Systemic iron reduction via an iron deficient diet decreases the severity of knee cartilage lesions in the Dunkin-Hartley guinea pig model of osteoarthritis.

OBJECTIVE: Iron accumulation is emerging as a player in aging-related disorders due to its propensity for generating reactive oxygen species (ROS). Studies investigating the role of iron in the pathogenesis of primary osteoarthritis (OA) are limited. We designed a proof-of-principle study to determine the effect of systemic iron deficiency, via an iron deficient diet, on knee OA in an animal model. METHODS: Twelve-week-old male Hartley guinea pigs received the standard diet (n = 6) or a diet devoid of iron (n = 6) for 19-weeks. Iron levels were determined in the serum, liver, and articular cartilage. Knees were collected to assess structural changes related to OA (microcomputed tomography, histopathology). Immunohistochemistry was performed to evaluate the presence and distribution of a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) and ROS-driven 4-hydroxynonenal (4-HNE)-induced protein adducts. Transcript expression was also assessed. RESULTS: Relative to control animals, an iron deficient diet reduced the concentration of this mineral in serum, liver, and articular cartilage. Iron deficient animals had lower histologic OA scores; decreased subchondral bone mineral density was also noted. This reduction in knee joint pathology was accompanied by a decrease in: ADAMTS4 in synovium; and 4-HNE protein adducts from lipid peroxidation in both the menisci and articular cartilage of iron deficient animals. Expression of iron-related genes in these tissues was also altered in treated animals. CONCLUSIONS: Results from this study suggest that systemic iron levels may play a role in knee OA pathogenesis, with a short-term deficit in dietary iron reducing the severity of knee cartilage lesions.

Clinical and Histologic Manifestations of a Novel Rectus Femoris Myotendinous Junction Injury in Rats.

Background: Animal models of muscle injury have primarily relied on methods which do not mimic the chronic scarring that typically occurs adjacent to the myotendinous junction (MTJ). The goal of this study was three-fold: (i) to create a strain-induced in vivo model of rectus femoris MTJ injury in rats; (ii) to document clinical manifestations of injury using longitudinal tracking of individual animals via voluntary and compulsory (treadmill) mobility analyses and (iii) to validate and assess the model for persistent scarring through serial histologic assessment and development of a semi-quantitative grading scheme to characterize injury response over time. Methods: Strain-induced MTJ injury was generated in male Sprague Dawley rats via needle tension directed along the transverse axis between the rectus femoris muscle and distal tendon that attaches to the patella. Animals received mobility assessments (gait analysis using a DigiGait Treadmill System and weight bearing using a Tekscan Rodent Walkway System) at days 0, 1, 3, 6, 13, 20, and 27 of the experimental protocol. Rats were euthanized at 1, 3, 7, 14, and 28 days post-injury (n = 6 rats per time-point) and hindlimbs were processed for histology. Results: Significant changes in locomotor parameters included injured and contralateral limb paw area, max dA/dt (limb deceleration/breaking time), stride time, stance time, force time impulse, and fore/hind symmetry, and injured limb maximum force. The most significant and consistent histologic finding was a pathologic fibrotic adhesive lesion at the muscle and tendon interface along the proximal aspect of the patella just distal to the injury site. This lesion was composed of reactive fibroblasts, disorganized collagen fibers, vascular profiles, and a myxomatous ground substance stroma. Conclusions: This work is the first to characterize the clinical and pathologic development of a chronic model of rectus femoris MTJ injury, which resulted in altered mobility likely caused by a strain-induced fibrotic scar along the anterior patella. Notably, both the functional and pathologic changes recapitulated the course of injury progression similar to what is described in humans. This work provides a unique model to study MTJ injury mechanisms for the identification of enhanced treatment options for patients who suffer from activity-related muscle conditions.

Systemic iron overload exacerbates osteoarthritis in the strain 13 guinea pig.

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.

Quantitative Gait Analysis Detects Significant Differences in Movement between Osteoarthritic and Nonosteoarthritic Guinea Pig Strains before and after Treatment with Flunixin Meglumine.

