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Osteoarthritis (OA) is a leading cause of morbidity among aging populations, yet symptom and/or disease-modification remains elusive. Adipose-derived mesenchymal stromal cells (adMSCs) have demonstrated immunomodulatory and anti-inflammatory properties that may alleviate clinical signs and interrupt disease onset and progression. Indeed, multiple manuscripts have evaluated intra-articular administration of adMSCs as a therapeutic; however, comparatively few evaluations of systemic delivery methods have been published. Therefore, the aim of this study was to evaluate the short-term impact of intravenous (IV) delivery of allogeneic adMSCs in an established model of spontaneous OA, the Hartley guinea pig. Animals with moderate OA received once weekly injections of 2 × 10

Older age is the primary risk factor for most chronic diseases, including Alzheimer’s disease (AD). Current preclinical models to study brain aging and AD are mainly transgenic and harbor mutations intended to mirror brain pathologies associated with human brain aging/AD (eg, by increasing production of the amyloid precursor protein, amyloid beta [Aβ], and/or phosphorylated tau, all of which are key pathological mediators of AD). Although these models may provide insight on pathophysiological processes in AD, none completely recapitulate the disease and its strong age-dependence, and there has been limited success in translating preclinical results and treatments to humans. Here, we describe 2 nontransgenic guinea pig (GP) models, a standard PigmEnTed (PET) strain, and lesser-studied Dunkin-Hartley (DH) strain, that may naturally mimic key features of brain aging and AD in humans. We show that brain aging in PET GP is transcriptomically similar to human brain aging, whereas older DH brains are transcriptomically more similar to human AD. Both strains/models also exhibit increased neurofilament light chain (NFL, a marker of neuronal damage) with aging, and DH animals display greater S100 calcium-binding protein B (S100β), ionized calcium-binding adapter molecule 1 (Iba1), and Aβ and phosphorylated tau—which are all important markers of neuroinflammation-associated AD. Collectively, our results suggest that both the PET and DH GP may be useful, nontransgenic models to study brain aging and AD, respectively.

Iron has been emerging as a key contributor to aging-associated, chronic disorders due to the propensity for generating reactive oxygen species. To date, there are a limited number of publications exploring the role of iron in the pathogenesis of primary/age-related osteoarthritis (OA). The objective of this study was to determine whether reduced iron via pharmacologic iron chelation with deferoxamine (DFO) affected the development and/or severity of cartilage lesions in a primary OA model. At 12-weeks-of-age, 15 male Dunkin-Hartley guinea pigs received either 46 mg/kg DFO (n = 8) or vehicle control (n = 7) injected subcutaneously twice daily for five days each week. Movement changes, captured via overhead enclosure monitoring, were also determined. Termination occurred at 30-weeks-of-age. Iron was quantified in serum, urine, liver, and femoral head articular cartilage. Left knees were evaluated for: structural changes using histopathology guidelines; and immunohistochemistry. Gene expression analysis was conducted on right knee articular cartilage. DFO reduced iron levels in femoral head articular cartilage (p = 0.0006) and liver (p = 0.02), and increased iron within urine (p = 0.04) and serum (p = 0.0009). Mobility of control animals declined, while the DFO group maintained activity levels similar to the first month of treatment (p = 0.05). OA-associated cartilage lesions were reduced in knees of DFO animals (p = 0.0001), with chondrocyte hypocellularity a key histologic difference between groups (p 0.0001). DFO-receiving animals had increased immunostaining for phosphorylated adenosine monophosphate activated protein kinase alpha within knee articular cartilage; lower transcript counts of several proapoptotic genes (p = 0.04-0.0004) and matrix-degrading enzymes (p = 0.02-0.0001), and increased expression of the anti-apoptotic gene Bcl-2 (p 0.0001) and a tissue inhibitor of matrix-metalloproteinases (p = 0.03) were also observed. These results suggest that iron chelation delayed the progression of primary OA in an animal model and could hold potential as a translational intervention. These findings provide expanded insight into factors that may contribute to the pathogenesis of primary OA.

Background The infrapatellar fat pad (IFP) is the largest adipose deposit in the knee; however, its contributions to the homeostasis of this organ remain undefined. To determine the influence of the IFP and its associated synovium (IFP/synovium complex or IFP/SC) on joint health, this study evaluated the progression of osteoarthritis (OA) following excision of this unit in a rodent model of naturally-occurring disease. Methods Male Dunkin-Hartley guinea pigs (n=18) received surgical removal of the IFP in one knee at 3 months of age; contralateral knees received sham surgery as matched internal controls. Mobility and gait assessments were performed prior to IFP/SC removal and monthly thereafter. Animals were harvested at 7 months of age. Ten set of these knees were processed for microcomputed tomography (microCT), histopathology, transcript expression analyses, and immunohistochemistry (IHC); 8 sets of knees were dedicated to microCT and biomechanical testing (material properties of knee joints tissues and anterior drawer laxity). Results Fibrous connective tissue (FCT) developed in place of the native adipose depot. Gait demonstrated no significant differences between IFP/SC removal and contralateral hindlimbs. MicroCT OA scores were improved in knees containing the FCT. Quantitatively, IFP/SC-containing knees had more osteophyte development and increased trabecular volume bone mineral density (vBMD) in femora and tibiae. Histopathology confirmed maintenance of articular cartilage structure, proteoglycan content, and chondrocyte cellularity in FCT-containing knees. Transcript analyses revealed decreased expression of adipose-related molecules and select inflammatory mediators in FCTs compared to IFP/SCs. This was verified via IHC for two key inflammatory agents. The medial articular cartilage in knees with native IFP/SCs showed an increase in equilibrium modulus, which correlated with increased amounts of magnesium and phosphorus. Discussion/conclusion Formation of the FCT resulted in reduced OA-associated changes in both bone and cartilage. This benefit may be associated with: a decrease in inflammatory mediators at transcript and protein levels; and/or improved biomechanical properties. Thus, the IFP/SC may play a role in the pathogenesis of knee OA in this strain, with removal prior to disease onset appearing to have short-term benefits.

Cannabidiol (CBD) has gained widespread popularity as a treatment for osteoarthritis (OA) in pets; however, there is minimal scientific evidence regarding safe and effective dosing. This study determined plasma and tissue pharmacokinetics after oral CBD oil suspension administration in Hartley guinea pigs (Cavia porcellus), which spontaneously develop OA at 3 months of age. Ten, 5-month-old, male guinea pigs were randomly assigned to receive 25 (n = 5) or 50 mg/kg (n = 5) CBD oil once orally. Blood samples were collected at 0, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h timepoints. Open-field enclosure monitoring revealed no adverse effects. After euthanasia, stifle cartilage and infrapatellar fat pads were collected to quantitate CBD. CBD concentrations were determined using a validated liquid chromatography-mass spectrometry method, and pharmacokinetic parameters were calculated using noncompartmental analysis. The area under the plasma concentration-versus-time curve was 379.5 and 873.7 h*ng/mL, maximum plasma concentration was 42 and 96.8 ng/mL, time to maximum plasma concentration was 1.6 and 4.8 h, and terminal phase half-life was 8.1 and 10.8 h for the 25 and 50 mg/kg doses, respectively. CBD was detected in joint tissues of all animals. Further studies, including work in female guinea pigs, are needed to determine the efficacy of CBD for OA.

