Objective: To determine whether angiogenesis and microglial activation were related to seizure-induced neuronal death in the cerebral cortex of Shetland Sheepdogs with familial epilepsy. Animals: Cadavers of 10 Shetland Sheepdogs from the same family (6 dogs with seizures and 4 dogs without seizures) and 4 age-matched unrelated Shetland Sheepdogs. Procedures: Samples of brain tissues were collected after euthanasia and then fixed in neutral phosphate–buffered 10% formalin and routinely embedded in paraffin. The fixed samples were sectioned for H&E staining and immunohistochemical analysis. Results: Evidence of seizure-induced neuronal death was detected exclusively in samples of cerebral cortical tissue from the dogs with familial epilepsy in which seizures had been observed. The seizure-induced neuronal death was restricted to tissues from the cingulate cortex and sulci surrounding the cerebral cortex. In almost the same locations as where seizure-induced neuronal death was identified, microvessels appeared longer and more tortuous and the number of microvessels was greater than in the dogs without seizures and control dogs. Occasionally, the microvessels were surrounded by oval to flat cells, which had positive immunohistochemical results for von Willebrand factor. Immunohistochemical results for neurons and glial cells (astrocytes and microglia) were positive for vascular endothelial growth factor, and microglia positive for ionized calcium–binding adapter molecule 1 were activated (ie, had swollen cell bodies and long processes) in almost all the same locations as where seizure-induced neuronal death was detected. Double-label immunofluorescence techniques revealed that the activated microglia had positive results for tumor necrosis factor-α, interleukin-6, and vascular endothelial growth factor receptor 1. These findings were not observed in the cerebrum of dogs without seizures, whether the dogs were from the same family as those with epilepsy or were unrelated to them. Conclusions and Clinical Relevance: Signs of angiogenesis and microglial activation corresponded with seizure-induced neuronal death in the cerebral cortex of Shetland Sheepdogs with familial epilepsy. Microglial activation induced by vascular endothelial growth factor and associated proinflammatory cytokine production may accelerate seizure-induced neuronal death in dogs with epilepsy.

Objective: To evaluate first-intention healing of CO2 laser, 4.0-MHz radiowave radiosurgery (RWRS), and scalpel incisions in ball pythons (Python regius). Animals: 6 healthy adult ball pythons. Procedures: A skin biopsy sample was collected, and 2-cm skin incisions (4/modality) were made in each snake under anesthesia and closed with surgical staples on day 0. Incision sites were grossly evaluated and scored daily. One skin biopsy sample per incision type per snake was obtained on days 2, 7, 14, and 30. Necrotic and fibroplastic tissue was measured in histologic sections; samples were assessed and scored for total inflammation, histologic response (based on the measurement of necrotic and fibroplastic tissues and total inflammation score), and other variables. Frequency distributions of gross and histologic variables associated with wound healing were calculated. Results: Gross wound scores were significantly greater (indicating greater separation of wound edges) for laser incisions than for RWRS and scalpel incisions at all evaluated time points. Necrosis was significantly greater in laser and RWRS incisions than in scalpel incision sites on days 2 and 14 and days 2 and 7, respectively; fibroplasia was significantly greater in laser than in scalpel incision sites on day 30. Histologic response scores were significantly lower for scalpel than for other incision modalities on days 2, 14, and 30. Conclusions and Clinical Relevance: In snakes, skin incisions made with a scalpel generally had less necrotic tissue than did CO2 laser and RWRS incisions. Comparison of the 3 modalities on the basis of histologic response scores indicated that use of a scalpel was preferable, followed by RWRS and then laser.

Objective-To investigate histomorphometric changes in the cartilage and subchondral bone of the third carpal bone associated with conditioning exercise in young Thoroughbreds. Animals-Nine 18-month-old Thoroughbreds. Procedures-Both third carpal bones of 9 horses (4 exercised spontaneously at pasture only and 5 given additional conditioning exercise beginning at a mean age of 3 weeks) were evaluated. Histomorphometric variables (hyaline and calcified cartilage thickness and collagen orientation; vascular channel area, number, and orientation; and osteochondral junction rugosity) of the third carpal bone, sampled at 4 dorsopalmar sites in the radial facet, were compared between the exercised and nonexercised groups. Results-The vascular channel area measured at the 4 dorsopalmar sites was larger in the exercised group than in the control group, but none of the variables were significantly different between groups. Both groups had significant site-specific variations in all measured variables. Most importantly, the vascular channel area was highest in the most dorsal aspect. Conclusions and Clinical Relevance-Results suggested that the mild exercise imposed in both groups during the developmental period appeared to be associated with an increase in the vascular channel area beneath the calcified cartilage layer in the third carpal bone. This increased vascular channel area could also be associated with high stress in the dorsal aspect of the radial facet, a region that is known to be vulnerable to osteochondral fragmentation.

