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 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 determine kinematic changes to the hoof of horses at a walk after induction of unilateral, weight-bearing forelimb lameness and to determine whether hoof kinematics return to prelameness (baseline) values after perineural anesthesia. Animals-6 clinically normal Quarter Horses. Procedures-For each horse, a sole-pressure model was used to induce 3 grades of lameness in the right forelimb, after which perineural anesthesia was administered to eliminate lameness. Optical kinematics were obtained for both forelimbs with the horse walking before (baseline) and after induction of each grade of lameness and after perineural anesthesia. Linear acceleration profiles were used to identify hoof events, and each stride was divided into hoof-contact, break-over, initial-swing, terminal-swing, and total-swing segments. Kinematic variables were compared within and between limbs for each segment by use of mixed repeated-measures ANOVA. Results-During the hoof-contact and terminal-swing segments, the hoof of the left (nonlame) forelimb had greater sagittal-plane orientation than did the hoof of the right (lame) forelimb. For the lame limb following lameness induction, the break-over duration and maximum cranial acceleration were increased from baseline. After perineural anesthesia, break-over duration for the lame limb returned to a value similar to that at baseline, and orientation of the hoof during the terminal-swing segment did not differ between the lame and nonlame limbs. Conclusions and Clinical Relevance-Subclinical unilateral forelimb lameness resulted in significant alterations to hoof kinematics in horses that are walking, and the use of hoof kinematics may be beneficial for the detection of subclinical lameness in horses.

Objective-To qualitatively describe lung CT images obtained from sedated healthy equine neonates (≤ 14 days of age), use quantitative analysis of CT images to characterize attenuation and distribution of gas and tissue volumes within the lungs, and identify differences between lung characteristics of foals ≤ 7 days of age and foals > 7 days of age. Animals-10 Standardbred foals between 2.5 and 13 days of age. Procedures-Foals were sedated with butorphanol, midazolam, and propofol and positioned in sternal recumbency for thoracic CT. Image analysis software was used to exclude lung from nonlung structures. Lung attenuation was measured in Hounsfield units (HU) for analysis of whole lung and regional changes in attenuation and lung gas and tissue components. Degree of lung attenuation was classified as follows: hyperinflated or emphysema, −1,000 to −901 HU; well aerated, −900 to −501 HU; poorly aerated, −500 to −101 HU; and nonaerated, > −100 HU. Results-Qualitative evidence of an increase in lung attenuation and patchy alveolar patterns in the ventral lung region were more pronounced in foals ≤ 7 days of age than in older foals. Quantitative analysis revealed that mean +/- SD lung attenuation was greater in foals ≤ 7 days of age (−442 +/- 28 HU) than in foals > 7 days of age (−521 +/- 24 HU). Lung aeration and gas volumes were lower than in other regions ventrally and in the mid lung region caudal to the heart. Conclusions and Clinical Relevance-Identified radiographic patterns and changes in attenuation were most consistent with atelectasis and appeared more severe in foals ≤ 7 days of age than in older neonatal foals. Recognition of these changes may have implications for accurate CT interpretation in sedated neonatal foals with pulmonary disease.