Background: The infrapatellar fat pad (IFP) is the largest adipose deposit in the knee; however, its contributions to the homeostasis of this organ remain undefined. To determine the influence of IFP on joint health, this study evaluated the progression of osteoarthritis (OA) following excision of the IFP in a rodent model of naturally-occurring disease. Methods: Male Dunkin-Hartley guinea pigs (n=18) received surgical removal of the IFP in one knee at 3 months of age; contralateral knees received sham surgery as matched internal controls. Mobility and gait assessments were performed prior to IFP removal and monthly thereafter. Animals were harvested at 7 months of age. Ten set of these knees were processed for microcomputed tomography (microCT), histopathology, transcript expression analyses, and immunohistochemistry (IHC); 8 sets of knees were dedicated to microCT and biomechanical testing (material properties of knee joints tissues and anterior drawer laxity). Results: Fibrous connective tissue (FCT) developed in place of the native adipose depot. Gait demonstrated no significant differences between IFP removal and contralateral hindlimbs. MicroCT OA scores were improved in knees containing the FCT. Quantitatively, IFP-containing knees had more osteophyte development and increased trabecular volume bone mineral density (vBMD) in femora and tibiae. Histopathology confirmed maintenance of articular cartilage structure, proteoglycan content, and chondrocyte cellularity in FCT-containing knees. Transcript analyses revealed decreased expression of adipose-related molecules and select inflammatory mediators in FCTs compared to IFPs. This was verified via IHC for inflammatory mediators. The medial articular cartilage in knees with native IFPs showed an increase in equilibrium modulus, which correlated with increased amounts of magnesium and phosphorus. Discussion/Conclusion: Formation of the FCT resulted in reduced OA-associated changes in both bone and cartilage. This benefit may be associated with: a decrease in inflammatory mediators at transcript and protein levels; and/or improved biomechanical properties. Thus, the IFP may play a role in the pathogenesis of knee OA in this strain, with removal prior to disease onset appearing to have short-term benefits.

The infrapatellar fat pad (IFP) is the largest adipose deposit in the knee; however, its contributions to the homeostasis of this organ remain undefined. To determine the influence of the IFP and its associated synovium (IFP/synovium complex or IFP/SC) on joint health, this study evaluated the progression of osteoarthritis (OA) following excision of this unit in a rodent model of naturally-occurring disease.Male Dunkin-Hartley guinea pigs (n=18) received surgical removal of the IFP in one knee at 3 months of age; contralateral knees received sham surgery as matched internal controls. Mobility and gait assessments were performed prior to IFP/SC removal and monthly thereafter. Animals were harvested at 7 months of age. Ten set of these knees were processed for microcomputed tomography (microCT), histopathology, transcript expression analyses, and immunohistochemistry (IHC); 8 sets of knees were dedicated to microCT and biomechanical testing (material properties of knee joints tissues and anterior drawer laxity).Fibrous connective tissue (FCT) developed in place of the native adipose depot. Gait demonstrated no significant differences between IFP/SC removal and contralateral hindlimbs. MicroCT OA scores were improved in knees containing the FCT. Quantitatively, IFP/SC-containing knees had more osteophyte development and increased trabecular volume bone mineral density (vBMD) in femora and tibiae. Histopathology confirmed maintenance of articular cartilage structure, proteoglycan content, and chondrocyte cellularity in FCT-containing knees. Transcript analyses revealed decreased expression of adipose-related molecules and select inflammatory mediators in FCTs compared to IFP/SCs. This was verified via IHC for two key inflammatory agents. The medial articular cartilage in knees with native IFP/SCs showed an increase in equilibrium modulus, which correlated with increased amounts of magnesium and phosphorus.Formation of the FCT resulted in reduced OA-associated changes in both bone and cartilage. This benefit may be associated with: a decrease in inflammatory mediators at transcript and protein levels; and/or improved biomechanical properties. Thus, the IFP/SC may play a role in the pathogenesis of knee OA in this strain, with removal prior to disease onset appearing to have short-term benefits.

Summary Objective Iron is emerging as a key player in aging-associated diseases due to its propensity for driving free radical formation. Studies examining the role of iron in the pathogenesis of primary osteoarthritis (OA) are limited. Our objective was to establish a direct relationship between excess iron and OA by administering iron dextran to a guinea pig strain with decreased propensity for developing this disease. Design Twenty, 12-week-old Strain 13 guinea pigs received either iron dextran or dextran control intraperitoneally once weekly for 4 weeks; termination occurred at 16 weeks of age. Iron levels were determined systemically (serum and liver) and within diarthrodial joints [femoral head articular cartilage and infrapatellar fat pads (IFPs) of knee joints]. One knee was collected to score structural changes associated with OA via microcomputed tomography (microCT) and histology using published grading schemes. Articular cartilage and IFPs were harvested from contralateral knees for gene expression analyses. Results Iron overload was confirmed systemically via increased serum iron and liver iron concentration. Articular cartilage and IFPs in the iron dextran group also had higher levels of iron. Excess iron worsened knee OA using both microCT and histologic scoring systems. Gene analyses revealed that exogenous iron altered the expression of iron trafficking proteins, select cytokines, and structural components of cartilage. Conclusion These results demonstrate that systemic iron overload caused cellular iron accumulation in the knee joint. This excess iron is associated with increased expression of local inflammatory mediators and early onset and progression of knee joint OA in Strain 13 animals.

The anterior cruciate ligament (ACL) is the most commonly injured knee ligament. Surgical reconstruction is the gold standard treatment for ACL ruptures, but 20-50% of patients develop post-traumatic osteoarthritis (PTOA). ACL rupture is thus a well-recognized etiology of PTOA; however, little is known about the initial relationship between ligamentous injury and subsequent PTOA. The goals of this project were to: (1) develop both partial and full models of mid-substance ACL rupture in male and female mice using non-invasive mechanical methods by means of tibial displacement; and (2) to characterize early PTOA changes in the full ACL rupture model. A custom material testing system was utilized to induce either partial or full ACL rupture by means of tibial displacement at 1.6 or 2.0 mm, respectively. Mice were euthanized either (i) immediately post-injury to determine rupture success rates or (ii) 14 days post-injury to evaluate early PTOA progression following full ACL rupture. Our models demonstrated high efficacy in inciting either full or partial ACL rupture in male and female mice within the mid-substance of the ACL. These tools can be utilized for preclinical testing of potential therapeutics and to further our understanding of PTOA following ACL rupture.

