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  • Author or Editor: Akikazu Ishihara x
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Abstract

Objective—To evaluate host cell permissiveness and cytotoxic effects of recombinant and modified adenoviral vectors in equine chondrocytes, synovial cells, and bone marrow–derived mesenchymal stem cells (BMD-MSCs).

Sample Population—Articular cartilage, synovium, and bone marrow from 15 adult horses.

Procedures—Equine chondrocytes, synovial cells, and BMD-MSCs and human carcinoma (HeLa) cells were cultured and infected with an E-1–deficient adenovirus vector encoding the β-galactosidase gene or the green fluorescent protein gene (Ad-GFP) and with a modified E-1–deficient vector with the arg-gly-asp capsid peptide insertion and containing the GFP gene (Ad-RGD-GFP). Percentages of transduced cells, total and transduced cell counts, and cell viability were assessed 2 and 7 days after infection.

Results—Permissiveness to adenoviral vector infection was significantly different among cell types and was ranked in decreasing order as follows: HeLa cells > BMD-MSCs > chondrocytes > synovial cells. Morphologic signs of cytotoxicity were evident in HeLa cells but not in equine cells. Numbers of transduced cells decreased by day 7 in all cell types except equine BMD-MSCs. Transduction efficiency was not significantly different between the Ad-GFP and Ad-RGD-GFP vectors.

Conclusion and Clinical Relevance—Sufficient gene transfer may be achieved by use of an adenovirus vector in equine cells. High vector doses can be used in equine cells because of relative resistance to cytotoxic effects in those cells. Greater permissiveness and sustained expression of transgenes in BMD-MSCs make them a preferential cell target for gene therapy in horses.

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in American Journal of Veterinary Research

Abstract

Objective—To evaluate the association between subjective lameness grades and kinetic gait parameters and assess the variability in kinetic parameters in horses with experimentally induced forelimb lameness.

Animals—32 horses.

Procedures—Forelimb lameness was induced in each horse via injection of lipopolysaccharide into 1 metacarpophalangeal joint (40 experimental trials). Subjective lameness grading and 13 kinetic gait parameters (force plate analysis) were assessed before (baseline) and at 12, 18, and 24 hours after lipopolysaccharide injection. While horses were trotting, kinetic gait analysis was performed for 8 valid repetitions at each time point. Repeated-measures analyses were performed with 8 repetitions for each kinetic parameter as the outcome, and lameness grades, time points after lipopolysaccharide injection, and repetition order as explanatory variables. Sensitivity and specificity of kinetic parameters for classification of horses as sound or lame (in relation to subjective lameness scores) were calculated. Between- and within-horse variabilities of the 13 kinetic parameters were assessed by calculation of coefficients of variation.

Results—Subjective lameness grades were significantly associated with most of the kinetic parameters. Vertical force peak and impulse had the lowest between- and within-horse coefficients of variation and the highest correlations with subjective lameness grade. Vertical force peak had the highest sensitivity and specificity for lameness classification. Vertical force peak and impulse were significantly decreased even in horses with mild or unobservable lameness.

Conclusions and Clinical Relevance—Among the kinetic gait parameters, vertical force peak and impulse had the best potential to reflect lameness severity and identify subclinical forelimb gait abnormalities. (Am J Vet Res 2005;66:1805–1815)

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in American Journal of Veterinary Research

Abstract

OBJECTIVE To assess efficiency of gravity filtration to enhance recovery of equine bone marrow elements including stem and progenitor cells.

ANIMALS 12 healthy adult horses.

PROCEDURES Bone marrow aspirates were collected from the fifth sternebral body and filtered by gravitational flow to obtain bone marrow elements. Raw and harvested bone marrow and marrow effluent were evaluated for WBC and platelet counts, automated and cytomorphologic cell differential counts, mesenchymal stem cell CFUs, cell viability, and differentiation capacity. Isolated cells were analyzed for CD90 and major histocompatibility complex (MHC) class I and II antigens.

