EFs (1 V/cm) of 1 Hz, 10 Hz, and 50 Hz for 3, 7, and 14 days and compared to control cultures. Immunocytochemistry was performed to evaluate the expression of neural markers. pNPCs grew uniformly with no evidence of alignment to the EFs and no change in cell numbers when compared with controls. Nestin expression was shown in all groups at 3 and 7 days, but not at 14 days. NG2 expression was low in all groups. Co-expression of glial fibrillary acidic protein (GFAP) and TUJ1 was significantly higher in the cultures exposed to 10- and 50-Hz EFs than the controls. In summary, sinusoidal AC EFs via IDEs did not alter the alignment and proliferation of pNPCs, but higher frequency stimulation appeared to delay differentiation into mature astrocytes.

Growth requirements and chromosomal instability of induced pluripotent stem cells generated from adult canine fibroblasts.

In mice and humans, it has been shown that embryonic and adult fibroblasts can be reprogrammed into pluripotency by introducing 4 transcription factors, Oct3/4, Klf4, Sox2, and c-Myc (OKSM). Here, we report the derivation of induced pluripotent stem cells (iPSCs) from adult canine fibroblasts by retroviral OKSM transduction. The isolated canine iPSCs (ciPSCs) were expanded in 3 different culture media [fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), or FGF2 plus LIF]. Cells cultured in both FGF2 and LIF expressed pluripotency markers [POU5F1 (OCT4), SOX2, NANOG, and LIN28] and embryonic stem cell (ESC)-specific genes (PODXL, DPPA5, FGF5, REX1, and LAMP1) and showed strong levels of alkaline phosphatase expression. In vitro differentiation by formation of embryoid bodies and by directed differentiation generated cell derivatives of all 3 germ layers as confirmed by mRNA and protein expression. In vivo, the ciPSCs created solid tumors, which failed to reach epithelial structure formation, but expressed markers for all 3 germ layers. Array comparative genomic hybridization and chromosomal fluorescence in situ hybridization analyses revealed that while retroviral transduction per se did not result in significant DNA copy number imbalance, there was evidence for the emergence of low-level aneuploidy during prolonged culture or tumor formation. In summary, we were able to derive ciPSCs from adult fibroblasts by using 4 transcription factors. The isolated iPSCs have similar characteristics to ESCs from other species, but the exact cellular mechanisms behind their unique co-dependency on both FGF2 and LIF are still unknown.

Isolation and characterization of neural progenitor cells from adult canine brains.

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 ß-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.

The cutaneous trunci muscle reflex: a predictor of recovery in dogs with acute thoracolumbar myelopathies caused by intervertebral disc extrusions.

OBJECTIVE: To determine whether changes in the cutaneous trunci muscle (CTM) reflex are an early predictor of outcome in dogs with severe acute thoracolumbar intervertebral disc extrusion (IVDE). STUDY DESIGN: Multicenter prospective cohort study. ANIMALS: Dogs (n = 36) with acute IVDE causing paraplegia, loss of nociception in pelvic limbs and tail, and an abnormal CTM reflex postoperatively. METHODS: The caudal border of the CTM reflex was established 24 hours after surgery and at discharge, and was reported as moving cranially, caudally, or staying static. Dogs were re-evaluated at 12-20 weeks and at 7-36 months postoperatively. Outcome was classified as improved or unimproved, successful or unsuccessful, and ascending myelomalacia or not, and compared with early movement of the CTM reflex by construction of contingency tables and performing a Fisher's exact test. RESULTS: By discharge (mean, 4.7 days; SD = 2.10), CTM reflex progression was caudal in 19 dogs, static in 11, and cranial in 6. Five of 6 dogs with cranial movement developed ascending myelomalacia (P < .0001). Seventeen of 19 dogs with caudal movement showed an improvement by 12-20 weeks (P = .0046) and none developed ascending myelomalacia (P = .0013). CONCLUSIONS: Postoperative changes of the caudal border of the CTM reflex are an early indicator of outcome in dogs with severe acute IVDE. Cranial movement of the CTM reflex is significantly associated with the development of ascending myelomalacia. Caudal movement is significantly associated with improvement, but not associated with a long-term successful outcome.

The sensory field and repeatability of the cutaneous trunci muscle reflex of the dog.