BackgroundThe infrapatellar fat pad (IFP) is the largest adipose deposit in the knee; however, its contributions to the homeostasis of this organ remain unknown. To determine the influence of this depot on joint health, this study determined the progression of osteoarthritis (OA) following excision of the IFP in a rodent model of naturally-occurring disease.MethodsMale Dunkin-Hartley guinea pigs (n=10) received surgical removal of the IFP in one knee at 3 months of age; contralateral knees received sham surgery as matched internal controls. Treadmill-based gait analysis was performed prior to IFP removal and monthly thereafter. Animals were harvested at 7 months of age. Both knees were processed for microcomputed tomography (microCT), histopathology, transcript expression analyses, and immunohistochemistry (IHC).ResultsFibrous connective tissue (FCT) developed in place of the native fat pad. Gait demonstrated no significant differences between IFP removal and contralateral limbs. MicroCT OA scores were improved in knees with the FCT. Histopathology confirmed maintenance of articular cartilage structure, proteoglycan content, and chondrocyte cellularity in FCT-containing knees. Transcript analyses revealed decreased expression of adipose-related molecules and inflammatory mediators in FCTs compared to IFPs. This was corroborated via IHC for select inflammatory mediators.Discussion/ConclusionFormation of the FCT resulted in reduced OA-associated changes in both bone and cartilage. A decrease in inflammatory mediators at transcript and protein levels may be associated with these improvements. The IFP may therefore play a role in the pathogenesis of knee OA in this strain, with removal prior to disease onset appearing to have short-term benefits.

Objectives The aim of this study was to evaluate the safety and collect pilot data measuring clinical effects of intravenously administered, adipose-derived, culture-expanded, allogeneic mesenchymal stem cells in dogs with elbow osteoarthritis. Materials and Methods Dogs (n = 13) with naturally occurring elbow osteoarthritis received three intravenous doses of allogeneic canine mesenchymal stem cells via an open-label clinical trial. Primary outcome measures collected over a 6-month study period included objective gait analysis, accelerometry, owner questionnaires and joint fluid analysis. Results No acute adverse events were observed following repeated intravenous treatment with allogeneic mesenchymal stem cells. A significant improvement in mean client-specific outcome measure (CSOM) activity score and CSOM behaviour score was observed when pre-treatment values were compared with post-treatment values (day >28). In contrast, mean peak vertical force significantly decreased from baseline to post-treatment (>day 28). Weekly activity counts did not show a significant difference between baseline to post-treatment time points. Synovial fluid biomarkers did not change during treatment, and labelled mesenchymal stem cells were rarely detected in synovial fluid samples collected after mesenchymal stem cell administration. Clinical Significance For dogs with naturally occurring elbow osteoarthritis, intravenous administration of mesenchymal stem cells was clinically well tolerated. While some subjective outcome measures showed significant improvements, objective outcome measures did not confirm similar changes. Further research is needed before intravenous mesenchymal stem cells can be recommended as a treatment for elbow osteoarthritis in dogs.

Musculoskeletal dysfunction is an age-related syndrome associated with impaired mitochondrial function and proteostasis. However, few interventions have tested targeting two drivers of musculoskeletal decline. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that stimulates transcription of cytoprotective genes and improves mitochondrial function. We hypothesized daily treatment with a Nrf2 activator in Hartley guinea pigs, a model of age-related musculoskeletal dysfunction, attenuates the progression of skeletal muscle mitochondrial dysfunction and impaired proteostasis, preserving musculoskeletal function. We treated 2-month- and 5-month-old male and female Hartley guinea pigs for 3 and 10 months, respectively, with the phytochemical Nrf2 activator PB125 (Nrf2a). Longitudinal assessments of voluntary mobility were measured using Any-Maze™ open-field enclosure monitoring. Cumulative skeletal muscle protein synthesis rates were measured using deuterium oxide over the final 30 days of treatment. Mitochondrial oxygen consumption in permeabilized soleus muscles was measured using ex vivo high resolution respirometry. In both sexes, Nrf2a 1) increased electron transfer system capacity; 2) attenuated the disease/age-related decline in coupled and uncoupled mitochondrial respiration; and 3) attenuated declines in protein synthesis in the myofibrillar, mitochondrial, and cytosolic subfractions of the soleus. These improvements were not associated with statistically significant prolonged maintenance of voluntary mobility in guinea pigs. Collectively, these results demonstrate that treatment with an oral Nrf2 activator contributes to maintenance of skeletal muscle mitochondrial function and proteostasis in a pre-clinical model of musculoskeletal decline. Further investigation is necessary to determine if these improvements are also accompanied by slowed progression of other aspects of musculoskeletal decline.

Objective: Faced with the frustration of chronic discomfort and restricted mobility due to osteoarthritis (OA), many individuals have turned to acupuncture for relief. However, the efficacy of acupuncture for OA is uncertain, as much of the evidence is inconclusive. The purpose of this study was to evaluate electroacupuncture (EA) in a rodent model of OA such that conclusions regarding its effectiveness for symptom or disease modification could be drawn. Methods: Ten 12-month-old male Hartley guinea pigs—which characteristically have moderate to advanced OA at this age—were randomly assigned to receive EA for knee OA (n = 5) or anesthesia only (control group, n = 5). Treatments were performed three times weekly for 3 weeks, followed by euthanasia 2 weeks later. Gait analysis and enclosure monitoring were performed weekly to evaluate changes in movement. Serum was collected for inflammatory biomarker testing. Knee joints were collected for histology and gene expression. Results: Animals receiving EA had significantly greater changes in movement parameters compared to those receiving anesthesia only. There was a tendency toward decreased serum protein concentrations of complement component 3 (C3) in the EA group compared to the control group. Structural and antioxidant gene transcripts in articular cartilage were increased by EA. There was no significant difference in total joint histology scores between groups. Conclusion: This study provides evidence that EA has a positive effect on symptom, but not disease, modification in a rodent model of OA. Further investigations into mechanistic pathways that may explain the efficacy of EA in this animal model are needed.

Skeletal muscle dysfunction, articular cartilage degeneration, and bone loss occur essentially in parallel during aging. Mechanisms contributing to this systemic musculoskeletal decline remain incompletely understood, limiting progress toward developing effective therapeutics. Because the progression of human musculoskeletal aging is slow, researchers rely on rodent models to identify mechanisms and test interventions. The Dunkin Hartley guinea pig is an outbred strain that begins developing primary osteoarthritis by 4 months of age with a progression and pathology similar to aging humans. The purpose of this study was to determine if skeletal muscle remodeling during the progression of osteoarthritis in these guinea pigs resembles musculoskeletal aging in humans. We compared Dunkin Hartley guinea pigs to Strain 13 guinea pigs, which develop osteoarthritis much later in the lifespan. We measured myofiber type and size, muscle density, and long-term fractional protein synthesis rates of the gastrocnemius and soleus muscles in 5, 9, and 15-month-old guinea pigs. There was an age-related decline in skeletal muscle density, a greater proportion of smaller myofibers, and a decline in type II concomitant with a rise in type I myofibers in the gastrocnemius muscles from Dunkin Hartley guinea pigs only. These changes were accompanied by age-related declines in myofibrillar and mitochondrial protein synthesis in the gastrocnemius and soleus. Collectively, these findings suggest Dunkin Hartley guinea pigs experience myofiber remodeling alongside the progression of osteoarthritis, consistent with human musculoskeletal aging. Thus, Dunkin Hartley guinea pigs may be a model to advance discovery and therapeutic development for human musculoskeletal aging.

