Physical inactivity and knee osteoarthritis in guinea pigs.

OBJECTIVE: To investigate whether and how a sedentary lifestyle contributes to knee osteoarthritis (OA) incidence and severity. DESIGN: An experiment was conducted using Hartley guinea pigs, an established idiopathic knee OA model. To simulate a sedentary lifestyle, growing animals (n = 18) were housed for 22 weeks in small cages that restricted their mobility, while another group of animals (n = 17) received daily treadmill exercise to simulate moderate physical activity. After the experiment, histological assessments, biochemical assays, and mechanical testing were conducted to compare tibial articular cartilage structure, strength, and degree of OA degeneration between sedentary and physically active animals. Groups were also compared based on body weight and composition, as well as gut microbial community composition assessed using fecal 16S rRNA gene sequencing. RESULTS: Prevalence of knee OA was similar between sedentary and physically active animals, but severity of the disease (cartilage lesion depth) was substantially greater in the sedentary group (P = 0.02). In addition, during the experiment, sedentary animals developed cartilage with lower aggrecan quantity (P = 0.03) and accumulated more body weight (P = 0.005) and visceral adiposity (P = 0.007). Groups did not differ greatly, however, in terms of cartilage thickness, collagen quantity, or stiffness, nor in terms of muscle weight, subcutaneous adiposity, or gut microbial community composition. CONCLUSIONS: Our findings indicate that a sedentary lifestyle promotes the development of knee OA, particularly by enhancing disease severity rather than risk of onset, and this potentially occurs through multiple pathways including by engendering growth of functionally deficient joint tissues and the accumulation of excess body weight and adiposity.

Sustained efficacy of a single intra-articular dose of FX006 in a rat model of repeated localized knee arthritis.

OBJECTIVE: To evaluate the efficacy of a single intra-articular (IA) dose of FX006, an extended-release formulation of triamcinolone acetonide (TCA) in poly(lactic-co-glycolic acid) (PLGA) microspheres, on the sequelae of repeated episodes of synovitis. DESIGN: Three flares of localized synovitis in the right knee of rats were induced over 4 weeks following a single IA injection of various doses of FX006, Kenalog(®) (TCA immediate release or TCA IR), or vehicle. Gait scores were employed to assess analgesic effect, and the joints were evaluated by histology at the end of the study. TCA plasma concentrations and corticosterone levels were monitored through the study. RESULTS: A single IA dose of 0.28 mg FX006 significantly improved gait scores through all three reactivations. TCA IR at 0.06 mg (providing comparable plasma TCA exposure, 10-fold higher Cmax) demonstrated comparable benefit through the first reactivation only and reduced-to-no efficacy thereafter. Significantly improved histological joint scores were observed with effective doses of FX006 but not with TCA IR. Corticosterone levels were initially decreased following both TCA IR and FX006 treatment, but recovered by Day 14. CONCLUSIONS: In localized, repeated synovitis in rats, sustained release of TCA following a single IA injection of FX006 significantly prolonged analgesia relative to TCA IR and significantly improved histological scores with no adverse effect on the HPA axis. Since synovitis can contribute to the pathophysiology of multiple joint diseases such as osteoarthritis (OA), RA and gout, FX006 may be an important treatment option for these conditions.

The OARSI histopathology initiative - recommendations for histological assessments of osteoarthritis in the rat.

OBJECTIVE: During the development of disease-modifying osteoarthritis (OA) drugs, rat models of OA are frequently used for a first assessment of in vivo efficacy. The most efficacious compound in the rat model may then be tested in a larger animal model before entering human trials. The aim of this study was to describe a histologic scoring system for use in different models of OA in rats that allows standardization and comparison of results obtained by different investigators. METHODS: The experience of the authors with current scoring systems and the range of lesions observed in rat and human OA studies were considered in recommending this common paradigm for rat histologic scoring. Considerations were made for reproducibility and ease of use for new scorers. Additional scoring paradigms may be employed to further identify specific effects of some disease-modifying drugs. RESULTS: Although the described scoring system is more complex than the modified Mankin scores, which are recommended for some other species, the reliability study showed that it is easily understood and can be reproducibly used, even by inexperienced scorers. CONCLUSIONS: The scoring paradigm described here has been found to be sufficiently sensitive to discriminate between treatments and to have high reproducibility. Therefore we recommend its use for evaluation of different rat OA models as well as assessment of disease-modifying effects of treatments in these models.