OBJECTIVE: To describe the cutaneous trunci muscle (CTM) reflex in dogs. STUDY DESIGN: Prospective descriptive study. ANIMALS: Normal dogs (n = 155) and 10 dogs with thoracolumbar myelopathies. METHODS: The CTM reflex caudal border was assessed from the ilial crests moving cranially until a CTM contraction was elicited. The lateral borders were evaluated at 4 levels and the distance from the midline to the lateral border was expressed asa percentage of the trunk hemicircumference. The caudal border was assessed in 10 dogs with myelopathies by 4 different observers and by 1 observer on 3 occasions;the inter- and intraobserver kappa coefficient was calculated. RESULTS: The CTM reflex was elicited in all dogs. Its caudal border was at L5 or L6 in 153 dogs and at L1 and L3 in 2 dogs. The lateral field of the reflex occupied>50%of the hemicircumference of the trunk at each level tested. The mean difference in measurement of the reflex caudal border was 0.55 cm between observers and 0.28 cm for the same observer. The inter- and intraobserver kappa coefficient was 0.67 and 0.87, respectively. CONCLUSIONS: The CTM reflex is elicited caudal to L5 in most normal dogs, and the lateral sensory field extends to 50% or more of the circumference of the trunk.Inter- and intraobserver differences in assessment of the caudal border are low.

Development of a model of sacrocaudal spinal cord injury in cloned Yucatan minipigs for cellular transplantation research.

Research into transplantation strategies to treat spinal cord injury (SCI) is frequently performed in rodents, but translation of results to clinical patients can be poor and a large mammalian model of severe SCI is needed. The pig has been considered an optimal model species in which to perform preclinical testing, and the Yucatan minipig can be cloned successfully utilizing somatic cell nuclear transfer (SCNT). However, induction of paralysis in pigs poses significant welfare and nursing challenges. The present study was conducted to determine whether Yucatan SCNT clones could be used to develop an SCI animal model for cellular transplantation research. First, we demonstrated that transection of the sacrocaudal spinal cord in Yucatan SCNT clones produces profound, quantifiable neurological deficits restricted to the tail. We then established that neurospheres could be isolated from brain tissue of green fluorescence protein (GFP) transfected SCNT clones. Finally, we confirmed survival of transplanted GFP-expressing neural stem cells in the SCI lesion and their differentiation into glial and neuronal lineages for up to 4 weeks without immunosuppression. We conclude that this model of sacrocaudal SCI in Yucatan SCNT clones represents a powerful research tool to investigate the effect of cellular transplantation on axonal regeneration and functional recovery.

Generation and characterization of neurospheres from canine adipose tissue-derived stromal cells.

Adipose tissue-derived stromal cells (ADSCs) have been identified as a powerful stem cell source for cellular transplantation therapy. The dog is increasingly used as a model of human neurological disease; however, few studies have reported induction of canine ADSCs to neural lineages. We characterized canine ADSCs and investigated whether they could be induced to differentiate into neural lineages. Subcutaneous adipose tissue collected from the dorsal epaxial region of adult dogs aged from 1 to 6 years was cultured to produce ADSCs that were then induced to neural lineages. RT-PCR, flow cytometry, and immunocytochemistry were performed to characterize these cell populations. Morphologically fibroblast-like ADSCs were isolated and had similar characteristics to mesenchymal stem cells. Under neurogenic conditions containing basic fibroblast growth factor and epidermal growth factor, ADSCs formed spherical cellular aggregates that resembled neurospheres. RT-PCR confirmed expression of Sox2 and CD90 by these aggregates. Expression of neural stem/progenitor markers (Nestin, Sox2, Vimentin) and neural lineage markers (A2B5, GFAP, Tuj1) was shown on immunocytochemistry. After differentiation, 60% of the cells were Tuj1 positive. In conclusion, we isolated and generated neural progenitor cells from canine ADSCs. ADSCs have potential for future autologous cell transplantation therapy for neurological disorders.

Functional recovery and neural differentiation after transplantation of allogenic adipose-derived stem cells in a canine model of acute spinal cord injury.

In this study, we evaluated if the implantation of allogenic adipose-derived stem cells (ASCs) improved neurological function in a canine spinal cord injury model. Eleven adult dogs were assigned to three groups according to treatment after spinal cord injury by epidural balloon compression: C group (no ASCs treatment as control), V group (vehicle treatment with PBS), and ASC group (ASCs treatment). ASCs or vehicle were injected directly into the injured site 1 week after spinal cord injury. Pelvic limb function after transplantation was evaluated by Olby score. Magnetic resonance imaging, somatosensory evoked potential (SEP), histopathologic and immunohistichemical examinations were also performed. Olby scores in the ASC group increased from 2 weeks after transplantation and were significantly higher than C and V groups until 8 weeks (p < 0.05). However, there were no significant differences between the C and V groups. Nerve conduction velocity based on SEP was significantly improved in the ASC group compared to C and V groups (p < 0.05). Positive areas for Luxol fast blue staining were located at the injured site in the ASC group. Also, GFAP, Tuj-1 and NF160 were observed immunohistochemically in cells derived from implanted ASCs. These results suggested that improvement in neurological function by the transplantation of ASCs in dogs with spinal cord injury may be partially due to the neural differentiation of implanted stem cells.

