Antegrade and retrograde CT urethrography with virtual urethroscopy in a dog with urethral narrowing after bilateral triple pelvic osteotomy.

An 8-month-old, castrated male Golden Retriever was unable to urinate without catheterization after a single-session bilateral triple pelvic osteotomy. To determine the cause, a retrograde urethrography was performed, but the results were equivocal. Antegrade (voiding by abdominal compression with heavy material) and retrograde CT urethrography were performed with virtual urethroscopy and revealed that the urethral diameter was narrowed near the pubic bone remnants due to pelvic canal narrowing. After corrective surgery, the patient was able to urinate normally. A combination of antegrade and retrograde CT urethrography with virtual urethroscopy was helpful for guiding surgical decision-making in this patient.

Ectopic Cushing's syndrome associated with a pheochromocytoma in a dog: a case report.

BACKGROUND: Ectopic Cushing's syndrome (ECS) associated with malignant tumors, such as small cell lung carcinoma, bronchial carcinoids, and pheochromocytoma, has been reported in human medicine. However, ECS related to pheochromocytoma has not been reported in dogs. CASE PRESENTATION: An 11-year-old castrated, male Scottish terrier was diagnosed with a left adrenal mass. Cushing's syndrome was suspected based on clinical signs, including pot belly, polyuria, polydipsia, bilateral alopecia, recurrent pyoderma, and calcinosis cutis. Cushing's syndrome was diagnosed on the basis of consistent clinical signs and repeated adrenocorticotropic hormone (ACTH) stimulation tests. In addition, tests for fractionated plasma metanephrine/normetanephrine suggested a pheochromocytoma. Unilateral adrenalectomy was performed after medical management with trilostane and phenoxybenzamine. Histopathology confirmed a diagnosis of pheochromocytoma without cortical lesions. After surgery, fractionated metanephrine/normetanephrine and the findings of low-dose dexamethasone suppression and ACTH stimulation tests were within the normal ranges without any medication. There were no clinical signs or evidence of recurrence and metastasis on thoracic and abdominal X-rays and ultrasonography up to 8 months after surgery. CONCLUSIONS: Pheochromocytoma should be considered a differential diagnosis for dogs with Cushing's syndrome with an adrenal tumor. A good prognosis can be expected with prompt diagnosis and surgical intervention.

Cryopreservation of heat-shocked canine adipose-derived mesenchymal stromal cells with 10% dimethyl sulfoxide and 40% serum results in better viability, proliferation, anti-oxidation, and in-vitro differentiation.

Cryopreserved canine adipose-derived mesenchymal stromal cells (Ad-MSCs) can be used instantly in dogs for clinical uses. However, cryopreservation results in a reduction of the cellular viability, proliferation, and anti-oxidation of post-thawed Ad-MSCs. Therefore, there is a need for in-vitro procedure to improve post-thawed Ad-MSCs' viability, proliferation, anti-oxidation, and differentiation capacity. In this study, fresh-Ad-MSCs were activated with heat shock, hypoxia (5% O2), or hypoxia (5% O2) + heat shock treatments. The results showed that compared to the other treatments, heat shock significantly improved the proliferation rate, anti-oxidation, heat shock proteins and growth factors expressions of canine-fresh-Ad-MSCs. Consequently, fresh-Ad-MSCs were heat-shocked and then cryopreserved with different combinations of dimethyl sulfoxide (Me2SO) and fetal bovine serum (FBS) to determine the combination that could effectively preserve the cellular viability, proliferation, anti-oxidation and differentiation capacity of Ad-MSCs after cryopreservation. We found that C-HST-Ad-MSCs cryopreserved with 10% Me2SO + 40% FBS presented significantly (p < 0.05) improved cellular viability, proliferation rate, anti-oxidant capacity, and differentiation potential as compared to C-HST-Ad-MSCs cryopreserved with 1% Me2SO + 10% FBS or 1% Me2SO alone or control. We concluded, heat shock treatment is much better to enhance the characteristics of fresh-Ad-MSCs than other treatments, moreover, C-HST-Ad-MSCs in 10% Me2SO + 40% FBS showed better results compared to other cryopreserved groups. However, future work is required to optimize the expression of heat shock proteins, which would further improve the characteristics of fresh- and cryopreserved-HST-Ad-MSCs and reduce the dependency on Me2SO and FBS.

