Comparative study of Monoiodoacetate (MIA) and Papain chemical model of osteoarthritis in Hartley strain guinea pigs

Introduction

Osteoarthritis (OA) is the most common arthritis which leads to chronic disability in both animals and humans. Rate of occurrence is higher in weight bearing joints at old age in human beings and animals [6] . Over 10% of the human population above 60 years is affected by OA, the impact of this health problem is still underestimated (10).Osteoarthritis (OA) is a systemic and chronic arthropathy characterized by the progressive breakdown of the articular cartilage (the end point of OA) along with changes in the subchondral bone, synovium (synovial inflammation), meniscus, tendons/ligaments, and muscles [13] . Animal models of OA are used extensively in search of pathogenesis of degenerative joint disease and in search of potential disease modifying anti-OA drugs. Lab Animal models, although imperfect, exhibit many of the pathologic features that characterize the canine or human disease. For induction of OA, chemical and surgical methods have been employed widely due to faster onset of disease and reproducible induction of arthritic change. Intra-articular injection of chemicals such as Mono-iodoacetate (MIA) or papain can cause the degeneration of cartilage and the development of osteoarthritis by inhibition of the activity of glyceraldehyde-3phosphate dehydrogenase in chondrocytes or by induction of synovial inflammation and degeneration of supporting structure and resultant instability, respectively. The aim of the present study was to evaluate the comparative efficacy of two chemicals MIA and papain in inducing osteoarthritis when injected intra articularly in knee joints of guinea pigs.

Experimental design

Two groups having 9 animals each were formed out of eighteen selected guinea pigs to develop osteoarthritis (OA). All the animals were anaesthetized with xylazine 6 mg/kg body weight intramuscular and 10 minutes after by ketamine 60 mg/kg body weight intramuscularly [1] . After appropriate anesthesia each animal was positioned on their back and the required limb was flexed by 90° at the knee. The patellar ligament was palpated below the patella and the injection was made into this region. Care was taken not to advance the needle too far into the cruciate ligaments. For induction of monoiodoacetate (MIA) induced arthritis, single intraarticular injection of 1 mg of monosodium iodoacetate (MIA; Sigma, St. Louis, MO, USA; cat #I2512) was administered through the infrapatellar ligament of the right knee [7] designated as group MIA (R). MIA was dissolved in physiologic saline and administered in a volume of 100 μl using a 27-gauge, 0.5-inch needle. The left contra lateral knees of the same animals were injected with 100 μl of physiologic saline to act as control designated as group MIA (L). In second batch of 9 animals, Papain enzyme (Sigma, Cat # P 3125) at dose rate of 10mg in 0.05M sodium acetate (pH 4.5) with enzymatic activity of 31 I.U/mg was injected intra-articularly in right knee joint of each animal designated as group Papain(R) while as left knee joints were injected with 100ul of sterile physiological saline solution (0.9%) designated as group Papain (L).

Clinical assessment of the joint was recorded on alternate days till two weeks of the intraarticular injection

Histopathology and Histochemistry

Three guinea pigs were sacrificed from both groups under high dose of intracardiac thiopentone on 7, 14 and 28th day post MIA or papain injection. Femorotibial joints of guinea pigs were separated with sharp knife on each sampling day and specimens were fixed in 10% neutral buffer formalin, decalcified in Gooding and Stewart solution and processed for paraffin embedding; frontal parts of tibial end were sectioned at 5um thickness and subsequently stained with haematoxylin-eosin (H&E) and by toluidine blue for proteoglycan. Semi-quantitative histopathological grading was performed in accordance with modified histological scoring system of Mankin's et al. [14] to evaluate guinea pig knee OA. Overall histological score of the tissue in left and right knees of both groups was calculated by summation of scores of each parameter and compared.

Estimation of Biochemical markers IL-6, IL-1β and TNF-α by ELISA kit methods

For estimation of biochemical markers, blood samples were collected directly from the heart before euthanizing the guinea pigs and from 3 separate animals to act as control for serum markers. Serum was separated and preserved at-20 °C until analysis.

Statistical analysis

The means of parametric observations were compared by repeated measures analysis of variance (ANOVA) and significance of differences was determined by post-hoc testing with Bonferroni's method. In each group base values

were compared with values at different intervals with paired't' test using SPSS software. Non-parametric observations were compared by Kruskal-Wallis test [19] .

