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Abstract

OBJECTIVE To evaluate effects of various concentrations of collagenase and dimethyl sulfoxide (DMSO) on yield of equine adipose-derived multipotent stromal cells (ASCs) before and after cryopreservation.

SAMPLE Supragluteal subcutaneous adipose tissue from 7 Thoroughbreds.

PROCEDURES Tissues were incubated with digests containing 0.1%, 0.05%, or 0.025% type I collagenase. Part of each resulting stromal vascular fraction was cryopreserved in 80% fetal bovine serum (FBS), 10% DMSO, and 10% Dulbecco modified Eagle medium F-12 and in 95% FBS and 5% DMSO. Half of each fresh and cryopreserved heterogeneous cell population was not immunophenotyped (unsorted) or was immunophenotyped for CD44+, CD105+, and major histocompatability complex class II (MHCII; CD44+-CD105+-MHCII+ cells and CD44+-CD105+-MHCII cells). Cell proliferation (cell viability assay), plasticity (CFU frequency), and lineage-specific target gene and oncogene expression (reverse transcriptase PCR assays) were determined in passage 1 cells before and after culture in induction media.

RESULTS Digestion with 0.1% collagenase yielded the highest number of nucleated cells. Cell surface marker expression and proliferation rate were not affected by collagenase concentration. Cryopreservation reduced cell expansion rate and CD44+-CD105+-MHCII CFUs; it also reduced osteogenic plasticity of unsorted cells. However, effects appeared to be unrelated to DMSO concentrations. There were also variable effects on primordial gene expression among cell isolates.

CONCLUSIONS AND CLINICAL RELEVANCE Results supported the use of 0.1% collagenase in an adipose tissue digest and 5% DMSO in cryopreservation medium for isolation and cryopreservation, respectively, of equine ASCs. These results may be used as guidelines for standardization of isolation and cryopreservation procedures for equine ASCs.

Full access
in American Journal of Veterinary Research
in Journal of the American Veterinary Medical Association
in Journal of the American Veterinary Medical Association

Abstract

Objective—To evaluate correlations among measurements on radiographic and computed tomography (CT) images with articular cartilage microdamage in lax hip joints of dogs.

Animals—12 adult mixed-breed hounds.

Procedures—Pelvic CT and radiography were performed. Hip joints were harvested following euthanasia. Orthopedic Foundation for Animals (OFA) and PennHIP radiograph reports were obtained. Norberg angle (NA) and radiographic percentage femoral head coverage (RPC) were determined. Center-edge angle (CEA), horizontal toit externe angle (HTEA), ventral acetabular sector angle (VASA), dorsal acetabular sector angle (DASA), horizontal acetabular sector angle (HASA), acetabular index (AI), and CT percentage femoral head coverage (CPC) were measured on 2-dimensional CT images. Femoral head–acetabular shelf percentage was measured on sagittal 3-dimensional CT (SCT) and transverse 3-dimensional CT (TCT) images. Light microscopy was used to score joint cartilage. Relationships of OFA confirmation and PennHIP osteoarthritis scores with radiography, CT, and cartilage variables and relationships of cartilage scores with radiography and CT measurements were evaluated with Spearman rank correlations. Pearson correlation was used for relationships of distraction index (DI) with radiography, CT, and cartilage variables.

Results—Significant relationships included PennHIP osteoarthritis score with cartilage score, CEA, HTEA, DASA, AI, CPC, and TCT; OFA confirmation score with cartilage score, NA, RPC, CEA, HTEA, DASA, AI, CPC, and TCT; cartilage score with NA, RPC, CEA, HTEA, DASA, HASA, AI, and TCT; and DI with cartilage score, CEA, HTEA, DASA, HASA, AI, and CPC.

Conclusions and Clinical Relevance—CT appeared to be a valuable imaging modality for predicting cartilage microdamage in canine hip joints.

Full access
in American Journal of Veterinary Research

Abstract

OBJECTIVE

To evaluate differentiation of canine adipose–derived multipotent stromal cells (ASCs) into ligamentoblasts on tensioned collagen type I (Col1) templates in a perfusion culture system.