There is potential discrepancy between human and laboratory animal studies of osteoarthritis (OA), as radiographic assessment is the hallmark of the former and histopathology the standard for the latter. This suggests a need to evaluate OA in animal models in a manner similar to that utilized in people. Our study aimed to develop a whole joint grading scheme for microcomputed tomography (microCT) images in Hartley guinea pigs, a strain that recapitulates joint changes highlighted in human spontaneous OA.Knees from animals aged 2, 3, 5, 9, and 15 months were evaluated via whole joint microCT and standard histologic scoring. Quantitative microCT parameters, such as bone volume/total volume were also collected.Both whole joint microCT and histologic scores increased with advancing age and showed strong correlation (r = 0.89. p 0.0001). Histologic scores, which focus on cartilage changes, increased progressively with age. Whole joint microCT scores, which characterize bony changes, followed a stepwise pattern: scores increased between 3 and 5 months of age, stayed consistent between 5 and 9 months, and worsened again between 9 and 15 months.This work provides data that advocates the use of a whole joint microCT scoring system in guinea pig studies of OA, as it provides important information regarding bony changes that occur at a different rate than articular cartilage changes. This grading scheme, in conjunction with histology and quantitative microCT measurements, may enhance the translational value of this animal model as it pertains to human work.

Background Feline Infectious Peritonitis (FIP) is a fatal disease of cats that can be very difficult to definitively diagnose antemortem. Multiplex fluorescent immunocytochemical (MF-ICC) assays are emerging as useful diagnostic tests in veterinary medicine, particularly for fluid samples. Objective We aimed to develop and optimize an MF-ICC assay to detect feline coronavirus within macrophages, with the primary goal of determining the allowable/recommended sample storage conditions for clinical use of this assay. Methods A feline macrophage cell line was infected with the FIP virus. Following harvest into EDTA tubes (simulating typical clinical collection of effusion), cells were stored at 4℃, 22℃, and 37℃. For each temperature condition, slides for MF-ICC were made at 0, 1, 2, 3, and 5 days post-collection. To assess the stability of immunoreactivity following fixation, freshly harvested infected cells were fixed onto slides and maintained at 4℃ for 1, 2, 4, and 12 weeks. All slides were analyzed by MF-ICC for the presence of mononuclear cells with co-expression of vimentin and coronaviral antigen. Results MF-ICC confirmed that cells tested positive for coronavirus at 4℃ through 3 days post-harvest, 22℃ through 48 hours post-harvest, and 37℃ through 24 hours post-harvest. The MF-ICC assay was successfully performed on fixed slides through the 12-week time point. This assay also demonstrated positive results on a clinical sample of abdominal fluid from a cat later confirmed to have FIP. Conclusions The MF-ICC assay described here offers a potentially specific and relatively stable antemortem diagnostic test for feline infectious peritonitis. Evaluation of this assay in clinical samples is ongoing.

BACKGROUND Biochemistry analyzers in many high-throughput laboratories use indirect potentiometry to determine serum electrolyte concentrations, which involves a pre-analytical dilution step that may be associated with artifactual increases or decreases in electrolyte concentrations under circumstances of altered serum water fraction (SWF). Severe hypo- and hyperproteinemia, conditions that cause altered SWF, are recognized but under-emphasized causes of falsely measured serum sodium concentrations. OBJECTIVES The goals of this study were to determine the average actual SWF (SWFA ) and establish formulae to correct serum sodium concentration measured by indirect potentiometry in hypo- and hyperproteinemic cats. METHODS Serum samples from 112 feline patients were analyzed for electrolytes (measured by both indirect and direct potentiometry), total protein, albumin, triglycerides, and cholesterol. Each serum sample was also lyophilized to determine the SWFA . A feline-specific formula to estimate SWF (SWFE-FEL ) was developed and evaluated with a multivariable linear model. RESULTS The mean SWFA in this population of cats was 91.2%, which was significantly different (P

Synovial fluid analysis is a key component of the minimum database needed to diagnose and manage primary and secondary articular joint disorders. Unfortunately, preanalytical variables can drastically alter samples submitted for evaluation to veterinary laboratories and it is considered the stage at which most laboratory error occurs. This article addresses common sources of preanalytical variability and error that are seen in veterinary medicine. With consistent quality control and reporting of specimens, downstream clinical decision making and management of patients can be accelerated and improved.

A 6-year-old, male castrated, mixed-breed dog was referred to the James L. Voss Veterinary Teaching Hospital at Colorado State University for bicavitary effusion. On examination, the dog was tachycardic and tachypneic with bilaterally decreased lung sounds. Thoracic and abdominal ultrasonic examination revealed pleural and peritoneal effusions, which were aspirated and submitted for fluid analysis and cytology. Both cavity fluids were classified as exudates with a large population of vacuolated mononuclear cells. Multiplex immunocytochemistry (ICC) for cytokeratin and vimentin demonstrated exclusively cytokeratin expression, indicating these cells were of epithelial origin. A full diagnostic evaluation was performed, including CBC, clinical chemistry, a pet-side test for heartworm disease, ehrlichiosis, Lyme disease, and anaplasmosis, imaging modalities of thorax, abdomen, and heart, urinalysis, and fine-needle aspirations of spleen, liver, and popliteal lymph nodes. The dog was diagnosed with pleural and peritoneal carcinoma with presumed carcinomatosis. A single dose of intracavitary carboplatin was administered before discharge, and over a period of 2 weeks, 5 thoracocenteses were performed. A subcutaneous mass was noted at a thoracocentesis site one week after initial presentation. Cytology of the mass was consistent with carcinoma, and neoplastic seeding of the tumor cells from the thoracocentesis was suspected. The dog was euthanized 15 days after the first visit, and a necropsy was performed. Findings were consistent with carcinomatosis secondary to anaplastic pulmonary carcinoma with transient subcutaneous seeding of neoplastic cells during routine thoracocentesis. This case demonstrates the utility of multiplex ICC in the clinical setting.