Changes in pre- and postoperative serum leptin concentrations in dogs with gallbladder mucocele and cholelithiasis.

BACKGROUND: Leptin has been shown to have various physiological and pathological roles in the canine gallbladder. In this study, we performed pre- and postoperative short-term follow-up analyses to confirm changes in serum leptin levels before and after cholecystectomy due to gallbladder mucocele (GBM) or cholelithiasis in dogs. RESULTS: Twenty-six cholecystectomized dogs (GBM: n = 14; cholelithiasis: n = 12) for prophylactic or clinical symptom relief were enrolled in the present study. Dogs were subgrouped according to clinical symptoms and prognosis after surgery as follows: 1) asymptomatic group (n = 13), 2) recovery group (n = 8), and 3) death group (n = 5). Liver enzymes, total bilirubin, lipid profiles, and leptin concentrations were determined from sera on the pre-operative day and at 1, 3, and 7 days postoperation. Serum leptin concentrations were gradually but significantly decreased in the asymptomatic group (p = 0.008, 0.004, and 0.004 on days 1, 3, and 7, respectively, compared with that before surgery) and the recovery group (p = 0.048 and 0.048 on days 3 and 7, respectively, compared with that before surgery). However, in the death group, leptin concentrations did not differ significantly over time (p = 0.564). Additionally, serum leptin levels in the recovery group (p = 0.006) and death group (p = 0.021) were significantly higher than those in the asymptomatic group. Liver enzymes and total bilirubin (T-Bil) were significantly decreased only in the recovery group, particularly on day 7. In the asymptomatic group, liver enzymes and T-Bil were not changed significantly over time, and in the death group, only T-Bil was significantly decreased on day 7. Total cholesterol and triglyceride levels were not significantly decreased over time in all groups. CONCLUSIONS: These results indicate that leptin is a potential biomarker reflecting the severity and prognosis of GBM and cholelithiasis both before and after cholecystectomy in dogs.

Evaluation of Mesenchymal Stem Cell Sheets Overexpressing BMP-7 in Canine Critical-Sized Bone Defects.

The aim of this study was to investigate the in vitro osteogenic capacity of bone morphogenetic protein 7 (BMP-7) overexpressing adipose-derived (Ad-) mesenchymal stem cells (MSCs) sheets (BMP-7-CS). In addition, BMP-7-CS were transplanted into critical-sized bone defects and osteogenesis was assessed. BMP-7 gene expressing lentivirus particles were transduced into Ad-MSCs. BMP-7, at the mRNA and protein level, was up-regulated in BMP-7-MSCs compared to expression in Ad-MSCs. Osteogenic and vascular-related gene expressions were up-regulated in BMP-7-CS compared to Ad-MSCs and Ad-MSC sheets. In a segmental bone-defect model, newly formed bone and neovascularization were enhanced with BMP-7-CS, or with a combination of BMP-7-CS and demineralized bone matrix (DBM), compared to those in control groups. These results demonstrate that lentiviral-mediated gene transfer of BMP-7 into Ad-MSCs allows for stable BMP-7 production. BMP-7-CS displayed higher osteogenic capacity than Ad-MSCs and Ad-MSC sheets. In addition, BMP-7-CS combined with demineralized bone matrix (DBM) stimulated new bone and blood vessel formation in a canine critical-sized bone defect. The BMP-7-CS not only provides BMP-7 producing MSCs but also produce osteogenic and vascular trophic factors. Thus, BMP-7-CS and DBM have therapeutic potential for the treatment of critical-sized bone defects and could be used to further enhance clinical outcomes during bone-defect treatment.