SAMPLES

Infrapatellar fat pad ASCs from healthy stifle joints of 6 female mixed-breed dogs.

PROCEDURES

Third-passage ASCs (6 × 106 cells/template) were loaded onto suture-augmented Col1 templates under 15% static strain in perfusion bioreactors. Forty-eight ASC-Col1 constructs were incubated with ligamentogenic (ligamentogenic constructs; n = 24) or stromal medium (stromal constructs; 24) for up to 21 days. Specimens were collected from each construct after 2 hours (day 0) and 7, 14, and 21 days of culture. Cell number, viability, distribution, and morphology; construct collagen content; culture medium procollagen-I-N-terminal peptide concentration; and gene expression were compared between ligamentogenic and stromal constructs.

RESULTS

ASCs adhered to collagen fibers. Cell numbers increased from days 0 to 7 and days 14 to 21 for both construct types. Relative to stromal constructs, cell morphology and extracellular matrix were more mature and collagen content on day 21 and procollagen-I-N-terminal peptide concentration on days 7 and 21 were greater for ligamentogenic constructs. Ligamentogenic constructs had increased expression of the genes biglycan on day 7, decorin throughout the culture period, and Col1, tenomodulin, fibronectin, and tenascin-c on day 21; expression of Col1, tenomodulin, and tenascin-c increased between days 7 and 21.

CONCLUSIONS AND CLINICAL RELEVANCE

Ligamentogenic medium was superior to stromal medium for differentiation of ASCs to ligamentoblasts on suture-augmented Col1 scaffolds. Customized ligament neotissue may augment treatment options for dogs with cranial cruciate ligament rupture.

Free access
in American Journal of Veterinary Research

Abstract

OBJECTIVE To evaluate associations of measures assessed by radiography, 2-D CT, and 3-D CT of the hip joints of immature dogs with osteoarthritis in the same joints at maturity.

ANIMALS 46 hound-type dogs from a colony predisposed to osteoarthritis.

PROCEDURES Images of hip joints (1/dog) were obtained at 16, 32, and 104 weeks of age. Radiographic measures included Norberg angle, distraction index, and osteoarthritis score. Two-dimensional CT measures included acetabular index, percentage of femoral head coverage, and center edge, horizontal toit externe, acetabular anteversion, and ventral, dorsal, and horizontal acetabular sector angles. Three-dimensional CT measures were femoral head and neck volume, femoral neck angle, and femoral head and neck radius. Differences among measures at 16 and 32 weeks in dogs with different osteoarthritis scores at later time points, relationships among variables at each time point, and relationships of single and combined measures with the presence of osteoarthritis at 104 weeks were evaluated.

RESULTS The 16- and 32-week distraction index, center edge angle, dorsal acetabular sector angle, horizontal acetabular sector angle, percentage of femoral head coverage, acetabular index, and Norberg angle and the 32-week femoral neck angle varied significantly with osteoarthritis severity at 104 weeks. Presence of osteoarthritis in mature dogs was most strongly associated with 16-week combined measures of distraction index and center edge angle and 32-week combined measures of dorsal acetabular sector angle and Norberg angle.

CONCLUSIONS AND CLINICAL RELEVANCE Changes in hip joint morphology associated with radiographic signs of osteoarthritis were detectable as early as 16 weeks of age and varied with osteoarthritis severity in adult dogs. The use of combined hip joint measures may improve early identification of dogs predisposed to hip joint osteoarthritis.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To characterize ground reaction forces (GRFs) and determine whether there were correlations between forces and passive coxofemoral joint laxity in puppies.

Animals—Fifty-one 16-week-old hound-breed dogs.

Procedure—Force-plate gait evaluation and distraction radiographic imaging were performed. Ground reaction forces evaluated included x (mediolateral), y (craniocaudal breaking and propulsion), and z (vertical) peak force and impulse. Z-plane limb loading and unloading rates, loading interval, and weight distribution and y-plane stance time breaking and propulsion percentages were calculated. One-way ANOVA with the Duncan multiple range test was used to evaluate differences in gait variables among limbs. The relationships of left, right, highest, and mean distraction index (DI) with individual limb data of each dog were evaluated with the Spearman rank correlation. Left and right DIs were compared by means of linear regression analysis.