Objective-To determine the effects of 2 weeks of intense exercise on expression of markers of pulmonary venous remodeling in the caudodorsal and cranioventral regions of the lungs of horses. Animals-6 horses. Procedures-Tissue samples of the caudodorsal and cranioventral regions of lungs were obtained before and after conditioning and 2 weeks of intense exercise. Pulmonary veins were isolated, and a quantitative real-time PCR assay was used to determine mRNA expression of matrix metalloproteinase-2 and −9, tissue inhibitor of metalloproteinase-1 and −2, collagen type I, tenascin-C, endothelin-1, platelet-derived growth factor, transforming growth factor (TGF)-β, and vascular endothelial growth factor (VEGF). Protein expression of collagen (via morphometric analysis) and tenascin-C, TGF-β, and VEGF (via immunohistochemistry) was determined. Results-Exercise-induced pulmonary hemorrhage was detected in 2 horses after exercise. The mRNA expression of matrix metalloproteinase-2 and −9, tissue inhibitor of metalloproteinase-2, TGF-β, and VEGF was significantly lower in pulmonary veins obtained after exercise versus those obtained before exercise for both the caudodorsal and cranioventral regions of the lungs. Collagen content was significantly higher in tissue samples obtained from the caudodorsal regions of the lungs versus content in samples obtained from the cranioventral regions of the lungs both before and after exercise. Exercise did not alter protein expression of tenascin-C, TGF-β, or VEGF. Conclusions and Clinical Relevance-Results of this study indicated 2 weeks of intense exercise did not alter expression of marker genes in a manner expected to favor venous remodeling. Pulmonary venous remodeling is complex, and > 2 weeks of intense exercise may be required to induce such remodeling.

Objective-To determine the efficiency of a novel point-of-care gravitational marrow separator and bone marrow aspiration needle for concentrated bone marrow production and bone marrow-derived mesenchymal stem cell (MSC) separation and assess the effect of repeated bone marrow collections in horses. Animals-8 healthy adult horses. Procedures-Bone marrow aspiration was performed twice (1 month apart) from sternebral bodies with a standard or prototype multidirectional needle. Concentrated bone marrow was obtained by gravitational marrow separation and evaluated for WBC and platelet counts, automated and cytomorphologic cell differential counts, MSCs, and cell viability. Results-Concentrated bone marrow samples obtained with the marrow separator had 5- to 19-fold bone marrow-derived MSC, WBC, and platelet counts, compared with original bone marrow samples. Use of a multidirectional needle increased the frequency of obtaining MSC-richer concentrated bone marrow. Repeating bone marrow aspiration at 1 month yielded greater MSC numbers but slightly lower cell viability after processing. Conclusions and Clinical Relevance-The gravitational bone marrow separator and multidirectional needle were used to effectively harvest bone marrow and improve the quality of concentrated bone marrow. Comparable, or even greater, numbers of bone marrow-derived MSCs were collected by repeated bone marrow aspiration after a 1-month interval from the same aspiration sites. Use of the marrow separator and multidirectional bone marrow aspiration needle can facilitate a 1-step, point-of-care, nonlaboratory method to obtain concentrated bone marrow as a mixture of bone marrow-derived MSCs and growth factors from platelets and plasma.

Objective: To evaluate protein expression in bronchoalveolar lavage fluid (BALF) obtained from West Highland White Terriers with idiopathic pulmonary fibrosis (IPF), dogs with chronic bronchitis, and healthy control dogs to identify potential biomarkers for IPF. Samples: BALF samples obtained from 6 West Highland White Terriers with histologically confirmed IPF, 5 dogs with chronic bronchitis, and 4 healthy Beagles. Procedures: Equal amounts of proteins in concentrated BALF samples were separated via 2-D differential gel electrophoresis. Proteins that were differentially expressed relative to results for healthy control dogs were identified with mass spectrometry and further verified via western blotting. Results: Expression of 6 proteins was upregulated and that of 1 protein was downregulated in dogs with IPF or chronic bronchitis, compared with results for healthy dogs. Expression of proteins β-actin, complement C3, α-1-antitrypsin, apolipoprotein A-1, haptoglobin, and transketolase was upregulated, whereas expression of lysozyme C was downregulated. Conclusions and Clinical Relevance: Proteomics can be used to search for biomarkers and to reveal disease-specific mechanisms. The quantitative comparison of proteomes for BALF obtained from dogs with IPF and chronic bronchitis and healthy dogs revealed similar changes for the dogs with IPF and chronic bronchitis, which suggested a common response to disease processes in otherwise different lung diseases. Specific biomarkers for IPF were not identified.