Protective effects of a cathepsin K inhibitor, SB-553484, in the canine partial medial meniscectomy model of osteoarthritis.

OBJECTIVE: Cathepsin K (cat K), a cysteine protease expressed in osteoclasts, chondrocytes and synovial fibroblasts, degrades several bone and cartilage matrix components suggesting its potential role in osteoarthritis (OA). We investigated the effects of SB-553484, an inhibitor of cat K, on lesion severity and biomarkers of collagen degradation in the canine partial medial meniscectomy model. METHODS: A partial medial meniscectomy was performed in mature female beagle dogs. Animals were dosed orally with vehicle or SB-553484 at 50mg/kg BID for 28 days. The femorotibial joints were evaluated for gross and microscopic histological changes. Biomarkers of collagen degradation were also analyzed. RESULTS: In dogs treated with SB-553484, subjective gross and calculated degeneration scores decreased significantly by 29% and 46%, respectively. Histopathologic evaluation demonstrated that the summed tibial degeneration score decreased significantly by 21%. Inhibition of tibial cartilage degeneration was significant in zone 1 (32%) and the depth ratio of any tibial matrix change was decreased significantly by 28%. Urinary biomarkers of bone and cartilage degradation were also significantly reduced. CONCLUSION: Treatment with SB-553484 resulted in mild to moderate beneficial effects on gross and histopathological parameters. Reduction of biomarkers of collagen type I and II degradation indicated a direct effect of the compound on bone and cartilage. These data suggest that the prevention of cartilage degradation by cat K inhibition may represent a valid strategy for pharmacological intervention in OA and that monitoring collagen degradation biomarkers may provide an indication of the protective effects of inhibition of bone and cartilage degradation.

Ochratoxin A carcinogenesis in the (C57BL/6J X C3H)F1 mouse.

The potential carcinogenic effects of the mycotoxin ochratoxin A [(OA); CAS: 303-47-9] were assessed in a 24-month feeding study in male and female (C57BL/6J X C3H)F1 (B6C3F1) mice. The mice were assigned to 3 groups of 50 males and 50 females each; group 1 mice were the controls, group 2 mice were fed 1 ppm OA, and group 3 mice were fed 40 ppm OA. Renal neoplasms, both carcinomas and adenomas, were found only in male mice of the 40-ppm dose group. Fourteen of 49 animals that survived at least 20 months had neoplasms morphologically consistent with renal carcinoma. Renal adenomas were present in some of these mice and in other 40-ppm-group males, making a total of 26 mice with renal adenomas. All male mice of the 40-ppm dose group had nephropathy characterized by varying degrees of renal tubular dilation, attenuation and hyperplasia of lining epithelium, and proliferation of regenerative tubules. Females of the 40-ppm dose group had similar but less severe renal changes but no carcinomas or adenomas. Compound-related renal lesions were absent in the 1-ppm dose group. The incidence of hepatocellular neoplasms was slightly increased in male and female mice fed diets containing OA. These results indicate that OA is a renal carcinogen in male B6C3F1 mice and a hepatic carcinogen in female mice of this strain.

Pharmacological and histological examinations of regional differences of guinea-pig lung: a role of pleural surface smooth muscle in lung strip contraction.