Whole transferrin receptor (TfR) is present in reticulocyte exosomes. Soluble transferrin receptor (sTfR) is cleaved from whole TfR in human plasma, with the remnant cytoplasmic domain (cTfR) remaining membrane associated. In humans, sTfR is a biomarker that can detect iron deficiency in the presence of inflammatory disease. This condition is still a diagnostic dilemma in veterinary species. We aimed to (1) confirm the presence of exosomes and exosome-associated TfR in the serum of dogs, cats, and horses; and (2) to assess and compare the proportion of cTfR to total (cTfR + whole) in exosomal membranes of healthy and diseased dogs and cats and in healthy horses to indirectly predict their anticipated levels of circulating sTfR. We used discarded serum and whole blood samples from canine and feline patients, separated into healthy and diseased groups based on the health status of each patient, and healthy equine participants from a previous study. Ultracentrifugation, followed in some experiments by OptiPrep discontinuous density gradient fractionation, was used to isolate exosomes. Exosomes and associated TfR were identified using TEM and Western blot for TfR, respectively. Densitometry tracings of Western blots of serum exosomes were used to measure the proportion of cTfR to total TfR. Extracellular vesicles compatible with exosomes were successfully isolated and expressed TfR. The proportion of cTfR in dogs was greater than 50%, indicating that a majority of the whole TfR was cleaved to produce sTfR (and remnant cTfR). There was significant interindividual variation and no significant difference between healthy and diseased animals. The proportion of cTfR in cats was very low at 11%, indicating that very little sTfR was likely produced. There was a small yet significant difference between healthy and diseased cats. Healthy horses do not appear to cleave exosome-associated TfR. Diagnosis of iron deficiency in the presence of inflammatory disease remains a challenge in veterinary medicine. Our results indicate that TfR is poorly or unpredictably cleaved in veterinary species, revealing that there are species differences in exosomal TfR handling. These data suggest that development of an assay for the detection and quantification of sTfR in the species investigated may not be warranted.

Background True and functional iron deficiency can result in anemia. Current tests to assess iron status often do not allow differentiation between these entities, which can affect optimal treatment. Previous work suggested low reticulocyte hemoglobin content (CHr) may be an early indicator of iron deficiency. Objective This study aimed to correlate several inflammation markers with CHr values in dogs. We hypothesize that dogs with low CHr values have hematologic and biochemical evidence of inflammation. Methods Animals with CHr values below the reference interval were included in the low CHr group, while dogs with normal or increased CHr were included in the control group. HCT, MCV, CHr, reticulocyte mean cell volume (MCVr), concentrations of serum iron, C-reactive protein (CRP), ferritin, and ceruloplasmin, and total iron-binding capacity (TIBC), percent transferrin saturation (% sat), and total WBC, neutrophil, and monocyte counts were determined. Nonparametric tests were performed; median values and percentage of abnormalities between each group were compared. Results Relative to control dogs, animals in the low CHr group had higher median values for CRP, ferritin, ceruloplasmin, and WBC concentration (P ≤ .05), and lower median values for HCT and MCV (P ≤ .0001). Higher frequencies of abnormalities for CRP, ferritin, WBC, neutrophil, and monocyte concentrations (P ≤ .02) were present in the low CHr group. Conclusions Dogs with low CHr values often have evidence of inflammation, but low CHr did not reliably predict Fe deficiency. Additional diagnostic tests are needed to differentiate true and functional iron deficiency.

Background Obesity is a global disease, affecting nearly half a billion people. Unfortunately, this trend is mirrored in our canine population. Objectives As obesity is a complex inflammatory disease, there is a need to determine whether routine medical screening tests may indicate, or be influenced by, its presence. The objective of the current study was to determine if significant differences exist between CBC and biochemical profile values from control vs overweight/obese, client-owned dogs considered clinically healthy. Methods Dogs presented for routine health examinations, including minor dental or elective surgical procedures, were retrospectively identified from a hospital population. Animals were allocated to 2 categories based on body condition score (BCS), and data were analyzed by Mann–Whitney nonparametric analysis with statistical significance at a P ≤ .05. Results After exclusions, 116 dogs were assigned to the overweight/obese group (BCS ≥ 7) and 240 dogs to the control group (BCS = 4–6). Overweight/obese dogs had higher total leukocyte counts and higher plasma protein and globulin concentrations. Other differences were attributed to decreased serum water fraction (increased sodium, albumin, calcium, and anion gap) in the overweight/obese group. Interestingly, chloride concentration was decreased (in the face of increased sodium) in the obese group. Conclusions There is CBC and biochemical evidence to support the concern that obesity influences laboratory values, even in dogs considered clinically healthy. Prospective studies aimed at characterizing these changes are needed to provide insight into the connection between obesity and its comorbidities.

Background Effects of aging on hematologic and biochemical variables are well described in people. Anemia of the elderly is attributed to iron deficiency, anemia of chronic disease, chronic kidney disease, myelodysplasia, or idiopathic causes. Limited studies have examined these variables in aging dogs, but they have typically examined single breeds in research settings. Objective The objective of this study was to identify differences in CBC and biochemistry values between adult and aged dogs of many breeds. Methods Dogs presenting for wellness examinations and minor dental/elective surgeries that were otherwise clinically healthy were retrospectively identified. Dogs were categorized by age: adult (1–7.9 years), senior (8–11.9 years), and geriatric (12+ years). Standard CBC and biochemistry data were collated. Asian breeds, Greyhounds, and dogs with data indicating overt underlying disease were excluded. The Kruskal–Wallis test was used to compare groups with statistical significance set at P ≤ .05. Results Hematocrit, MCV, and serum iron decreased with age, indicating possible iron-restricted erythropoiesis (IRE), due to iron deficiency or low-grade chronic inflammation. Total proteins, globulins, and platelet counts increased with age while albumin decreased, suggesting low-grade inflammation. Urea was increased in older dogs without a concurrent increase in creatinine, which points toward gastrointestinal bleeding or dehydration. Conclusion Clinically healthy, aging dogs have changes in laboratory variables that indicate altered physiologies compared to younger adult animals, including evidence of IRE, inflammation, and potential gastrointestinal bleeding, suggesting a similar trend to that of elderly human beings. Future studies will examine markers of iron metabolism and inflammation in aging dogs.