Feasibility of single-port retroperitoneoscopic adrenalectomy in dogs.

OBJECTIVE: To evaluate the feasibility of single-port retroperitoneoscopic adrenalectomy (SPRA) in dogs. STUDY DESIGN: A pilot experimental study. ANIMALS: Eight healthy beagle dogs. METHODS: SPRA was performed on the left and right sides (4 dogs each). Resection of the adrenal gland was performed through a SILS port using a retroperitoneal approach. Operative time was defined from skin incision to the completion of skin suture. Postoperative pain was evaluated by using 3 pain scores. Integrity of the adrenal gland capsule was evaluated by histologic assessment. RESULTS: Mean time taken to complete the SPRA was 44.1 minutes (range, 37-51) and was significantly longer on the right side than on the left side (P < .05). There were no complications intraoperatively or during 14 days of postoperative monitoring. The adrenal gland capsule was found to be injured in 3 of the 8 dogs by histologic assessment. CONCLUSION: This is the first report of SPRA in the veterinary literature. With this technique it is possible to perform adrenalectomy with some risk of capsule penetration and with excellent visibility. CLINICAL SIGNIFICANCE: This study suggests that SPRA is feasible and can be used to resect small adrenal tumors with minimal complications.

Impact of local injection of brain-derived neurotrophic factor-expressing mesenchymal stromal cells (MSCs) combined with intravenous MSC delivery in a canine model of chronic spinal cord injury.

BACKGROUND AIMS: The microenvironment of the chronically injured spinal cord does not allow for axonal regeneration due to glial scarring. To ameliorate this, several therapeutic strategies have been used. We investigated whether combined transplantation of chondroitinase ABC (chABC) and mesenchymal stromal cells (MSCs) genetically modified to secrete brain-derived neurotrophic factor (BDNF) with intravenous (IV) administration of MSCs can promote recovery of hindlimb function after chronic spinal cord injury (SCI). METHODS: Canine BDNF-expressing MSCs were generated using a lentivirus packaging protocol. Twelve beagle dogs with experimentally induced chronic SCI were divided into chABC/MSC-green fluorescent protein (GFP), chABC/MSC-BDNF and chABC/MSC-BDNF/IV groups. The MSCs (1 × 107 cells) and chABC were transplanted 3 weeks after SCI in all groups, and IV injection of MSC-GFP (1 × 107 cells) was performed 1 and 2 weeks after MSC transplantation in the chABC/MSC-BDNF/IV group. Spinal cords were harvested 8 weeks after transplantation. RESULTS: The dogs in the chABC/MSC-BDNF included groups had significantly improved functional recovery 8 weeks after transplantation compared with those in the chABC/MSC-GFP group. The animals in the chABC/MSC-BDNF/IV group showed significant improvements in functional recovery at 6, 7 and 8 weeks compared with those in the chABC/MSC-BDNF group. Fibrotic changes were significantly decreased in the chABC/MSC-BDNF/IV group. We also observed significant decreases in the expression levels of tumor necrosis factor-α, interleukin-6, COX-2, glial fibrillary acidic protein and GalC and increased expression levels of BDNF, ß3-tubulin neurofilament medium, and nestin in the chABC/MSC-BDNF/IV group. CONCLUSIONS: We suggest that transplantation of combined chABC and BDNF-expressing MSCs, along with IV injection of MSCs, is the optimal therapy for chronic SCI.

Retroperitoneal Laparoscopy in Dogs: Access Technique, Working Space, and Surgical Anatomy.