Results and Discussion

A major symptom of the patients with osteoarthritis (OA) is pain that is triggered by peripheral as well as central changes within the pain pathways. Assessment of pain is of critical importance for mechanistic studies as well as for the validation of drug targets. Right knee joints of both MIA (R) and Papain (R) groups had showen severe pain initially which decreased to moderate pain towards the end of the observation period whereas, their respective left joints exhibited moderate pain initially followed by no pain in the remaining period of the observation. Intra-articular injection of monosodium iodoacetate (MIA) in the knee joint disrupts chondrocyte metabolism resulting in cartilage degeneration and subsequent nociceptive behavior that has been described as a model of osteoarthritic (OA) pain. Transient synovial inflammation may be the underlying cause of pain during the first week following MIA injection, whereas pain sensation in later stages may be due to biomechanical changes affecting the articular cartilage and subchondral bone [2] . Papain is, a proteolytic enzyme when administered to cartilage causes its breakdown, producing inflammatory cytokines (TNF-α and IL-1β) [4] . The mild to moderate pain in the left joints for few days might be due to swelling caused by injection of normal saline which subsided very early as compared to right knee joints (table 3).

There was a significant increase in the joint diameter from its baseline value at all intervals till the end of observation period in MIA and Papain treated joints whereas, the corresponding left joints had initial significant increase followed by normal diameter. The increase in joint diameter of treatment joints could be due to inflammation caused by the chemicals through activation of chemokines leading to joint swelling. Swelling is the cardinal sign of inflammation which might be due to associated synovitis and inflammation of other joint structures. The initial increase in joint diameters in physiological saline treated joints might be due to joint manipulation at the time of injection and mild reaction to salt solution which subsided in few days.

MIA and Papain might have caused synovitis and release of inflammatory cytokines [4] which might have led to significant joint swelling which persisted for longer duration as compared to their control joints (table 4).

Radiography is the standard technique used to diagnose and chronicle the progression of osteoarthritis. Severity of radiographic OA can be estimated using semi-quantitative scoring systems. Radiographic features including narrowing of joint space, presence of osteophytes, sclerosis of subchondral bone and deformity of bone ends can be assessed, and scored in five grades of 0 (none), 1 (doubtful), 2 (minimal), 3 (moderate) and 4 (severe). MIA administration in joints of guinea pigs had resulted in mild and moderate osteoarthritis on day 14 and day 28, respectively (table 5 & fig 1). Papain treated joints had shown mild osteoarthritis until day 28 (table 5 & fig 2). Also joints of MIA treated animals revealed non significantly (p>0.05) higher scores on day 14 and 28 than papain treated joints. There was constant decrease in joint space along with cartilage loss and bone osteolysis in MIA treated joints but Papain treated joints showed only reduction in joint space with mild bone osteolysis on day 28 (Fig