Age is a primary risk factor for osteoarthritis (OA). The mechanisms that contribute to OA are poorly understood and disease modifying treatments have not been identified. A critical shortcoming in developing therapies is the limited number of translational models available to identify the causes of naturally occurring OA. Our goal is to use the common marmoset as a non-human primate (NHP) model of age-related OA. NHP are the closest evolutionary relative to humans and share many characteristics of human aging. The marmoset has advantages over other NHP for aging research because of their relatively short maximal lifespan and small size. Micro-computed tomography (uCT) was performed on whole-knee joints obtained from young (10 yrs, n=3) marmosets at necropsy. OA was evaluated using a clinical uCT scoring system and quantitative assessments of subchondral bone structure and ossified meniscal volume. Advancing age was positively correlated to increased uCT OA score (p

Current cartilage transplantation techniques achieve suboptimal restoration and rely on patient donor cells or living grafts of chondrocytes.We sought to enhance allogeneic grafts by testing mosaics of genetically engineered and naïve juvenile human chondrocytes (jCh).We obtained specimens from three humans and performed three experiments (two in vitro, one in vivo). We compared neocartilage with and without (1) supplemented serum-free medium (chondrocyte differentiation medium [CDM]), (2) adenoviral BMP-2 (AdBMP-2) transduction, and (3) varying ratios (0.1-1) of transduced and naïve jCh. We compared (4) healing with mosaic grafts with naïve neocartilage or marrow stimulation in immunosuppressed rats. For each of 10 in vitro treatment groups, we had six replicates for each human, and for each of three in vivo treatment groups, we had four replicates for one human. We scored the histology with the semiquantitative Bern score.AdBMP-2 and naïve neocartilage growth in CDM were histologically superior (Bern score, 5.2 versus 3.7; 8.0 versus 1.8) and size (8.0 versus 6.1; 7.9 versus 2.2 mg) to standard medium. In CDM, AdBMP-2 decreased viability (76% versus 90%), but increased BMP-2 production (619 ng/mL versus 43 pg/mL). Ten percent and 25% AdBMP-2 transduction had Bern scores of 6.8 and 6.5 and viability of 84% and 83%, respectively. Twenty-five percent mosaic grafts provided better healing histologically than marrow stimulation or naive neocartilage.Low-level AdBMP-2 and CDM augment neocartilage parameters in vitro and vivo.Genetic augmentation of jCh and creation of mosaic neocartilage may improve graft viability and articular healing compared with naïve neocartilage.

Summary Objective Diminish interleukin-1β (IL-1β) signaling in a model of primary osteoarthritis by RNA interference-based transcript reduction or receptor blockade, and quantify changes incurred on transcript expression of additional mediators. Methods Knees of Hartley guinea pigs were collected at 120 and 180 days of age following injection with viral vectors ( N = 4/treatment group/date) at 60 days. Two groups received either adeno-associated viral serotype 5 vector containing a knockdown sequence (TV), or adenoviral vector encoding for IL-1 receptor antagonist protein (Ad-IRAP); treatments were contrasted with opposite knees administered corresponding vector controls. A third group evaluated TV relative to saline-only injected knees. Chondropathy and immunohistochemistry findings were compared to untreated guinea pigs. Transcript expression levels in cartilage were calculated using the comparative CT (2 −ΔΔCT ) method and analyzed by one-way analysis of variance (ANOVA) with pairwise comparisons using Tukey 95% confidence intervals. Results Vector transduction was confirmed at both harvest dates. TV and Ad-IRAP, relative to vector controls, significantly decreased IL-1β. Inflammatory mediators [tumor necrosis factor-α (TNF-α), IL-8, interferon-γ (IFN-γ)], and catabolic matrix metalloproteinase 13 (MMP13) were also decreased, while anabolic transforming growth factor-β1 (TGF-β1) was increased. IL-1β was also decreased by TV vs saline, with a decrease in MMP13 and increase TGF-β1; TNF-α, IL-8, and IFN-γ were transiently increased. Conclusions This work confirmed that a reduction in IL-1β signaling was accomplished by either method, resulting in decreased expression of three inflammatory mediators and one catabolic agent, and increased expression of an anabolic molecule. Thus, evidence is provided that IL-1β serves a role in vivo in spontaneous osteoarthritis and that these translational tools may provide beneficial disease modification.

Objective—To determine effects of various concentrations of retinoic acid (RA) or a synthetic RA receptor antagonist (LE135) on equine chondrocytes or bone marrow—derived equine mesenchymal stem cells (BMDMSCs) in monolayer cultures. Sample—Articular cartilage and BMDMSCs from 5 clinically normal horses. Procedures—Monolayers of chondrocytes cultured in standard media and of BMDMSCs cultured in chondrogenic media were treated with RA at concentrations of 0, 0.1, 1, or 10μM or LE135 at concentrations of 0, 0.1, 1, or 10μM on day 0. On days 7 and 14, samples were analyzed for DNA concentration, chondrocyte morphology or features consistent with chondrogenesis (ie, chondral morphology [scored from 0 to 4]), and gene expression of collagen type Ia (CI), collagen type II (CII), and aggrecan. Results—Chondrocytes treated with RA had more mature chondral morphology (range of median scores, 3.0 to 4.0) than did untreated controls (range of median scores, 0.5 to 0.5). Chondrocytes treated with LE135 did not sustain chondrocyte morphology. All BMDMSCs had evidence of chondral morphology or high CII:CI ratio. Retinoic acid (1 or 10μM) or LE135 (10μM) treatment decreased DNA content of BMDMSC cultures. At 0.1 and 1μM concentrations, LE135 weakly but significantly increased chondral morphology scores, compared with untreated controls, but lack of aggrecan expression and lack of increased CII:CI ratio, compared with that of controls, did not affect chondrogenesis. Conclusions and Clinical Relevance—RA promoted maturation and hypertrophy in chondrocytes but not BMDMSCs in monolayer cultures. Deficiency or blockade of RA may prevent hypertrophy and maturation of differentiated chondrocytes.

Objective—To evaluate 2 commercially available transfection reagents for transfection efficiency and distribution of small interfering RNA (siRNA) molecules to chondrocytes in monolayer cultures and full-thickness cartilage explants from guinea pigs and horses. Sample—Cartilage explants from 5 one-month-old and 3 adult guinea pigs and 5 adult clinically normal horses. Procedures—Monolayer chondrocytes and uniform cartilage explants were exposed to 1 of 2 siRNA transfection complexes according to manufacturers' protocols (1μM [1×]). Additionally, monolayer chondrocytes were exposed to 2× the suggested amount of a proprietary siRNA molecule. Full-thickness cartilage explants were treated with 1× (1μM), 2× (2μM), and 4× (4μM) or 1× (0.13μM), 4× (0.52μM), and 8× (1.04μM) the recommended concentrations of the proprietary siRNA and the cationic liposome siRNA, respectively, in equivalent media volumes. Use of fluorescent siRNA duplexes allowed quantification of transfected cells via flow cytometry and direct visualization of the depth and distribution of in situ transfection via fluorescent microscopy. Results—With both transfection reagents, > 90% of monolayer chondrocytes were transfected. In explants, only use of the proprietary molecule achieved > 50% transfection efficiency, whereas use of the cationic liposome achieved < 20%. Only the proprietary molecule-treated cartilage consistently contained fluorescent cells throughout all zones; the cationic liposome-transfected chondrocytes were restricted to explant surfaces. Conclusions and Clinical Relevance—Robust transfection of chondrocytes in monolayer was achieved with both reagents, but only use of the proprietary molecule attained effective full-thickness transfection of explants that may allow relevant transcript reduction via RNAi.