1. Parenchymal lung strip preparations have been widely used as an in vitro model of peripheral airway smooth muscle. The present study examined functional responses of 4 consecutive guinea-pig lung parenchymal strips isolated from the central region (segment 1) to the distal edge (segment 4) of the lower lung lobe. The middle two segments were designated as segments 2 and 3. 2. Lung segments 1 and 4 exhibited significantly greater contraction than the other 2 segments to KCl when responses were expressed as mg force per mg tissue weight. Contractile responses to bronchospastic agents including histamine, carbachol, endothelin-1, leukotrienes (LT) B4 and D4, and the thromboxane A2-mimetic U46619 demonstrated no significant difference in EC50 values among the 4 lung segments. 3. Contractile responses of segments 1 and 4 to antigen-challenge (ovalbumin), ionophore A23187 and substance P were significantly greater than the other 2 segments with respect to either sensitivity or maximum responsiveness. 4. U46619-induced contractions of the 4 lung segments were relaxed in similar manner by papaverine and theophylline up to 100%, salbutamol up to 80%, and sodium nitroprusside by only 20%. In contrast, sodium nitroprusside markedly reversed U46619-induced contraction of pulmonary arterial rings and bronchial rings. 5. Histological studies identified 2-4 layers of smooth muscle cells underlying the lung pleural surface. Mast cells were prominent in this area. Moreover, morphometric studies showed that segment 4 possessed the least amount of smooth muscle structures from bronchial/bronchiolar wall and vasculatures as compared to the other 3 segments, and a significant difference in this respect was evident between segment 1 and segment 4.6. Since lung segments 1 and 4 are covered with larger surface area of lung pleura, the present results suggest that the significantly greater intrinsic contractile responses of segments 1 and 4 are associated with the presence of increased lung pleural surface possibly together with more mast cells. Thus, a primary contribution to the net contraction of the lung parenchymal strips may be smooth muscle from the lung pleura, alveolar ducts and interstitial contractile cells rather than from bronchi/bronchioles and microvasculatures.

Aerosolized LTB4 produces delayed onset increases in pulmonary gas trapping.

Airway obstruction, as measured by increases in postmortem pulmonary gas trapping, and lung inflammatory changes were examined in guinea pigs exposed for up to 4 h to aerosols of leukotriene B4 (LTB4) or its non-chemotactic isomer, 6-trans-12-epi-LTB4. Airway obstruction and cytological responses in isomer-exposed animals were similar to those of unexposed control animals. LTB4-exposed animals had minimal inflammatory changes at 0.5 h but became dyspneic by 2 h and had increased airway obstruction, bronchoalveolar lavage neutrophils and eosinophils, and pulmonary tissue granulocyte scores. The LTB4-induced effects at 4 h were similar to those 2 h, except for further increase in BAL neutrophils and eosinophils. LTB4-induced airway obstructive and inflammatory changes were prevented by pretreatment with the LTB4 receptor antagonist SC-41930, but were unaffected by indomethacin. Thus, prolonged LTB4 inhalation can produce delayed onset airway obstruction that is stereospecific, cyclooxygenase-independent, and temporally associated with the influx of granulocytes into lung airways.

Effect of dexamethasone on A23187-induced airway responses in the guinea pig.

We examined the effect of dexamethasone on A23187-induced bronchospasm, pulmonary inflammation and airway responses to substance P. Guinea pigs, dosed orally once a day for 4 days with dexamethasone (3.0, 10.0 or 30.0 mg/kg) or saline, were exposed to an aerosol of A23187 for 12 min or until labored breathing began. Postmortem pulmonary gas trapping was used as an indicator of in vivo airway obstruction and changes in bronchial responses. Dexamethasone did not alter airway obstruction or inflammation 1 h after A23187 exposure. However, dexamethasone reduced the enhanced airway responses to substance P and bronchiolar/peribronchiolar inflammation 24 h post-A23187. It is possible that glucocorticosteroid suppression of A23187-induced pulmonary inflammation was important in reducing the increased airway responses to substance P.

Pharmacologic actions of the second generation leukotriene B4 receptor antagonist LY29311: in vivo pulmonary studies.