RESULTS Mean cell viability of harvested bone marrow was 95.9%. Total WBCs and platelets were efficiently captured on the filter (> 95%), and mean recovery in harvested bone marrow was 30%. Cytologic cell differential counts indicated that the percentage of neutrophils was significantly less and the progenitor cell population was significantly higher and concentrated 1.56-fold in harvested bone marrow, compared with results for raw bone marrow. Flow cytometry and cell culture were used to characterize harvested bone marrow cells as positive for expression of CD90 and negative for MHCI and MHCII, which indicated stem cells with a multipotent phenotype that differentiated into chondrocytes, osteocytes, adipocytes, and tenocytes.

CONCLUSIONS AND CLINICAL RELEVANCE Gravitational filtration of bone marrow efficiently yielded platelets and cells and produced a progenitor-enriched, leukocyte-reduced product, compared with raw bone marrow.

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in American Journal of Veterinary Research

Abstract

Objective—To evaluate the effects of triamcinolone acetonide (TA), sodium hyaluronate (HA), amikacin sulfate (AS), and mepivacaine hydrochloride (MC) on articular cartilage morphology and matrix composition in lipopolysaccharide (LPS)-challenged and unchallenged equine articular cartilage explants.

Sample Population—96 articular cartilage explants from 4 femoropatellar joints of 2 adult horses.

Procedures—Articular cartilage explants were challenged with LPS (100 ng/mL) or unchallenged for 48 hours, then treated with TA, HA, AS, and MC alone or in combination for 96 hours or left untreated. Cartilage extracts were analyzed for glycosaminoglycan (GAG) content by dimethyl-methylene blue assay (ng/mg of dry wt). Histomorphometric quantification of total lacunae, empty lacunae, and lacunae with pyknotic nuclei was recorded for superficial, middle, and deep cartilage zones.

Results—LPS induced a significant increase in pyknotic nuclei and empty lacunae. Treatment with TA or HA significantly decreased empty lacunae (TA and HA), compared with groups without TA or HA, and significantly decreased empty lacunae of LPS-challenged explants, compared with untreated explants. Treatment with AS or MC significantly increased empty lacunae in unchallenged explants, and these effects were attenuated by TA. Treatment with MC significantly increased empty lacunae and pyknotic nuclei and, in combination with LPS, could not be attenuated by TA. Content of GAG did not differ between unchallenged and LPS-challenged explants or among treatments.

Conclusions and Clinical Relevance—Treatment with TA or HA supported chondrocyte morphology in culture and protected chondrocytes from toxic effects exerted by LPS, AS, and MC.

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in American Journal of Veterinary Research

Abstract

Objective—To determine analgesic effects of intraneural injection of ethyl alcohol or formaldehyde in the palmar digital nerves of horses.

Animals—6 horses.

Procedures—Ethyl alcohol was injected in the medial palmar digital nerve of 1 forelimb, and formaldehyde was injected in the contralateral nerve. The lateral palmar digital nerve in 1 forelimb was surgically exposed, but not injected, and the contralateral lateral palmar digital nerve was not treated. For each heel, severity of lameness in response to experimentally induced heel pain (lameness score and peak vertical force), thermal reaction time, and heel skin sensitivity scores were recorded. Heel pain was experimentally induced by advancing a threaded bolt through a custom-made horseshoe to apply pressure to the palmar horned aspect of the hoof. Horses were followed up for 112 days, when a subset of nerves was sampled for histologic analysis.

Results—Alcohol and formaldehyde significantly reduced all measures of heel pain, and analgesia was evident over the 112 days of the study. Pastern circumference was significantly greater for formaldehyde-treated than for alcohol-treated limbs. Histologic evaluation showed preservation of nerve fiber alignment with an intact epineurium, loss of axons, axon degeneration, fibrosis, and inflammation in alcohol-treated and formaldehyde-treated nerves.

Conclusions and Clinical Relevance—Results suggested that intraneural injection of either ethyl alcohol or formaldehyde in the palmar digital nerves of horses resulted in substantial, but partial, heel analgesia that persisted for at least 112 days. No advantage of formaldehyde over alcohol was found, and formaldehyde resulted in greater soft tissue inflammation.

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in American Journal of Veterinary Research

Abstract

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.

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in American Journal of Veterinary Research

Abstract

Objective—To assess analgesia, inflammation, potency, and duration of action associated with intra-articular injection of triamcinolone acetonide (TA), mepivacaine hydrochloride, or both in metacarpophalangeal (MCP) joints of horses with experimentally induced acute synovitis.