Osteoarthritis (OA) is a debilitating condition characterized by inflammation, breakdown, and consequent loss of cartilage of the joints. Epidemiological studies indicate obesity is an important risk factor involved in OA initiation and progression. Traditional views propose OA to be a biomechanical consequence of excess weight on weight-bearing joints; however, emerging data demonstrates that systemic and local factors released from white adipose depots play a role. Hence, current views characterize OA as a condition exacerbated by a metabolic link related to adipose tissue, and not solely related to redistributed/altered weight load. Factors demonstrated to influence cartilage and bone homeostasis include adipocyte-derived hormones (“adipokines”) and adipose depot released cytokines. Epidemiological studies demonstrate a positive relation between systemic circulating cytokines, leptin, and resistin with OA types, while the association with adiponectin is controversial. Local factors in joints have also been shown to play a role in OA. In particular, this includes the knee, a weight-bearing joint that encloses a relatively large adipose depot, the infrapatellar fat pad (IFP), which serves as a source of local inflammatory factors. This review summarizes the relation of obesity and OA as it specifically relates to the IFP and other integral supporting structures. Overall, studies support the concept that metabolic effects associated with systemic obesity also extend to the IFP, which promotes inflammation, pain, and cartilage destruction within the local knee joint environment, thus contributing to development and progression of OA.

Platelet rich plasma (PRP) has recently been investigated for use in tissue regeneration studies that seek to utilize the numerous growth factors released from platelet alpha-granules. This study examined gene expression patterns, DNA, and collagen content of equine flexor digitorum superficialis tendon (SDFT) explants cultured in media consisting of PRP and other blood products. Blood and bone marrow aspirate (BMA) were collected from horses and processed to obtain plasma, PRP, and platelet poor plasma (PPP). IGF-I, TGF-beta1, and PDGF-BB were quantified in all blood products using ELISA. Tendons were cultured in explant fashion with blood, plasma, PRP, PPP, or BMA at concentrations of 100%, 50%, or 10% in serum-free DMEM with amino acids. Quantitative RT-PCR for expression of collagen type I (COL1A1), collagen type III (COL3A1), cartilage oligomeric matrix protein (COMP), decorin, matrix metalloproteinase-3 (MMP-3), and matrix metalloproteinase-13 (MMP-13) was performed as were DNA and total soluble collagen assays. TGF-beta1 and PDGF-BB concentrations were higher in PRP compared to all other blood products tested. Tendons cultured in 100% PRP showed enhanced gene expression of the matrix molecules COL1A1, COL3A1, and COMP with no concomitant increase in the catabolic molecules MMP-3 and MMP-13. These findings support in vivo investigation of PRP as an autogenous, patient-side treatment for tendonitis.

Background:Meniscal root tears cause menisci and their insertions to inadequately distribute loads and potentially leave underlying articular cartilage unprotected. Untreated meniscal root tears are becoming increasingly recognized to induce joint degradation; however, little information is known about anterior meniscal root tears and how they affect joint tissue.Purpose:To observe the early degenerative changes within the synovial fluid, menisci, tibial articular cartilage, and subchondral bone after arthroscopic creation of untreated anterior meniscal root tears.Study Design:Controlled laboratory study.Methods:Anterolateral meniscal root tears were created in 1 knee joint of 5 adult Flemish Giant rabbits, and anteromedial meniscal root tears were created in 4 additional rabbits. The contralateral limbs were used as nonoperated controls. The animals were euthanized at 8 weeks postoperatively; synovial fluid was aspirated, and tissue samples of menisci and tibial articular cartilage were collected and processed for multiple analyses to detect signs of early degeneration.Results:Significant changes were found within the synovial fluid, meniscal tissue, and tibial subchondral bone of the knees with anterior meniscal root tears when compared with controls. There were no significant changes identified in the tibial articular cartilage when comparing the tear groups with controls.Conclusion:This study demonstrated early degenerative changes within the synovial fluid, menisci, and tibial subchondral bone when leaving anterior meniscal root tears untreated for 8 weeks. The results suggest that meniscal tissue presents measurable, degenerative changes prior to changes within the articular cartilage after anterior meniscal root tears. Anterior destabilization of the meniscus arthroscopically may lead to measurable degenerative changes and be useful for future in vivo natural history and animal repair studies.Clinical Relevance:The present study is the first to investigate various tissue changes after anterior meniscal root tears of both the medial and lateral menisci. The results from this study suggest that degenerative changes occur within the synovial fluid, meniscus, and tibial subchondral bone prior to any measurable changes to the tibial articular cartilage. Further studies should expand on this study to evaluate how these components continue to progress when left untreated for long periods.

An 11-year-old male castrated mixed-breed dog was presented for exercise intolerance, tetraparesis, and persistent hypoglycemia. Abdominal ultrasound examination revealed 2 nodules within the right limb of the pancreas. Cytology from one nodule was consistent with a carcinoma of neuroendocrine origin, with a primary differential diagnosis of insulinoma. Histologic evaluation and immunohistochemistry for synaptophysin and insulin confirmed the diagnosis of insulinoma. Additionally, there was a solitary nodule of mineralized compact bone composing approximately 60% of the mass. To the authors' knowledge, this is the first report of osseous metaplasia within an insulinoma (islet cell carcinoma).

SummaryObjectiveTo provide a comprehensive immunohistochemical (IHC) map of the temporal expression and tissue distribution of interleukin-1β (IL-1β) through progression of osteoarthritis (OA) in two strains of guinea pigs with varying propensity for spontaneous knee joint disease.MethodsOA-prone Hartley and OA-resistant Strain 13 guinea pigs were collected at 60, 120, 180, 240, 360, and 480 days of age (N=4 animals per strain per date). IHC was performed on whole joint preparations; the distribution of IL-1β expression on coronal sections was mapped, semi-quantitatively scored, and correlated to OA grade using Mankin criteria with guinea pig-specific modifications. OA and IHC indices were compared among times and between strains using the Kruskal–Wallis one-way analysis of variance by ranks followed by Dunn’s post test.ResultsOA indices for both strains increased from 60 to 480 days of age; a statistically higher score (P≤0.01) was found in Hartley animals at 180, 240, 360, and 480 days. At 60 days of age, IL-1β expression was detected in cartilage, menisci, synovium, and subchondral bone in both strains. Persistent and statistically increased (P

Objective—To evaluate early cellular influences of bone morphogenetic protein (BMP)12 and BMP2 on equine superficial digital flexor tenocytes (SDFTNs) and equine bone marrow–derived mesenchymal stem cells (BMDMSCs). Animals—9 adult clinically normal horses. Procedures—BMDMSCs and SDFTNs were cultured in monolayer, either untreated or transduced with adenovirus encoding green fluorescent protein, adenovirus encoding BMP12, or adenovirus encoding BMP2. Cytomorphologic, cytochemical, immunocytochemical, and reverse transcriptase–quantitative PCR (RT-qPCR) analyses were performed on days 3 and 6. Genetic profiling for effects of BMP12 was evaluated by use of an equine gene expression microarray on day 6. Results—BMDMSCs and SDFTNs had high BMP12 gene expression and remained viable and healthy for at least 6 days. Type l collagen immunocytochemical staining for SDFTNs and tenocyte-like morphology for SDFTNs and BMDMSCs were greatest in BMP12 cells. Cartilage oligomeric matrix protein, as determined via RT-qPCR assay, and chondroitin sulfate, as determined via gene expression microarray analysis, were upregulated relative to control groups in SDFTN-BMP12 cells. The BMDMSCs and SDFTNs became mineralized with BMP2, but not BMP12. Superficial digital flexor tenocytes responded to BMP12 with upregulation of genes relevant to tendon healing and without mineralization as seen with BMP2. Conclusions and Clinical Relevance—Targeted equine SDFTNs may respond to BMP12 with improved tenocyte morphology and without mineralization, as seen with BMP2. Bone marrow–derived mesenchymal stem cells may be able to serve as a cell delivery method for BMP12.