A computer-aided gait analysis system was used to contrast two guinea pig strains with differing propensity for osteoarthritis (OA), with/without administration of a nonsteroidal anti-inflammatory drug. Walking speed and static/dynamic gait parameters were determined at baseline. Flunixin meglumine was given and animals were evaluated 4, 24, and 72 hours after treatment. Body weight was compared using unpaired t-tests. Knee joints were histologically evaluated using species-specific criteria; indices were analyzed using one-way ANOVA, Kruskal-Wallis test, followed by Dunn's multiple comparisons. A generalized linear model followed by Tukey's posttests juxtaposed gait parameters; walking speed was a covariate for other outcome measures. Body weight was not different between strains; OA-prone animals demonstrated more progressive chondropathy. At baseline, OA-prone animals had slower walking speeds, narrower hind limb bases of support, shorter stride lengths, and slower limb swing speeds relative to OA-resistant animals. These differences were not detected 4 or 24 hours after treatment. By 72 hours, OA-prone animals had returned to baseline values. These findings indicate a distinct voluntary gait pattern in a rodent model of bilateral primary OA, modification of which may allow rapid screening of novel therapies. Flunixin meglumine temporarily permitted OA-prone animals to move in a manner that was analogous to OA-resistant animals.

In vivo reduction or blockade of interleukin-1ß in primary osteoarthritis influences expression of mediators implicated in pathogenesis.

OBJECTIVE: Diminish interleukin-1ß (IL-1ß) 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-1ß. 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-ß1 (TGF-ß1) was increased. IL-1ß was also decreased by TV vs saline, with a decrease in MMP13 and increase TGF-ß1; TNF-α, IL-8, and IFN-γ were transiently increased. CONCLUSIONS: This work confirmed that a reduction in IL-1ß 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-1ß serves a role in vivo in spontaneous osteoarthritis and that these translational tools may provide beneficial disease modification.

Effective reduction of the interleukin-1ß transcript in osteoarthritis-prone guinea pig chondrocytes via short hairpin RNA mediated RNA interference influences gene expression of mediators implicated in disease pathogenesis.

OBJECTIVE: To ascertain a viral vector-based short hairpin RNA (shRNA) capable of reducing the interleukin-1ß (IL-1ß) 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-1ß 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-1ß 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-1ß 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-ß (TGF-ß) 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-1ß transcript relative to vehicle-only or non-targeting vector control exposed cartilage, respectively. CONCLUSIONS: Successful reduction of the IL-1ß 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-1ß transcript in vivo are warranted.

Temporal expression and tissue distribution of interleukin-1ß in two strains of guinea pigs with varying propensity for spontaneous knee osteoarthritis.

OBJECTIVE: To provide a comprehensive immunohistochemical (IHC) map of the temporal expression and tissue distribution of interleukin-1ß (IL-1ß) through progression of osteoarthritis (OA) in two strains of guinea pigs with varying propensity for spontaneous knee joint disease. METHODS: OA-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-1ß 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. RESULTS: OA 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-1ß expression was detected in cartilage, menisci, synovium, and subchondral bone in both strains. Persistent and statistically increased (P<0.05) IL-1ß expression was found in these same tissues in Hartley animals at 120 and 180 days, while Strain 13 animals demonstrated a significant reduction in positive immunostaining. Statistical differences in IHC indices between strains beyond 240 days of age were restricted to synovium (days 240 and 480) and subchondral bone (days 360 and 480). CONCLUSIONS: As expected, histologic OA proceeded in an accelerated manner in Hartley animals relative to Strain 13 animals. The OA-prone strain did not demonstrate reduced IL-1ß expression during adult maturity as occurred in the OA-resistant strain, and this persistent expression may have corresponded to early incidence of OA. Future interventional studies are warranted to explore whether dysregulation of IL-1ß expression may contribute to premature onset of spontaneous disease in the Hartley guinea pig.