OBJECTIVE: To develop and describe a laparoscopic retroperitoneal access technique, investigate working space establishment, and describe the surgical anatomy in the retroperitoneal space as an initial step for clinical application of retroperitoneal laparoscopy in dogs. STUDY DESIGN: Cadaveric and experimental study. ANIMALS: Cadaveric (n=8) and healthy (n=6) adult dogs. METHODS: The retroperitoneal access technique was developed in 3 cadavers based on the human technique and transperitoneal observation. Its application and working space establishment with carbon dioxide (CO2 ) insufflation alone was evaluated in 5 cadavers by observing with a transperitoneal telescope and in 6 live dogs by repeated computed tomography (CT) scans at pressure of 0, 5, 10, and 15 mmHg. Recordings of retroperitoneoscopy as well as working space volume and linear dimensions measured on CT images were analyzed. RESULTS: Retroperitoneal access and working space establishment with CO2 insufflation alone were successfully performed in all 6 live dogs. The only complication observed was in 1 dog that developed subclinical pneumomediastinum. As pressure increased, working space was established from the ipsilateral to the contralateral side, and peritoneal tearing eventually developed. Working space volume increased significantly from 5 mmHg and linear dimensions increased significantly from 0 to 10 mmHg. With pneumo-retroperitoneum above 5 mmHg, retroperitoneal organs, including kidneys and adrenal glands, were easily visualized. CONCLUSION: The retroperitoneal access technique and working space establishment with CO2 insufflation starting with 5 mmHg and increasing to 10 mmHg provided adequate working space and visualization of retroperitoneal organs, which may allow direct access for retroperitoneal laparoscopy in dogs.

Effect of the combination of mesenchymal stromal cells and chondroitinase ABC on chronic spinal cord injury.

Transplantation of mesenchymal stromal cells (MSCs) has been identified as a potential therapeutic modality for treating spinal cord injury (SCI). Degradation of chondroitin sulfate proteoglycans (CSPGs) using the enzyme chondroitinase ABC (chABC) can promote functional recovery after SCI. The effect of the simultaneous administration of MSCs and chABC on chronic SCI was investigated. Sixteen dogs were assigned to one of the following four groups: (i) canine adipose tissue-derived MSCs (cADMSCs), (ii) chABC, (iii) cADMSCs + chABC and (iv) control. Treatments were carried out 3 weeks after SCI; cADMSCs (1 × 10(7) cells suspended in 150 µL of PBS), chABC (5 U/mL, 150 µL), cADMSCs + chABC (1 × 10(7) cells suspended in 150 µL of chABC), or phosphate-buffered saline (150 µL) were injected into the spinal cord at three locations to a depth of 3 mm using a 30-gauge needle. The spinal cord was harvested 8 weeks after transplantation. In a behavioral assessment, dogs treated with cADMSCs + chABC and cADMSCs alone showed significantly better functional recovery 8 weeks after transplantation compared with the control and chABC groups (P < 0.05). In addition, the combination of cADMSCs and chABC increased the expression of digested CSPGs (2B6), ß3 tubulin, and NF-M. However, the levels of COX2 (P < 0.05), and tumor necrosis factor-α was higher in the treatment groups than in the control. In conclusion, transplantation of cADMSCs + chABC was more effective in improving clinical signs and neural regeneration, but a strategy for anti-inflammation after the treatment for chronic SCI would be needed for further improvement.

Functional recovery after spinal cord injury in dogs treated with a combination of Matrigel and neural-induced adipose-derived mesenchymal Stem cells.