Objective-To compare effects of anesthetic induction with midazolam-propofol or midazolam-etomidate on intraocular pressure (IOP), pupillary diameter (PD), pulse rate, blood pressure, and respiratory rate in clinically normal dogs. Animals-18 dogs. Procedures-Dogs undergoing ophthalmic surgery received midazolam (0.2 mg/kg, IV) and either propofol or etomidate (IV) until intubatable. For all dogs, results of physical examinations, ophthalmic examinations of the nonoperated eye, and preanesthetic blood analyses were normal. Intraocular pressure, PD, blood pressure, pulse rate, and respiratory rate were measured in the nonoperated eye at 5 time points: just prior to the anesthetic induction sequence, after 5 minutes of preanesthetic oxygenation via face mask, after IV administration of midazolam, after IV anesthetic induction, and after endotracheal intubation. Results-PD decreased significantly from baseline by 4.4 +/- 0.4 mm (mean +/- SD) after anesthetic induction and 5.3 +/- 0.4 mm after intubation in the etomidate group and by 1. 2 +/- 0.4 mm after intubation in the propofol group. Intraocular pressure was increased significantly from baseline by 3.2 +/- 1.0 mm Hg after anesthetic induction in the etomidate group and by 4.7 +/- 1.2 mm Hg after anesthetic induction and 4.5 +/- 1. 2 mm Hg after intubation in the propofol group. Pulse rate was significantly lower by 28.6 +/- 12.6 beats/min after anesthetic induction in the etomidate group, compared with the propofol group. Conclusions and Clinical Relevance-At the studied doses, midazolam-etomidate caused clinically important miosis and increased IOP. Midazolam-propofol caused an even greater increase in IOP but had minimal effects on PD.

Objective—To determine relative concentrations of selected major brain tissue metabolites and their ratios and lobar variations by use of 3-T proton (hydrogen 1 [1H]) magnetic resonance spectroscopy (MRS) of the brain of healthy dogs. Animals—10 healthy Beagles. Procedures—3-T 1H MRS at echo times of 144 and 35 milliseconds was performed on 5 transverse slices and 1 sagittal slice of representative brain lobe regions. Intravoxel parenchyma was classified as white matter, gray matter, or mixed (gray and white) and analyzed for relative concentrations (in arbitrary units) of N-acetylaspartate (NAA), choline, and creatine (ie, height at position of peak on MRS graph) as well as their ratios (NAA-to-choline, NAA-to-creatine, and choline-to-creatine ratios). Peak heights for metabolites were compared between echo times. Peak heights for metabolites and their ratios were correlated and evaluated among matter types. Yield was calculated as interpretable voxels divided by available lobar voxels. Results—Reference ranges of the metabolite concentration ratios were determined at an echo time of 35 milliseconds (NAA-to-choline ratio, 1.055 to 2.224; NAA-to-creatine ratio, 1.103 to 2.161; choline-to-creatine ratio, 0.759 to 1.332) and 144 milliseconds (NAA-to-choline ratio, 0.687 to 1.788; NAA-to-creatine ratio, 0.984 to 2.044; choline-to-creatine ratio, 0.828 to 1.853). Metabolite concentration ratios were greater in white matter than in gray matter. Voxel yields ranged from 43% for the temporal lobe to 100% for the thalamus. Conclusions and Clinical Relevance—Metabolite concentrations and concentration ratios determined with 3-T 1H MRS were not identical to those in humans and were determined for clinical and research investigations of canine brain disease.

Objective—To investigate the in vitro differentiation of canine bone marrow stromal cells (BMSCs) into functional, mature neurons. Sample—Bone marrow from 6 adult dogs. Procedures—BMSCs were isolated from bone marrow and chemically induced to develop into neurons. The morphology of the BMSCs during neuronal induction was monitored, and immunocytochemical analyses for neuron markers were performed after the induction. Real-time PCR methods were used to evaluate the mRNA expression levels of markers for neural stem or progenitor cells, neurons, and ion channels, and western blotting was used to assess the expression of neuronal proteins before and after neuronal induction. The electrophysiological properties of the neuron-like cells induced from canine BMSCs were evaluated with fluorescent dye to monitor Ca2+ influx. Results—Canine BMSCs developed a neuron-like morphology after neuronal induction. Immunocytochemical analysis revealed that these neuron-like cells were positive for neuron markers. After induction, the cells’ mRNA expression levels of almost all neuron and ion channel markers increased, and the protein expression levels of nestin and neurofilament-L increased significantly. However, the neuron-like cells derived from canine BMSCs did not have the Ca2+ influx characteristic of spiking neurons. Conclusions and Clinical Relevance—Although canine BMSCs had neuron-like morphological and biochemical properties after induction, they did not develop the electrophysiological characteristics of neurons. Thus, these results have suggested that canine BMSCs could have the capacity to differentiate into a neuronal lineage, but the differentiation protocol used may have been insufficient to induce development into functional neurons.