This study quantified and compared the transduction efficiencies of adenoviral (Ad), Arg-Gly-Asp (RGD)-modified Ad, adeno-associated viral serotype 2 (AAV2), and self-complementary AAV2 (scAAV2) vectors within full-thickness osteoarthritic (OA) and unaffected canine cartilage explants in vitro. Intraarticular administration of Ad and scAAV2 vectors was performed to determine the ability of these vectors to transduce unaffected guinea pig cartilage in vivo. Following explant exposure to vector treatment or control, the onset and surface distribution of reporter gene expression was monitored daily with fluorescent microscopy. At termination, explants were divided: one half was digested for analysis using flow cytometry; the remaining portion was used for histology and immunohistochemistry (IHC). Intact articular joints were collected for real-time RT-PCR and IHC to detect reporter gene expression following injection of selected vectors. Ad vector transduced focal areas along the perimeters of explants; the remaining vectors transduced chondrocytes across 100% of the surface. Greater mean transduction efficiencies were found with both AAV2 vectors as compared to the Ad vector (p < or = 0.026). Ad and Ad-RGD vectors transduced only superficial chondrocytes of OA and unaffected cartilage. Uniform reporter gene expression from AAV2 and scAAV2 was detected in the tangential and transitional zones of OA cartilage, but not deeper zones. AAV2 and scAAV2 vectors achieved partial and full-thickness transduction of unaffected cartilage. In vivo work revealed that scAAV2 vector, but not Ad vector, transduced deeper zones of cartilage and menisci. This study demonstrates that AAV2 and scAAV2 are reliable vectors for use in cartilage in vitro and in vivo.

Numerous investigations have reported the efficacy of exogenous hyaluronan (HA) in modulating acute and chronic inflammation. The current study was performed to determine the in vitro effects of lower and higher molecular weight HA on lipopolysaccharide (LPS)-challenged fibroblast-like synovial cells. Normal synovial fibroblasts were cultured in triplicate to one of four groups: group 1, unchallenged; group 2, LPS-challenged (20 ng/ml); group 3, LPS-challenged following preteatment and sustained treatment with lower molecular weight HA; and group 4, LPS-challenged following pretreatment and sustained treatment with higher molecular weight HA. The response to LPS challenge and the influence of HA were compared among the four groups using cellular morphology scoring, cell number, cell viability, prostaglandin E2 (PGE2) production, IL-6 production, matrix metalloproteinase 3 (MMP3) production, and gene expression microarray analysis. As expected, our results demonstrated that LPS challenge induced a loss of characteristic fibroblast-like synovial cell culture morphology (P < 0.05), decreased the cell number (P < 0.05), increased PGE2 production 1,000-fold (P < 0.05), increased IL-6 production 15-fold (P < 0.05), increased MMP3 production threefold (P < 0.05), and generated a profile of gene expression changes typical of LPS (P < 0.005). Importantly, LPS exposure at this concentration did not alter the cell viability. Higher molecular weight HA decreased the morphologic change (P < 0.05) associated with LPS exposure. Both lower and higher molecular weight HA significantly altered a similar set of 21 probe sets (P < 0.005), which represented decreased expression of inflammatory genes (PGE2, IL-6) and catabolic genes (MMP3) and represented increased expression of anti-inflammatory and anabolic genes. The molecular weight of the HA product did not affect the cell number, the cell viability or the PGE2, IL-6, or MMP3 production. Taken together, the anti-inflammatory and anticatabolic gene expression profiles of fibroblast-like synovial cells treated with HA and subsequently challenged with LPS support the pharmacologic benefits of treatment with HA regardless of molecular weight. The higher molecular weight HA product provided a cellular protective effect not seen with the lower molecular weight HA product.

Summary Objective To ascertain a viral vector-based short hairpin RNA (shRNA) capable of reducing the interleukin-1β (IL-1β) transcript in osteoarthritis (OA)-prone chondrocytes and detect corresponding changes in the expression patterns of several critical disease mediators. Methods Cultured chondrocytes from 2-month-old Hartley guinea pigs were screened for reduction of the IL-1β transcript following plasmid-based delivery of U6-driven shRNA sequences. A successful plasmid/shRNA knockdown combination was identified and used to construct an adeno-associated virus serotype 5 (AAV5) vector for further evaluation. Relative real-time reverse transcription polymerase chain reaction (RT-PCR) was used to quantify in vitro transcript changes of IL-1β and an additional nine genes following transduction with this targeting knockdown vector. To validate in vitro findings, this AAV5 vector was injected into one knee, while either an equivalent volume of saline vehicle (three animals) or non-targeting control vector (three animals) were injected into opposite knees. Fold differences and subsequent percent gene expression levels relative to control groups were calculated using the comparative CT (2 −ΔΔCT ) method. Results Statistically significant decreases in IL-1β expression were achieved by the targeting knockdown vector relative to both the mock-transduced control and non-targeting vector control groups in vitro . Transcript levels of anabolic transforming growth factor-β (TGF-β) were significantly increased by use of this targeting knockdown vector. Transduction with this targeting AAV5 vector also significantly decreased the transcript levels of key inflammatory cytokines [tumor necrosis factor-α (TNF-α), IL-2, IL-8, and IL-12] and catabolic agents [matrix metalloproteinase (MMP)13, MMP2, interferon-γ (IFN-γ), and inducible nitrous oxide synthase (iNOS)] relative to both mock-transduced and non-targeting vector control groups. In vivo application of this targeting knockdown vector resulted in a >50% reduction ( P =0.0045) or >90% ( P =0.0001) of the IL-1β transcript relative to vehicle-only or non-targeting vector control exposed cartilage, respectively. Conclusions Successful reduction of the IL-1β transcript was achieved via RNA interference (RNAi) techniques. Importantly, this alteration significantly influenced the transcript levels of several major players involved in OA pathogenesis in the direction of disease modification. Investigations to characterize additional gene expression changes influenced by targeting knockdown AAV5 vector-based diminution of the IL-1β transcript in vivo are warranted.