BACKGROUND AIMS: Previous studies have reported that scaffold or cell-based transplantation may improve functional recovery following spinal cord injury (SCI), but these results were based on neuronal regeneration and cell replacement. In this study, we investigated whether a combination of Matrigel and neural-induced mesenchymal stem cells (NMSC) improved hindlimb function in dogs with SCI, and what mechanisms were involved. METHODS: We pre-differentiated canine adipose-derived mesenchymal stem cells into NMSC. A total of 12 dogs subjected to SCI procedures were assigned to one of the following three transplantation treatment groups: phosphate-buffered saline (PBS); Matrigel; or Matrigel seeded with NMSC. Treatment occurred 1 week after SCI. Basso, Beattie and Bresnahan (B.B.B.) and Tarlov scores, histopathology, immunofluorescence staining and Western blot analysis were used to evaluate the treatment effects. RESULTS: Compared with dogs administered PBS or Matrigel alone, dogs treated with Matrigel + NMSC showed significantly better functional recovery 8 weeks after transplantation. Histology and immunochemical analysis revealed that the combination of Matrigel + NMSC reduced fibrosis from secondary injury processes and improved neuronal regeneration more than the other treatments. In addition, the combination of Matrigel + NMSC decreased the expression of inflammation and/or astrogliosis markers. Increased expressions of intracellular molecules related to neuronal extension, neuronal markers and neurotrophic factors were also found in the Matrigel + NMSC group. However, the expression of nestin as a neural stem cell marker was increased with Matrigel alone. CONCLUSIONS: The combination of Matrigel + NMSC produced beneficial effects in dogs with regard to functional recovery following SCI through enhancement of anti-inflammation, anti-astrogliosis, neuronal extension and neuronal regeneration effects.

Heat-Shock Proteins Can Potentiate the Therapeutic Ability of Cryopreserved Mesenchymal Stem Cells for the Treatment of Acute Spinal Cord Injury in Dogs.

BACKGROUND: Mesenchymal stem cells (MSCs) are applied in the treatment of spinal cord injury (SCI) because of their neural tissue restoring ability. In the clinical setting, intravenous injection of cryopreserved cells is essential for the immediate treatment of SCI, exhibiting the disadvantage of reduced cell properties. METHODS: In this study, we potentiated the characteristics of cryopreserved MSCs by heat-shock (HS) treatment to induce the expression of HS protein (HSP) HSP70/HSP27 and further improved antioxidant capacity by overexpressing HSP32 (heme oxygenase-1 [HO-1]). We randomly assigned 12 beagle dogs with acute SCI into three groups and transplanted cells intravenously: (i) F-MSCs (MSCs in frozen/thawed conditions); (ii) F-HSP-MSCs (HS-treated MSCs in frozen/thawed conditions); and (iii) F-HSP-HO-MSCs (HO-1-overexpressing and HS-treated MSCs in frozen/thawed conditions). RESULTS: The potentiated MSCs exhibited increased growth factor-, anti-inflammatory-, antioxidant-, homing- and stemness-related gene expression. In the animal experiments, the HSP-induced groups showed significant improvement in hind-limb locomotion, highly expressed neural markers, less intervened fibrotic changes, and improved myelination. In particular, the HO-1-overexpression group was more prominent, controlling the initial inflammatory response with high antioxidant capabilities, suggesting that antioxidation was important to prevent secondary injury. Accordingly, HSPs not only successfully increased the ability of frozen MSCs but also demonstrated excellent neural protection and regeneration capacity in the case of acute SCI. CONCLUSIONS: The application of HSP-induced cryopreserved MSCs in first-aid treatment for acute SCI is considered to help early neural sparing and further hind-limb motor function restoration.

Role of laminin-111 in neurotrophin-3 production of canine adipose-derived stem cells: involvement of Akt, mTOR, and p70S6K.