. Left joints (control) of both groups showed no change in joint space and no bone osteolysis. Few osteophytes were also present in one animal at the end of observation period in MIA treated joint (MIA(R) group).The findings of the present study substantiate the fact that MIA and Papain can damage the cartilage. MIA causes degenerative changes in articular cartilage by direct interference with chondrocyte metabolism [12] and has been used to induce degenerative joint disease in the rat [7] , mouse [12] , and guinea pig. Papain is the proteolytic enzyme and causes osteoarthritis by releasing chondroitin sulphate from the protein polysaccharide complex of the matrix of articular cartilage [9] . However it doesn't induce direct effect on chondrocytes and collagen [8] . For the histological characterization of the lesions, the majority of published animal studies used a scoring system of evaluation. We have used Mankin's histopathological score method which combines all the changes in cartilage structure, proteoglycan content as measured by toluidine blue method, cellularity and tidemark integrity with additional features of osteophyte development in one score to evaluate the histopathological changes in osteoarthritis in guinea pigs. The same scoring has been used in cartilage studies by [11] . At the end of the study period i.e. day 28, higher osteoarthritic scores were recorded in MIA (R) treated joints than Papain (R) treated joints. It might be due to more damaging effect of MIA than papain causing earlier onset of histopathological changes in MIA group than that in Papain group. Earlier studies have reported that multiple doses of Papain are needed to cause higher grades of osteoarthritis [15] . Normal saline treated joints of both groups had milder changes in cartilage structure (only undulations present) and minimal decrease in proteoglycan content. Osteophytes were not seen in any of the joints treated with normal saline in both groups. Histopathological features of OA, such as loss of proteoglycan, cartilage degeneration, osteophyte formation, a dose dependent reduction in spontaneous locomotion after monoiodoacetate injection into the knee of rats has been reported in earlier study [7] . The injection of iodoacetate induces the loss of cartilage proteoglycan as measured by safranin O-fast green staining. The loss of proteoglycan content has been reported in guinea pigs injected with iodoacetate [21,22] . Histochemical examination of the sections from the experimental joints indicated a loss and degradation of the sulfated glycosaminoglycans by administration of papain in the guinea pig knee joint [20] . Our finding were in accordance with the observation of [12] who observed distinct depletion of stain and cytolysis post papain and iodoacetate injection which was comparable with bovine serum albumin induced OA. [18] reported death of chondrocytes in patches which was due to variable sensitivity of chondrocytes in different areas of the cartilage. Areas of hypocellularity and hypercellularity observed with progression of OA post papain injection in the present work were confirmed to the results of [17,3] . Proliferative response of chondrocytes could be due to depletion of proteoglycans initially and later on lowered number was observed possibly due to lowered metabolic activity of chondrocytes. Similar findings have been reported by [12] . In the present study highest histological lesion score (11.00±3.51) was observed in MIA cartilages on 28th day post MIA injection and (8.00±1.15) in case of Papain treated joints. So, it was concluded that severity of lesions progressed with passage of time and maximum lesions were observed on day 28th that seem to be consistent with early OA changes in humans.

Different cytokines take part in pathogenesis of osteoarthritis. There is a growing need to identify and validate reliable biochemical biomarkers that can provide information on the status and progression of joint destruction in OA. IL-1β and IL-6 levels remained higher at all time intervals in both MIA and Papain groups from the control animal values but the increase was more significant in MIA group than Papain group. IL-1β levels in MIA group were significantly (p<0.05) higher than Papain group on day 14 and IL-6 levels on day 7 respectively. TNF-α showed non-significant increase in both groups. The monosodium iodoacetate (MIA) model has been well characterized for the evaluation of OA pain. The iodoacetate creates local inflammation in the knee from fluid expansion of the synovial membrane, which is then followed by the production of inflammatory mediators (TNF-α, IL-1β, IL-6).

These mediators further contribute to OA pathogenesis by increasing cartilage degradation. The cartilage damage and degradation leads to chronic neuronal damage with neuropathic characteristics. The MIA-OA model is thought to contain both inflammatory and neuropathic pain-related states by affecting local tissues and sensory innervations of the peripheral nervous system. Cytokines such as IL-1β and TNF-α produced by activated synoviocytes, mononuclear cells or by articular cartilage itself significantly up-regulate metalloproteinases (MMP) gene expression [5] . Cytokines also blunt chondrocytes compensatory synthesis pathways required to restore the integrity of the degraded extracellular matrix. Moreover, in OA synovium, a relative deficit in the production of natural antagonists of the IL-1 receptor (IL-1RA) has been demonstrated, and could possibly be related to an excess production of nitric oxide in OA tissues. This, coupled with an upregulation in the receptor level, has been

shown to be an additional enhancer of the catabolic effect of IL-1β inthis disease. IL-1β and TNF-alpha significantly upregulate MMP-3 steady-state mRNA derived from human synovium and chondrocytes. The proinflammatory cytokine interleukin 1beta (IL-1β) has several chemical and bioactive characteristics allowing this catabolic protein to be involved in initiation and progression of OA. IL-1β upregulation leads to cascade of intracellular events that can result in activation of proteinases, creation of a pro-destructive articular milieu, suppression of anabolic pathways, and a decrease in the synthesis of cartilage extracellular matrix [5] . IL-6 correlated positively with total histological score because IL-6 has been shown to use trans-signaling to regulate pre-B cell colony-enhancing factor (PBEF), which is involved in the progression of OA [16] .

Conclusion

On the basis of findings of the present study, both MIA and papain can be used to induce experimental osteoarthritis in Hartley strain guinea pigs but MIA induces faster and more severe osteoarthritic changes than papain.

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