Objective: To investigate effects of various imidazoline and nonimidazoline α-adrenergic agents on aggregation and antiaggregation of bovine and equine platelets. Sample: Blood samples obtained from 8 healthy adult cattle and 16 healthy adult Thoroughbreds. Procedures: Aggregation and antiaggregation effects of various imidazoline and nonimidazoline α-adrenergic agents on bovine and equine platelets were determined via a turbidimetric method. Collagen and ADP were used to initiate aggregation. Results: Adrenaline, noradrenaline, or α-adrenoceptor agents alone did not induce changes in aggregation of bovine or equine platelets or potentiate ADP- or collagen-induced platelet aggregation. Adrenaline and the α2-adrenoceptor agonist clonidine had an inhibitory effect on ADP- and collagen-induced aggregation of bovine platelets. The α2-adrenoceptor antagonists phentolamine and yohimbine also inhibited collagen-induced aggregation of bovine platelets. Noradrenaline, other α-adrenoceptor agonists (xylazine, oxymetazoline, and medetomidine), and α-adrenoceptor antagonists (atipamezole, idazoxan, tolazoline, and prazosin) were less effective or completely ineffective in inhibiting ADP- and collagen-induced aggregation of bovine platelets. The imidazoline α2-adrenoceptor agonist oxymetazoline submaximally inhibited collagen-induced aggregation of equine platelets, and the α2-adrenoceptor antagonist idazoxan, along with phentolamine and yohimbine, also inhibited collagen-induced aggregation of equine platelets. The imidazoline compound antazoline inhibited both ADP- and collagen-induced aggregation of equine platelets. Conclusions and Clinical Relevance: Several drugs had effects on aggregation of platelets of cattle and horses, and effective doses of ADP and collagen also differed between species. The α2-adrenoceptor agonists (xylazine and medetomidine) and antagonists (tolazoline and atipamezole) may be used by bovine and equine practitioners without concern for adverse effects on platelet function and hemostasis.

Objective-To investigate whether differences existed between clinically normal dogs and dogs with goniodysgenesis-related glaucoma (GDRG) in serum autoantibodies against optic nerve antigens. Animals-16 dogs with GDRG, 17 healthy dogs with unremarkable pectinate ligament and iridocorneal angle morphology, and 13 euthanized dogs with no major ocular abnormalities or underlying diseases. Procedures-Western blotting was performed with optic nerve extracts from the euthanized dogs as an antigen source and serum from clinically normal dogs and dogs with GDRG as a primary antibody (autoantibody) source. Blots were evaluated for presence and density of bands. Results-Multiple bands were identified on western blots from all dogs with GDRG and all clinically normal dogs, with a high degree of variability among individual dogs. Dogs with GDRG were significantly more likely than healthy dogs to have bands present at 38, 40, and 68 kDa. Dogs with GDRG had significant increases in autoreactivity at 40 and 53 kDa and a significant decrease in autoreactivity at 48 kDa. Conclusions and Clinical Relevance-Significant differences in serum autoantibodies against optic nerve antigens were found in dogs with versus without GDRG. Although it remains unclear whether these differences were part of the pathogenesis of disease or were sequelae to glaucomatous changes, these findings provide support for the hypothesis that immune-mediated mechanisms play a role in the development or progression of GDRG. However, the high degree of variability among individual dogs and the considerable overlap between groups suggest that the clinical usefulness of this technique for distinguishing dogs with GDRG from clinically normal dogs is likely limited.

Objective-To quantify left ventricle (LV) volumes by use of 1-D, 2-D, and 3-D echocardiography versus MRI in dogs. Animals-10 healthy Beagles. Procedures-During anesthesia, each dog underwent an echocardiographic examination via the Teichholz method, performed on the basis of standard M-mode frames (1-D); the monoplane Simpson method of disk (via 2-D loops); real-time triplane echocardiography (RTTPE) with a 3-D probe; and real-time 3-D echocardiography with a 3-D probe. Afterward, cardiac MRI was performed. Values for the LV end-diastolic volume (EDV), end-systolic volume (ESV), and ejection fraction (EF) were compared between each echocardiographic method and the reference method (cardiac MRI). Results-No significant differences for EDV, ESV, and EF were detected between RTTPE and cardiac MRI. Excellent correlations (r = 0.97, 0.98, and 0.95 for EDV, ESV, and EF, respectively) were found between RTTPE and values for cardiac MRI. The other echocardiographic methods yielded values significantly different from cardiac MRI and results correlated less well with results of cardiac MRI for EDV, ESV, and EF. Use of the Teichholz method resulted in LV volume overestimation, whereas the Simpson method of disk and real-time 3-D echocardiography significantly underestimated LV volumes. Conclusions and Clinical Relevance-Use of RTTPE yielded excellent correlations and nonsignificant differences with cardiac MRI and is a suitable method for routine veterinary cardiac examination.