Extracellular matrix (ECM) components play an important role in the regulation and maintenance of neural stem cells (NSCs). Laminin, an ECM component, is a key factor in promoting axonal regeneration and differentiation of NSCs. Since NSCs cannot be easily harvested with low morbidity, adipose-derived stem cells have been suggested for therapeutic applications of neural tissue damage. Therefore, the potential of laminin-111 to enhance the production of neurotrophin-3 (NT3) and its related signal pathways from canine adipose tissue-derived stem cells (cADSCs) was investigated. Laminin-111 enhanced NT3 production in neural induction medium (NIM). Treatment of NIM or laminin-111 on cADSCs distinctively changed integrin ß1 mRNA and protein expression levels. In addition, laminin-111-induced Akt phosphorylation was inhibited by integrin ß1 small interfering RNA (siRNA) and PI3K inhibitors (LY294002 and wortmannin). Furthermore, increased phosphorylations of mTOR and p70S6K by laminin-111 were blocked by inhibitors or specific siRNA, respectively. Moreover, laminin-111-induced NT3 production was blocked by these inhibitors. In experiments to induce the differentiation of cADSCs, laminin-111 increased the expression of neuronal markers ß 3 tubulin, MAP2, and NeuN, and decreased the expression of the NSC markers nestin and vimentin. In conclusion, laminin-111 increases NT3 production through Akt, mTOR, and p70S6K pathways via integrin ß1 in cADSCs cultured in NIM.

Paracrine effect of canine allogenic umbilical cord blood-derived mesenchymal stromal cells mixed with beta-tricalcium phosphate on bone regeneration in ectopic implantations.

BACKGROUND AIMS: The aim of this study was to evaluate the paracrine effects of canine umbilical cord blood (cUCB) mesenchymal stromal cells (MSC) mixed with beta-tricalcium phosphate (beta-TCP) on bone regeneration in ectopic implantation. METHODS: beta-TCP mixed with cUCB MSC (UCB-MSC group), cell lysates (cell lysate group) or a control (control group) were respectively implanted in a subcutaneous pouches in the back of beagle dogs . The implants were harvested 1, 4, 7, 14, 28, 56, 84 days after implantation. Histological findings and stain analyzes of tartrate-resistant acid phosphatase (TRACP) and assays of alkaline phosphatase (ALP) and TRACP were evaluated. The mRNA expression levels of interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), cyclooxygenase-2 (COX-2), vascular endothelial growth factor (VEGF) and transforming growth factor-beta (TGF-beta) were analyzed using semi-quantitative reverse transcription - polymerase chain reactions (RT-PCR). An enzyme-linked immunosorbent assay (ELISA) was used to confirm the protein expression levels of IL-6, COX-2, VEGF and TGF-beta. RESULTS: TRACP-positive cells were observed in all groups 7 days after implantation. ALP and TRACP activities in the UCB-MSC group 84 days after implantation were significantly higher than those of the control (P>0.05). Histologic findings after 84 days showed that the osteoid matrix area in the UCB-MSC group was significantly larger than that of the control (P<0.05). The mRNAs levels of IL-1, IL-6 and VEGF in UCB-MSC and cell lysate groups on day 1 were up-regulated compared with the control. The protein levels of IL-6 and VEGF in the UCB-MSC group at day 1 were significantly higher than that of the other groups (P<0.05). CONCLUSIONS: It is suggested that a significant release of cytokines by cUCB MSC, 1 day following implantation, could enhance bone regeneration.

Intravenous Administration of Heat Shock-Treated MSCs Can Improve Neuroprotection and Neuroregeneration in Canine Spinal Cord Injury Model.

Transplantation of mesenchymal stem cells (MSCs) is a promising treatment for spinal cord injury (SCI). However, many transplanted cells die within a few days, eventually limiting the efficacy of cellular therapy. To overcome this problem, we focused on the potential of heat shock (HS) proteins in facilitating recovery from cell damage and protecting against cytotoxicity. PCR results showed that the expression of neurotrophic factor, anti-inflammatory, stemness, and homing genes increased in HS-treated MSCs. We investigated whether HS-treated MSCs could promote recovery of hindlimb function in an acute canine SCI model. We compared the effects of intravenous transplantation with (i) lactated Ringer's solution as a control, (ii) green fluorescent protein-expressing MSCs (MSCs-GFP), and (iii) GFP-expressing and HS-treated MSCs (MSCs-GFP-HS). Spinal cords were harvested at four weeks and used for Western blot and histopathological analyses. The MSCs-GFP-HS group showed significant improvements in hindlimb function from weeks 3 and 4 compared with the other groups. This group also showed higher expression of neural markers, fewer intervening fibrotic changes, and pronounced myelination. These results suggest that induction of an HS response in MSCs could promote neural sparing. In conclusion, transplantation of HS-treated MSCs could improve neuroprotection and neuroregeneration in acute SCI.