Objective—To assess the anesthetic efficacy and local tolerance of topically applied 0.4% oxybuprocaine ophthalmic solution to in dogs and compare its effects with those of 1% tetracaine solution. Animals—34 ophthalmically normal Beagles. Procedures—Dogs were assigned to 2 groups, and baseline corneal touch threshold (CTT) was measured bilaterally with a Cochet-Bonnet aesthesiometer. Dogs of group 1 (n = 22) received a single drop of 0.4% oxybuprocaine ophthalmic solution in one eye and saline (0.9% NaCl) solution (control treatment) in the contralateral eye. Dogs of group 2 (n = 12) received a single drop of 0.4% oxybuprocaine ophthalmic solution in one eye and 1% tetracaine ophthalmic solution in the contralateral eye. The CTT of each eye was measured 1 and 5 minutes after topical application and then at 5-minute intervals until 75 minutes after topical application. Results—CTT changes over time differed significantly between oxybuprocaine-treated and control eyes. After instillation of oxybuprocaine, maximal corneal anesthesia (CTT = 0) was achieved within 1 minute, and CTT was significantly decreased from 1 to 45 minutes, compared with the baseline value. No significant difference in onset, depth, and duration of corneal anesthesia was found between oxybuprocaine-treated and tetracaine-treated eyes. Conjunctival hyperemia and chemosis were detected more frequently in tetracaine-treated eyes than in oxybuprocaine-treated eyes. Conclusions and Clinical Relevance—Topical application of oxybuprocaine and tetracaine similarly reduced corneal sensitivity in dogs, but oxybuprocaine was less irritating to the conjunctiva than was tetracaine.

In this study, to understand the pathogenesis of new rabbit hemorrhagic disease virus (RHDVa) serotype, we carried out to administrate RHDVa to rabbits, and to examine sequential electron microscopic changes and relationship between pathogenesis and apoptosis. TUNEL-positive cells began to be observed from 24 hours after inoculation (HAI) and the number of positive cells was slightly increased with the course of time. Whereas marked increase of positive cells was seen in the liver from the rabbits died acutely. Typical viral particles with cup-like projections and a diameter of 30~40 nm were detected in homogenized liver samples and tissues at 36 and 48, and 48 HAI, respectively. Ultrastructurally, glycogen deposition was observed from the first stage of hepatocellular degeneration by RHDVa infection and then, swelling and disruption of cristae of mitochondria by viral particles, swelling of smooth endoplasmic reticulum, vacuoles and vesicles were detected. Condensation, margination and fragmentation of chromatin were observed in degenerative hepatocytes at 36 and 48 HAI, indicating apoptotic bodies. These data offer that hepatocytic apoptosis by RHDV infection could be closely related with mitochondrial impairment in the hepatocytes.

To develop a live vaccine candidate using an attenuated strain of Salmonella Typhimurium (ST), biochemical properties, plasmid profile, PFGE patterns and pathogenic analysis of the ST isolate were carried out after sequential passage of the ST isolate in porcine neutrophils. By the passage, the ability of the neutrophil-adapted isolate to utilize d-xylose was lost, while the ability of the strain to ferment trehalose was delayed after 2 or more days of the culture. Also, changes including deletion of the gene fragments were observed in PFGE analysis of the neutrophil-adapted isolates. Two plasmids, 105kb and 50kb, were cured in the strain passaged over 15 times in porcine neutrophils. The 50% of lethal dose (LD50) of the parent strain was changed from 1 × 105 LD50 to 6 × 106 LD50 by the passage in intraperitoneal injection of the strains into mice. These results suggested that bacterial genotypic and phenotypic responses might be globally altered depending on the inside environment of neutrophils.