Removal of the infrapatellar fat pad and associated synovium benefits female guinea pigs in the Dunkin Hartley model of idiopathic osteoarthritis.

Background: Several tissues contribute to the onset and advancement of knee osteoarthritis (OA). One tissue type that is worthy of closer evaluation, particularly in the context of sex, is the infrapatellar fat pad (IFP). We previously demonstrated that removal of the IFP had short-term beneficial effects for a cohort of male Dunkin-Hartley guinea pigs. The present project was designed to elucidate the influence of IFP removal in females of this OA-prone strain. It was hypothesized that resection of the IFP would reduce the development of OA in knees of a rodent model predisposed to the disease. Methods: Female guinea pigs (n=16) were acquired at an age of 2.5 months. Surgical removal of the IFP and associated synovium complex (IFP/SC) was executed at 3 months of age. One knee had the IFP/SC resected; a comparable sham surgery was performed on the contralateral knee. All animals were subjected to voluntary enclosure monitoring and dynamic weight-bearing, as well as compulsory treadmill-based gait analysis monthly; baseline data was collected prior to surgery. Guinea pigs were euthanized at 7 months. Knees from eight animals were evaluated via histology, mRNA expression, and immunohistochemistry (IHC); knees from the remaining eight animals were allocated to microcomputed tomography (microCT), biomechanical analyses (whole joint testing and indentation relaxation testing), and atomic absorption spectroscopy (AAS). Results: Fibrous connective tissue (FCT) replaced the IFP/SC. Mobility/gait data indicated that unilateral IFP/SC removal did not affect bilateral hindlimb movement. MicroCT demonstrated that osteophytes were not a significant feature of OA in this sex; however, trabecular thickness (TbTh) in medial femorae decreased in knees containing the FCT. Histopathology scores were predominantly influenced by changes in the lateral tibia, which demonstrated that histologic signs of OA were increased in knees containing the native IFP/SC versus those with the FCT. Similarly, indentation testing demonstrated higher instantaneous and equilibrium moduli in the lateral tibial articular cartilage of control knees with native IFPs. AAS of multiple tissue types associated with the knee revealed that zinc was the major trace element influenced by removal of the IFP/SC. Conclusions: Our data suggest that the IFP/SC is a significant component driving knee OA in female guinea pigs and that resection of this tissue prior to disease has short-term benefits. Specifically, the formation of the FCT in place of the native tissue resulted in decreased cartilage-related OA changes, as demonstrated by reduced Osteoarthritis Research Society International (OARSI) histology scores, as well as changes in transcript, protein, and cartilage indentation analyses. Importantly, this model provides evidence that sex needs to be considered when investigating responses and associated mechanisms seen with this intervention.

Immunization against nucleus pulposus antigens to accelerate degenerative disc disease in a rabbit model.

Low back pain poses a significant societal burden, with progressive intervertebral disc degeneration (IDD) emerging as a pivotal contributor to chronic pain. Improved animal models of progressive IDD are needed to comprehensively investigate new diagnostic and therapeutic approaches to managing IDD. Recent studies underscore the immune system's involvement in IDD, particularly with regards to the role of immune privileged tissues such as the nucleus pulposus (NP) becoming an immune targeting following initial disc injury. We therefore hypothesized that generating an active immune response against NP antigens with an NP vaccine could significantly accelerate and refine an IDD animal model triggered by mechanical puncture of the disc. To address this question, rabbits were immunized against NP antigens following disc puncture, and the impact on development of progressive IDD was assessed radiographically, functionally, and histologically compared between vaccinated and non-vaccinated animals over a 12-week period. Immune responses to NP antigens were assessed by ELISA and Western blot. We found that the vaccine elicited strong immune responses against NP antigens, including a dominant ~37 kD antigen. Histologic evaluation revealed increases IDD in animals that received the NP vaccine plus disc puncture, compared to disc puncture and vaccine only animals. Imaging evaluation evidenced a decrease in disc height index and higher scores of disc degeneration in animals after disc punctures and in those animals that received the NP vaccine in addition to disc puncture. These findings therefore indicate that it is possible to elicit immune responses against NP antigens in adult animals, and that these immune responses may contribute to accelerated development of IDD in a novel immune-induced and accelerated IDD model.