Results—Mean ± SEM DI was 0.67 ± 0.02. Left and right DIs were strongly correlated, but there were no significant relationships between DIs and gait variables. Most fore- and hind limb gait variables differed significantly, whereas paired fore- and hind limb gait variables did not. Asymmetry was most pronounced in the x- and y-planes.

Conclusions and Clinical Relevance—GRFs were consistent with those of clinically normal mature dogs, supporting an absence of association between GRF and DI in young dogs. The GRFs and elucidation of the relationship between GRFs and DI may be useful for future studies in immature dogs.

Full access
in American Journal of Veterinary Research

Abstract

OBJECTIVE

To evaluate factors contributing to owner decision-making, satisfaction, and perception of quality of life (QOL) with treatment of dogs with nontraumatic hemoabdomen (NTH).

ANIMALS

132 client-owned dogs.

PROCEDURES

An electronic survey was administered to owners of 436 dogs that presented emergently with NTH to a single institution between January 2015 and May 2022. Following survey response, retrospective data collection was performed.

RESULTS

Owners reported QOL as the most important factor influencing their decision-making (92%), followed by risk of cancer (57%) or time remaining with their pet (56%). QOL scores were significantly higher with surgery versus those with palliative care (P = .007). Median survival time (MST) was 213 days with surgery and 39 days with palliative care (P = .049). Survival benefit of surgery was lost when considering only dogs with malignant histopathology (MST, 81 days; P = .305). Owners were more likely to be satisfied when they chose surgery over either euthanasia or palliative care (P = .039). Thirty-four owners (26%) second-guessed or were unsure of their decision.

CLINICAL RELEVANCE

Surgery resulted in the longest MST with greater perceived QOL and owner satisfaction compared with both palliative care and euthanasia and should therefore be considered highly. The importance of malignancy and survival time on owners’ decisions, along with the negative impact of metastasis on survival, underscores the importance of timely preoperative staging. The rate of second-guessing highlights the need for standardization of NTH discussions including treatment options and potential outcomes in order to effectively and efficiently guide treatment of patients with this common presentation.

Full access
in Journal of the American Veterinary Medical Association

Abstract

Objective

To determine the relation between epidural injectate volume (ml/kg of body weight) and its craniad migration in calves and pigs.

Animals

23 neonatal calves and 26 feeder pigs.

Procedure

Animals were randomly assigned to receive different volumes of new methylene blue (NMB, 1.2 mg/ml in 0.9% saline solution). Injections were made into the sacrococcygeal intervertebral space in calves and the lumbosacral intervertebral space in pigs, immediately after euthanasia. Sagittal sections of the spine were made at necropsy, and craniad migration of NMB was determined and rounded to the nearest intervertebral space.

Results

In calves treated with 0.05, 0.1, or 0.15 ml of NMB/kg, mean ± SEM number of stained spinal segments was 5 ± 0.3, 8 ± 0.6, and 8 ± 0.6, respectively. Craniad migration of NMB was significantly greater for 0.15 and 0.1 ml/kg volumes versus 0.05 ml/kg. In pigs treated with 0.05, 0.1, 0.2, or 0.3 ml of NMB/kg, mean number of stained spinal segments was 8 ± 1.1, 8 ± 0.9, 10 ± 1.2, and 18 ± 2.0. Craniad dye migration was significantly greater in the 0.3 ml/kg group versus the 3 lower volume groups. Linear regression performed on both sets of data after logarithmic transformation of spaces migrated to correct for non-normality was significant (P < 0.05), and R 2 values of 0.49 and 0.55 were obtained for calves and pigs, respectively.

Conclusions

There is a significant correlation between volume (ml/kg) of NMB injected in the epidural space and its craniad migration in calves and pigs.

Clinical Relevance

Results provide a basis for determination of volume of injectate to be given to reach a minimal desired level and should be a useful baseline for future investigations of epidural drug administration. (Am J Vet Res 1997;58:786–790)

Free access
in American Journal of Veterinary Research
in Journal of the American Veterinary Medical Association