Animals—18 horses.

Procedures—Both forelimbs of each horse were injected with lipopolysaccharide (LPS) 3 times. After the first LPS injection, 1 forelimb of each horse was treated with intra-articular injection of mepivacaine (80 mg; n = 6), TA (9 mg; 6), or mepivacaine with TA (same doses of each; 6) 12 hours after the initial LPS injection. Contralateral limbs served as control limbs. Joint pain was assessed via lameness score and measurements of vertical force peak and pain-free range of motion of the MCP joint. Periarticular edema was evaluated. Degree of synovial inflammation was determined via synovial fluid analysis for WBC count and total protein concentration. Samples of plasma and synovial fluid were analyzed for TA and mepivacaine concentrations.

Results—Each injection of LPS induced lameness and joint inflammation. Mepivacaine effectively eliminated lameness within 45 minutes after injection, regardless of whether TA was also administered, whereas TA reduced lameness, edema, and concentration of synovial fluid protein after the second LPS injection, regardless of whether mepivacaine was also injected. Treatment with TA also induced higher WBC counts and mepivacaine concentrations in synovial fluid, compared with results for mepivacaine alone.

Conclusions and Clinical Relevance—Results suggested TA is a potent analgesic and anti-inflammatory medication for acute synovitis in horses and that simultaneous administration of mepivacaine does not alter the potency or duration of action of TA.

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in American Journal of Veterinary Research

Abstract

Objective—To evaluate use of kinetic gait analysis for detection, quantification, and differentiation of hind limb lameness and spinal ataxia in horses.

Design—Prospective clinical study.

Animals—36 horses.

Procedures—Kinetic gait analysis with a force plate was performed for 12 clinically normal horses, 12 horses with hind limb lameness, and 12 horses with spinal ataxia. Kinetic variables were compared among groups, correlated to subjective grading, and used to build predictive models to assess the accuracy of discrimination.

Results—Subsets of kinetic variables were characteristically altered in ataxic and lame gaits. Ataxic horses had significantly increased lateral force peak and variation in vertical force peaks in both hind limbs. Lame horses had significantly decreased vertical force peak and increased variation in vertical force peaks only in the lame hind limb. These variables were used to differentiate between spinal ataxia and hind limb lameness with excellent accuracy. There were significant correlations between a subset of kinetic variables and subjective lameness and neurologic grades.

Conclusions and Clinical Relevance—Kinetic gait variables, specifically lateral force peak and the variation in vertical force, can be used to support the differential diagnosis between spinal ataxia and hind limb lameness in horses. Kinetic gait analysis may also be applied for quantification of equine hind limb gait abnormalities as well as confirming lack of lameness and ataxia in soundness examinations.

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in Journal of the American Veterinary Medical Association

Abstract

Objective—To evaluate intra-articular autologous protein solution (APS) for the treatment of osteoarthritis in horses.

Animals—40 client-owned horses with naturally occuring osteoarthritis.

Procedures—APS was generated from a dual-device system that concentrated plasma and WBC proteins and enriched platelet growth factors. Horses were randomly assigned to receive an intra-articular injection of 5 mL of saline (0.9% NaCl) solution (n = 20) or APS (20), exercised on a treadmill, and evaluated on the basis of lameness grades, kinetic gait analysis, joint circumference, and range of motion for 14 days. Horses that received saline solution were administered APS at termination of the study, and clients scored horses for lameness and discomfort before, 12 weeks after, and 52 weeks after the APS injection.

Results—The APS group had significant improvements in lameness grade, asymmetry indices of vertical peak force, and range of joint motion by 14 days, compared with baseline or control group values. No adverse effects associated with APS treatment were evident. Clients assessed lameness and comfort as improved at 12 and 52 weeks. The APS had greater likelihood (OR, 4.3 to 30.0) of a therapeutic response in horses with a lameness score < 4, < 10% vertical force asymmetry, or absence of marked osteophyte formation, subchondral sclerosis, or joint space narrowing. Concentration of interleukin-1 receptor antagonist in APS was 5.8 times that in blood.

Conclusions and Clinical Relevance—Intra-articular administration of APS can be considered an effective treatment option for equine osteoarthritis, with the potential for disease-modifying effects.

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in American Journal of Veterinary Research