Systemic administration of a pharmacologic iron chelator reduces cartilage lesion development in the Dunkin-Hartley model of primary osteoarthritis.

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.

Age- and sex-associated differences in hematology and biochemistry parameters of Dunkin Hartley guinea pigs (Cavia porcellus).

The Dunkin Hartley is the most common guinea pig strain used in biomedical research, particularly for studies of asthma, allergy, infectious disease, reproduction, and osteoarthritis. Minimally invasive blood tests, such as complete blood counts and serum biochemistry profiles, are often collected for diagnostics and laboratory analyses. However, reference intervals for these assays have not yet been well-documented in this strain. The purpose of this study was to establish reference intervals for hematologic and biochemical parameters of Dunkin Hartley guinea pigs and determine age- and sex-related differences. Hematologic and biochemical parameters were retrospectively obtained from 145 male and 68 female guinea pigs between 2 and 15 months of age. All blood parameters were analyzed by a veterinary clinical pathology laboratory. Reference intervals were established according to the American Society for Veterinary Clinical Pathology guidelines. Age- and sex-related differences were determined using unpaired t-tests or nonparametric Mann-Whitney tests. Hematocrit, red blood cell distribution width, mean platelet volume, white blood cell count, heterophils, monocytes, eosinophils, glucose, blood urea nitrogen, creatinine, calcium, magnesium, total protein, albumin, globulin, cholesterol, aspartate aminotransferase, gamma glutamyl transferase, and bicarbonate increased with age. Mean corpuscular hemoglobin concentration, cellular hemoglobin concentration mean, platelets, lymphocytes, phosphorus, albumin/globulin ratio, alkaline phosphatase, anion gap, and calculated osmolality decreased with age. Males had higher hemoglobin, hematocrit, red blood cell count, mean corpuscular hemoglobin concentration, white blood cell count, heterophils, Foa-Kurloff cells, alanine aminotransferase, and bicarbonate and lower mean corpuscular volume, red blood cell distribution width, platelets, mean platelet volume, eosinophils, total protein, albumin, globulin, cholesterol, potassium, anion gap, calculated osmolality, and iron compared to females. Establishing age and sex differences in hematologic and biochemical parameters of Dunkin Hartley guinea pigs provides valuable insight into their physiology to better evaluate diagnostics and experimental results.

A novel diindolylmethane analog, 1,1-bis(3'-indolyl)-1-(p-chlorophenyl) methane, inhibits the tumor necrosis factor-induced inflammatory response in primary murine synovial fibroblasts through a Nurr1-dependent mechanism.

The progression of rheumatoid arthritis involves the thickening of the synovial lining due to the proliferation of fibroblast-like synoviocytes (FLS) and infiltration by inflammatory cells. Tumor necrosis factor alpha (TNFα) is a pro-inflammatory cytokine involved in progression of the disease. Under rheumatoid conditions, FLS express the tumor necrosis factor (TNF)-recognition complex (TNFR1, TNFR2, VCAM-1 and ICAM-1), which induces local macrophage activation and leads to downstream nuclear factor κB (NF-κB) signaling. The NF-κB-regulated inflammatory gene, cyclooxygenase (COX), increases synthesis of prostaglandins that contribute to the propagation of inflammatory damage within the joint. Because the nuclear orphan receptor, NR4A2 (Nurr1), can negatively regulate NF-κB-dependent inflammatory gene expression in macrophages, we postulated that activation of this receptor by the Nurr1 ligand 1,1-bis(3'-indolyl)-1-(p-chlorophenyl) methane (C-DIM12) would modulate inflammatory gene expression in synovial fibroblasts by inhibiting NF-κB. Treatment with C-DIM12 suppressed TNFα-induced expression of adhesion molecules and NF-κB regulated genes in primary synovial fibroblasts including vascular adhesion molecule 1 (VCAM-1), PGE2 and COX-2. Immunofluorescence studies indicated that C-DIM12 did not prevent translocation of p65 and stabilized nuclear localization of Nurr1 in synovial fibroblasts. Knockdown of Nurr1 expression by RNA interference prevented the inhibitory effects of C-DIM12 on inflammatory gene expression, indicating that the anti-inflammatory effects of this compound are Nurr1-dependent. Collectively, these data suggest that this receptor may be a viable therapeutic target in RA.