Cardiopulmonary changes induced by retroperitoneal insufflation in healthy dogs in sternal recumbency with the abdomen unsupported.

This study assessed the effects of retroperitoneal carbon dioxide (CO2) insufflation on cardiopulmonary variables and intra-abdominal pressure (IAP) in mechanically ventilated dogs in sternal recumbency with the abdomen unsupported, following placement of a positioning kit and towels under the pectoral and pelvic regions. General anesthesia was induced in eight healthy adult male Beagles. A Swan-Ganz catheter was placed in the pulmonary artery via the jugular vein for cardiac output measurements. A Foley urethral catheter was placed to monitor transvesical IAP. A 10 mm balloon blunt-tip trocar was inserted into the retroperitoneal space. With a fixed respiratory rate and tidal volume by mechanical ventilation, insufflation pressure was sequentially increased from 0 to 10 mmHg in 5 mmHg increments, followed by desufflation. All variables were measured before insufflation, 5 min after the establishment of each insufflation pressure, and after desufflation. At 10 mmHg, the IAP was nearly equal to insufflation pressure. Cardiopulmonary function was not compromised at any point, although the cardiac index (CI), heart rate, mean arterial pressure (MAP), and mean pulmonary arterial pressure increased within normal ranges. End-tidal CO2 concentration, arterial CO2 partial pressure, and oxygen delivery index (DO2I) increased, whereas pH decreased, at 10 mmHg. CI, MAP, and DO2I did not recover to baseline after decompression. Thus, retroperitoneal CO2 insufflation up to 10 mmHg is well tolerated by mechanically ventilated dogs positioned in sternal recumbency with the abdomen unsupported, although sympathetic changes may occur with an insufflation pressure increase.

Discovery of N-glycan Biomarkers for the Canine Osteoarthritis.

Protein glycosylation is a post-translational modification that impacts on protein activity, stability, and interactions. It was sensitively altered by the cellular state and, therefore, is now used for a diagnostic or prognostic indicator of various human diseases such as cancer. To evaluate the clinical feasibility in the veterinary area, the N-glycan biomarkers were discovered from canine serum for the diagnosis of osteoarthritis (OA), which is one of the most common diseases of dogs. N-glycome was obtained from 20 µL of canine serum by the enzymatic cleavage followed by the purification and enrichment using solid-phase extraction. Independent compositions of 163 and 463 N-glycans were found from healthy control (n = 41) and osteoarthritis patients (n = 92), respectively. Initially, 31 of the potential biomarkers were screened by the p-values below 1.0 × 10-10 from ANOVA. Then, the area under the curve (AUC) and the intensity ratio between OA patient and healthy control (P/C ratio) were calculated. Considering the diagnostic efficacy, the AUC bigger than 0.9 and the P/C ratio larger than 3.0 were used to discover 16 N-glycans as diagnostic biomarkers. Particularly, five of the diagnostic biomarkers were AUC above 0.99 and three of N-glycans had AUC 1.0. The results suggest a clear possibility for N-glycan biomarkers to be used as a clinical tool in the veterinary medical area enabling to provide objective and non-invasive diagnostic information.

Therapeutic effects of platelet derived growth factor overexpressed-mesenchymal stromal cells and sheets in canine skin wound healing model.