We examined the in vivo actions of LY293111 sodium (2-[2-propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxyphenoxy]pro poxy]phenoxy] benzoic acid sodium salt). Guinea pigs were used to evaluate the effect of this agent on (1) acute airway obstruction produced by intravenous leukotriene B4, (2) pulmonary granulocyte infiltration and delayed onset airway obstruction resulting from a 4-h leukotriene B4 inhalation and (3) lung inflammation after aerosol challenge with the divalent cationic ionophore A23187 (6S-[6alpha(2S*,3S*),8beta(R*),9beta,11alpha]-5- (methylamino)-2-[[3,9,11-trimethyl-8-[1-methyl-2-oxo-2-(1H-pyrrol-2-yl)e thyl]-1,7-dioxaspiro[5.5]undec-2-yl]methyl]-4-benzoxazole carboxylic acid). Airway obstruction was quantitated using pulmonary gas trapping measurements and lung inflammation was evaluated by bronchoalveolar lavage (BAL) and histology. LY293111 sodium produced a dose-related inhibition of acute leukotriene B4-induced airway obstruction when administered i.v. (ED50=14 microg/kg) or p.o. (ED50=0.4 mg/kg). In contrast, LY293111 sodium did not inhibit the pulmonary gas trapping caused by aerosols of histamine, leukotriene D4, or the thromboxane mimetic U46619 (15 [(S)-hydroxy11a,9a-(epoxymethano)prosta-5Z,13E-dienoic acid]). Oral LY293111 sodium inhibited leukotriene B4-induced bronchoalveolar lavage granulocyte infiltration and delayed onset airway obstruction at doses as low as 0.3 mg/kg. In A23187-challenged animals, pulmonary inflammation was markedly inhibited at 1 h, but not 2 h and 4 h post-exposure. We conclude that LY293 11 sodium is a selective leukotriene B4 receptor antagonist with potent pulmonary anti-inflammatory activity.

Effects of PEGylated soluble tumor necrosis factor receptor type I (PEG sTNF-RI) alone and in combination with methotrexate in adjuvant arthritic rats.

OBJECTIVE: To determine the potential combination benefit of treatment with PEG sTNF-RI and methotrexate in adjuvant arthritic rats. METHODS: Lewis rats with adjuvant arthritis were treated by sc injections of either 3.0 or 0.3 mg/kg PEG sTNF-RI on days 9, 11, and 13 of adjuvant arthritis. The effects of PEG sTNF-RI treatment alone were compared to treatment with daily oral methotrexate (0.075, 0.06 or 0.045 mg/kg) or methotrexate in combination with PEG sTNF-RI. Efficacy was monitored by volume measurement of ankle joints, final paw weights and histologic evaluation with particular emphasis on bone lesions. RESULTS: Treatment with 3.0 or 0.3 mg/kg PEG sTNF-RI alone resulted in 52% or 28% inhibition, respectively, of paw swelling as assessed by final paw weight. Treatment with methotrexate at either 0.075, 0.06, or 0.045 mg/kg gave 84%, 51% or 18% inhibition and combination treatment resulted in additive inhibitory effects. Histologic evaluation of ankle joints demonstrated 68% or 25% inhibition of bone resorption with PEG sTNF-RI alone at 3.0 or 0.3 mg/kg. Treatment with 0.075, 0.06 or 0.045 mg/kg methotrexate resulted in 98%, 76% or 40% inhibition of bone resorption. Additive benefit was best seen with the lower doses of methotrexate. CONCLUSION: Combination therapy with PEG sTNF-RI and methotrexate results in additive benefit, with the final result being excellent inhibition of all arthritis parameters. Data from these studies supports the clinical investigation of the use of combination therapy of PEG sTNF-RI and methotrexate in rheumatoid arthritis patients.

Viridicatumtoxin mycotoxicosis in mice and rats.

LD50 studies of viridicatumtoxin were done in rats and mice using oral, intraperitoneal (i.p.), and subcutaneous (s.c.) routes. Mice were given oral doses of viridicatumtoxin up to 350 mg/kg body weight and rats were given doses of viridicatumtoxin up to 150 mg/kg. No deaths occurred in the animals dosed by the oral route. Hepatic alterations of hydropic change and necrosis of centrolobular hepatocytes were observed in mice given 250, 300, or 350 mg/kg viridicatum toxin and were more severe in the mice given the higher doses. No histopathologic alterations were present in the rats dosed orally. Mice were given doses of viridicatumtoxin up to 300 mg/kg, s.c., and rats up to 400 mg/kg. No deaths occurred in the animals dosed by this route. Microscopic alterations in both mice and rats were limited to the injection sites and consisted of coagulation necrosis. The single-dose, 72-h i.p. LD50 for the mouse and the rat was 70 mg/kg and 80 mg/kg respectively. Histopathologic alterations in mice given viridicatumtoxin i.p. included fibrinous peritonitis, large subcapsular areas of hepatic necrosis, single-cell hepatocytic necrosis, splenic lymphoid depletion, and vacuolar degeneration of the myocardium. Rats had splenic lymphoid depletion and fibrinous peritonitis.