Implantation of canine umbilical cord blood-derived mesenchymal stem cells mixed with beta-tricalcium phosphate enhances osteogenesis in bone defect model dogs.

This study was performed to evaluate the osteogenic effect of allogenic canine umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) mixed with beta-tricalcium phosphate (beta-TCP) in orthotopic implantation. Seven hundred milligrams of beta-TCP mixed with 1 x 10(6) UCB-MSCs diluted with 0.5 ml of saline (group CM) and mixed with the same volume of saline as control (group C) were implanted into a 1.5 cm diaphyseal defect and wrapped with PLGC membrane in the radius of Beagle dogs. Radiographs of the antebrachium were made after surgery. The implants were harvested 12 weeks after implantation and specimens were stained with H&E, toluidine blue and Villanueva-Goldner stains for histological examination and histomorphometric analysis of new bone formation. Additionally, UCB-MSCs were applied to a dog with non-union fracture. Radiographically, continuity between implant and host bone was evident at only one of six interfaces in group C by 12 weeks, but in three of six interfaces in group CM. Radiolucency was found only near the bone end in group C at 12 weeks after implantation, but in the entire graft in group CM. Histologically, bone formation was observed around beta-TCP in longitudinal sections of implant in both groups. Histomorphometric analysis revealed significantly increased new bone formation in group CM at 12 weeks after implantation (p < 0.05). When applied to the non-union fracture, fracture healing was identified by 6 weeks after injection of UCB-MSCs. The present study indicates that a mixture of UCB-MSCs and beta-TCP is a promising osteogenic material for repairing bone defects.

Cerebellar vermian hypoplasia in a Cocker Spaniel.

An eight-week-old female Cocker Spaniel was presented with ataxia, dysmetria and intention tremor. At 16 weeks, the clinical signs did not progress. Investigation including imaging studies of the skull and cerebrospinal fluid analysis were performed. The computed tomography revealed a cyst-like dilation at the level of the fourth ventricle associated with vermal defect in the cerebellum. After euthanasia, a cerebellar hypoplasia with vermal defect was identified on necropsy. A polymerase chain reaction amplification of cerebellar tissue revealed the absence of an in utero parvoviral infection. Therefore, the cerebellar hypoplasia in this puppy was consistent with diagnosis of primary cerebellar malformation comparable to Dandy-Walker syndrome in humans.

Transplantation of canine umbilical cord blood-derived mesenchymal stem cells in experimentally induced spinal cord injured dogs.

This study was to determine the effects of allogenic umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) and recombinant methionyl human granulocyte colony-stimulating factor (rmhGCSF) on a canine spinal cord injury model after balloon compression at the first lumbar vertebra. Twenty-five adult mongrel dogs were assigned to five groups according to treatment after a spinal cord injury: no treatment (CN); saline treatment (CP); rmhGCSF treatment (G); UCB-MSCs treatment (UCB-MSC); co-treatment (UCBG). The UCBMSCs isolated from cord blood of canine fetuses were prepared as 10(6) cells/150 microl saline. The UCB-MSCs were directly injected into the injured site of the spinal cord and rmhGCSF was administered subcutaneously 1 week after the induction of spinal cord injury. The Olby score, magnetic resonance imaging, somatosensory evoked potentials and histopathological examinations were used to evaluate the functional recovery after transplantation. The Olby scores of all groups were zero at the 0-week evaluation. At 2 week after the transplantation, the Olby scores in the groups with the UCB-MSC and UCBG were significantly higher than in the CN and CP groups. However, there were no significant differences between the UCB-MSC and UCBG groups, and between the CN and CP groups. These comparisons remained stable at 4 and 8 week after transplantation. There was significant improvement in the nerve conduction velocity based on the somatosensory evoked potentials. In addition, a distinct structural consistency of the nerve cell bodies was noted in the lesion of the spinal cord of the UCB-MSC and UCBG groups. These results suggest that transplantation of the UCB-MSCs resulted in recovery of nerve function in dogs with a spinal cord injury and may be considered as a therapeutic modality for spinal cord injury.