Adipose-derived mesenchymal stromal cells (Ad-MSCs) have excellent potential for skin wound repair. Moreover, platelet-derived growth factor (PDGF) has strong wound healing properties. The purpose of the present study was to compare the healing effects of PDGF-overexpressing canine allogeneic Ad-MSCs (PDGF-MSCs) and their cell sheets (PDGF-CSs) as compared to unexpressed Ad-MSCs (U-MSCs) and their cell sheets (UCSs) in a cutaneous wound healing model induced upon dogs. In in vitro study, the expression of immunomodulatory and growth factors was assessed by qRT-PCR. In in vivo study, cells and sheets were transplanted into a square-shaped full-thickness (1.5×1.5 cm) skin defect model created in 12 dogs. After 5 and 10 days, wounds were harvested and evaluated macroscopically and histopathologically. The qRT-PCR results showed that the PDGF-B gene was significantly upregulated (p<0.05) in PDGF-CS and PDGF-MSCs groups. Upon gross analysis of the wound, all stromal cells and their sheet groups showed accelerated (p<0.05) cutaneous wound healing compared to the negative control groups. As compared to U-MSCs and UCSs, the PDGF-MSCs showed significant epithelization on days 5 and 10 of healing, whereas PDGF-CSs showed improved epithelization only on day 10. In the granulation tissue analysis, PDGF-CSs and UCSs promoted more formation (p<0.05) of upper granulation tissue, collagen, and activated fibroblasts than PDGF-MSCs, and U-MSCs. Especially, the PDGF-CSs presented the highest formation and maturation of granulation tissue among all groups. All considered, PDGF overexpressed stromal cells or cells sheets can improve cutaneous wound healing in a canine model.

Therapeutic Effects of Intravenous Injection of Fresh and Frozen Thawed HO-1-Overexpressed Ad-MSCs in Dogs with Acute Spinal Cord Injury.

Owing to the antioxidant and anti-inflammatory functions of hemeoxygenase-1 (HO-1), HO-1-expressing canine adipose-derived mesenchymal stem cells (Ad-MSCs) could be efficacious in treating spinal cord injury (SCI). Further, frozen thawed HO-1 Ad-MSCs could be instantly available as an emergency treatment for SCI. We compared the effects of intravenous treatment with freshly cultured HO-1 Ad-MSCs (HO-1 MSCs), only green fluorescent protein-expressing Ad-MSCs (GFP MSCs), and frozen thawed HO-1 Ad-MSCs (FT-HO-1 MSCs) in dogs with acute SCI. For four weeks, dogs were evaluated for improvement in hind limb locomotion using a canine Basso Beattie Bresnahan (cBBB) score. Upon completion of the study, injured spinal cord segments were harvested and used for western blot and histopathological analyses. All cell types had migrated to the injured spinal cord segment. The group that received HO-1 MSCs showed significant improvement in the cBBB score within four weeks. This group also showed significantly higher expression of NF-M and reduced astrogliosis. There was reduced expression of proinflammatory cytokines (IL6, TNF-α, and IL-1ß) and increased expression of anti-inflammatory markers (IL-10, HO-1) in the HO-1 MSC group. Histopathological assessment revealed decreased fibrosis at the epicenter of the lesion and increased myelination in the HO-1 MSC group. Together, these data suggest that HO-1 MSCs could improve hind limb function by increasing the anti-inflammatory reaction, leading to neural sparing. Further, we found similar results between GFP MSCs and FT-HO-1 MSCs, which suggest that FT-HO-1 MSCs could be used as an emergency treatment for SCI.

Enterocutaneous fistula as a result of chronic bite wound repair in a dog.

A 6-year-old castrated male Maltese weighing 4.8 kg was presented with a non-healing wound exhibiting purulent discharge after surgery on scar tissue of a chronic twelve-month-old bite wound on the left caudal abdominal region. The dog had previously undergone four surgeries and had been on continuous antibiotic therapy for eight months. Following radiographic and ultrasonographic examinations, the problem was diagnosed as an enterocutaneous fistula of a herniated bowel loop under the skin. Surgical resection of the fistula involving the bowel loop resolved all symptoms.

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.