Animal models of osteoarthritis.

Animal models of osteoarthritis are used to study the pathogenesis of cartilage degeneration and to evaluate potential antiarthritic drugs for clinical use. Animal models of naturally occurring osteoarthritis (OA) occur in knee joints of guinea pigs, mice and other laboratory animal species. Transgenic models have been developed in mice. Commonly utilized surgical instability models include medial meniscal tear in guinea pigs and rats, medial or lateral partial meniscectomy in rabbits, medial partial or total meniscectomy or anterior cruciate transection in dogs. Additional models of cartilage degeneration can be induced by intra-articular iodoacetate injection or by administration of oral or parenteral quinolone antibiotics. None of these models have a proven track record of predicting efficacy in human disease since there are no agents that have been proven to provide anything other than symptomatic relief in human OA. However, agents that are active in these models are currently in clinical trials. Methodologies, gross and histopathologic features and comparisons to human disease will be discussed for the various models.

Animal models of osteoarthritis in an era of molecular biology.

Animal models of osteoarthritis (OA) are used to study the pathogenesis of cartilage degeneration and to evaluate potential anti-arthritic drugs for clinical use. In general, these models fall into 2 categories, spontaneous and induced (surgical instability or genetic manipulation). Animal models of naturally occurring OA occur in knee joints of guinea pigs, mice and Syrian hamsters. Commonly utilized surgical instability models include medial meniscal tear in guinea pigs and rats, medial or lateral partial meniscectomy in rabbits, medial partial or total meniscectomy or anterior cruciate transection in dogs. Transgenic models have been developed in mice. These models all have potential use in the study of molecular mechanisms associated with OA development via use of immunohistochemistry, biochemistry and molecular probes to identify altered matrix molecules at different stages in disease progression. Testing of specific types of inhibitors developed through evaluation of matrix changes in the disease process will ultimately help identify key processes which initiate and perpetuate the disease and will lead to discovery of new disease modifying pharmaceutical agents for OA patients. This paper will focus on the discussion of several models which are likely to be useful in the molecular dissection of processes involved in cartilage degeneration.

Evaluation of ochratoxin A for mutagenicity in a battery of bacterial and mammalian cell assays.

Ochratoxin A (OA), a nephrotoxic mycotoxin, was evaluated for genotoxic potential in a battery of in vitro and in vivo assays. OA was not mutagenic to Salmonella typhimurium, either with or without metabolic activation, in the plate incorporation (Ames) test at concentrations of 50-600 micrograms OA/plate or in the gradient plate assay at concentrations of 0.1-1000 micrograms OA/ml. No induction of unscheduled DNA synthesis was evident in primary cultures of rat hepatocytes exposed to concentrations of OA ranging from 0.000025 to 500 micrograms/ml. In the mouse lymphoma forward mutation assay, exposure of L5178Y TK+/- mouse lymphoma cells to OA did not increase the numbers of L5178Y TK-/- mutants. There was no significant difference between the numbers of sister-chromatid exchanges in cells from OA-treated Chinese hamsters and those in cells from the negative-control animals.

Fibroblast growth factor-18 stimulates chondrogenesis and cartilage repair in a rat model of injury-induced osteoarthritis.

OBJECTIVE: Osteoarthritis (OA) is the most common form of arthritis and a primary cause of disability, however, there are no treatments that can slow disease progression or repair damaged joint cartilage. Fibroblast growth factor-18 (FGF18) has been reported to have significant anabolic effects on cartilage. We therefore examined its effects on repair of cartilage damage in a rat meniscal tear model of OA. DESIGN: Surgical damage to the meniscus in rats leads to joint instability and significant damage to the articular cartilage at 3 weeks post-surgery. At this time, animals received bi-weekly intra-articular injections of FGF18 for 3 weeks, and the knee joints were then harvested for histologic examination. RESULTS: FGF18-induced dose-dependent increases in cartilage thickness of the tibial plateau, due to new cartilage formation at the articular surface and the joint periphery. The generation of new cartilage resulted in significant reductions in cartilage degeneration scores. The highest dose of FGF18 also induced an increase in chondrophyte size and increased remodeling of the subchondral bone. CONCLUSIONS: The results of this study demonstrate that FGF18 can stimulate repair of damaged cartilage in a setting of rapidly progressive OA in rats.

Progressive chronic osteoarthritis in femorotibial joints of partial medial meniscectomized guinea pigs.

The sequential histopathologic alterations in femorotibial joints of partial meniscectomized male and female guinea pigs were evaluated at 1, 2, 3, 6, and 12 weeks post-surgery. Foci of moderate to severe cartilage degeneration were present in the medial tibial plateau and femoral condyle of the operated leg by 1 week post-surgery. At 2 weeks post-surgery, the cartilage degeneration in the operated legs was more extensive and there was evidence of early chondrophyte formation on the medial side of either the femur or tibia in three animals. Changes were progressively more severe at 3, 6, and 12 weeks. Focal areas of minimal to mild cartilage degeneration were in the medial tibial plateau of the contralateral nonoperated leg in some animals at 3 weeks post-surgery and in all animals at 12 weeks post-surgery. Changes in the contralateral leg of meniscectomized guinea pigs have not been described previously. Since cartilage degeneration was often severe by 1 week post-surgery, the model has limited utility for testing agents designed to modify the degenerative process in the operated leg. Milder, more slowly progressive lesions in the contralateral leg may be amenable to therapeutic intervention.

Development of a rapid screen for detecting and differentiating immunomodulatory vs. anti-inflammatory compounds in rats.

Polyarthritis can be induced in rats using a synthetic adjuvant, N,N-dioctyldecyl-N', N-bis(2-hydroxyethyl) propanediamine (LA) suspended in oil. The disease is morphologically indistinguishable from the classic adjuvant arthritis induced by Freund's complete adjuvant (FCA). LA injection (7.5 mg/animal) consistently induced paw swelling, splenomegaly and fibrinogen level increases at certain time points. Studies evaluating various protocols and parameters determined that a 15 day assay where agents administered from days 9 through 13, would differentiate immunomodulatory and anti-inflammatory compounds. Parameters utilized were body weight, paw volumes, spleen weights, and fibrinogen levels. Immunomodulatory agents reduce paw swelling, splenomegaly and in some cases fibrinogen levels. NSAIDS reduce paw swelling, increase splenomegaly and have no effect on fibrinogen levels. These results indicate that compounds active in the traditional FCA assay can be detected and differentiated with respect to anti-inflammatory vs. immunomodulatory activity in a rapid screen.

Effects of body weight restriction on the development and progression of spontaneous osteoarthritis in guinea pigs.

Hartley albino guinea pigs develop spontaneous osteoarthritis (OA) of the knee joint. A study was done to determine the importance of body weight in the pathogenesis of this disease. Two groups of 20 male guinea pigs each were maintained on the same diets. The control group was allowed ad libitum feed consumption and the other group was restricted to 30-35 gm of feed per day. Ten animals from each group were killed at 9 months of age to evaluate histologic features of the knee joints. The severity of the OA lesions was reduced by 40%, in conjunction with a 28% decrease in body weight, in the diet-restricted group. The remaining animals were killed at 18 months of age. Those in the diet-restricted group had a 56% reduction in severity of lesions, with a 29% decrease in body weight. These results indicate that body mass in guinea pigs, as in humans, is an important predisposing factor for the development of spontaneous OA of the knee.

Incidence of obstructive uropathy in male B6C3F1 mice on a 24-month carcinogenicity study and its apparent prevention by ochratoxin A.

A 24-month study assessed the carcinogenic potential of the nephrotoxic mycotoxin ochratoxin A (OA) in B6C3F1 mice. Three groups of 50 males and 50 females were fed 0.1 or 40 ppm OA in the diet. Obstructive urinary tract disease (mouse urological syndrome [MUS]) accounted for the greatest number of spontaneous deaths in the male mice of control (12/50) and 1 ppm (13/50) dose groups, but the disease was not observed in the males fed 40 ppm OA. The earliest age of onset of clinical signs of MUS was 4 months and the average age of onset was 10.1 months. The first death from MUS was observed at 5 months and average age at death was 12.2 months. The mice were caged in groups of five mice per cage and clustering of cases of MUS was observed. Properties of OA which may be important to its preventive effect include inhibition of growth of gram positive bacteria and the production of polyuria as a result of renal proximal tubular damage.