Knee Joints Of Wistar Rats Research Articles

Effect of Low-Intensity Pulsed Ultrasound on Macrophage Properties and Fibrosis in the Infrapatellar Fat Pad in a Carrageenan-Induced Knee Osteoarthritis Rat Model.

In the progression of knee osteoarthritis (KOA), fibrosis of the infrapatellar fat pad (IFP) is a key pathological change. Low-intensity pulsed ultrasound (LIPUS) inhibits IFP fibrosis by decreasing the gene expression and activity of hypoxia-inducible factor (HIF-1α), which is a protein involved in IFP fibrosis in KOA rat models. On the other hand, macrophages play an important role in the progression of fibrosis in various tissues, and LIPUS irradiation suppresses macrophage infiltration and inflammatory cytokine secretion. However, whether LIPUS suppresses macrophage polarity and IFP fibrosis in KOA remains unclear. Therefore, we investigated the effect of LIPUS on macrophage polarity and IFP fibrosis. A KOA model was created by injecting carrageenin into the bilateral knee joints of Wistar rats (eight weeks old). Tissues were harvested over time for histological and molecular biological analysis. The KOA model was also subjected to LIPUS irradiation for two weeks following the injection of carrageenin. RM-4-positive cells were widely distributed in IFP two weeks after carrageenin administration, but M2 macrophages were significantly increased, and the Sirius red area was decreased in the LIPUS-irradiated group compared with those in the non-irradiated group. The gene expression of M1 macrophage markers was significantly decreased and that of M2 macrophage markers was significantly increased in the LIPUS-irradiated group. The expression of transforming growth factor-β (TGF-β) and type 1 collagen was also significantly decreased. These results suggest that LIPUS may serve as a novel approach for the treatment of KOA through its effect on M1 macrophages and suppression of TGF-β expression.

MiR-18a-3p improves cartilage matrix remodeling and inhibits inflammation in osteoarthritis by suppressing PDP1

Osteoarthritis (OA) is a degenerative disease characterized by synovial inflammation. MiR-18a-3p was reported to be downregulated in knee anterior cruciate ligament of OA patients. In the present study, the specific functions and mechanism of miR-18a-3p in OA were explored. An in vitro model of OA was established using 10 ng/ml IL-1β to treat ATDC5 cells, and medial meniscus instability surgery was performed on Wistar rats to establish in vivo rat model of OA. RT-qPCR revealed that miR-18a-3p was downregulated in IL-1β-stimulated ATDC5 cells. MiR-18a-3p overexpression inhibited secretion of inflammatory cytokines and concentration of matrix metalloproteinases, as shown by ELISA and western blotting. The binding relation between miR-18a-3p and pyruvate dehydrogenase phosphatase catalytic subunit 1 (PDP1) was detected by luciferase reporter assays. MiR-18a-3p targeted PDP1 and negatively regulated PDP1 expression. Results of rescue assays revealed that PDP1 upregulation reserved the suppressive effect of miR-18a-3p overexpression on levels of inflammatory cytokines and matrix metalloproteinases in IL-1β-stimulated ATDC5 cells. H&E staining was used to observe pathological changes of synovial tissues in the knee joint of Wistar rats. Safranin O-fast green/hematoxylin was used to stain cartilage samples of knee joints. MiR-18a-3p overexpression suppressed OA progression in vivo. Overall, miR-18a-3p improves cartilage matrix remodeling and suppresses inflammation in OA by targeting PDP1.

177Lu–labeled micro liposomes as a potential radiosynoviorthesis therapeutic agent

Micro–sized multivesicular liposomes were prepared, radiolabeled with 177Lu, and tested in vitro and in vivo to evaluate the potential of 177Lu–labeled micro liposomes in radiosynoviorthesis (RSO) therapy. A standard reverse–phase procedure of liposome preparation with a lipid mixture of DPPC: CHOL (80:20%) was used for the synthesis. TEM and fluorescence microscopy imaging were performed to determine the size, shape, and structure of the prepared liposomes. Both measurements are in good agreement while TEM micrographs additionally indicate to a large multivesicular inner structure of prepared liposomes. A simple and straightforward procedure was used for liposome radiolabeling with 177Lu, a well–known and commonly used radionuclide in radiotherapy with favorable properties, that can be exploited in RSO therapy. Radiolabeled 177Lu–liposomes were tested in vitro for stability and then injected into the knee joints of Wistar rats where liposome in vivo behavior was followed up to 30 days post injection. Results from both ex vivo biodistribution and in vivo imaging studies presented a high stability and retention (>94 %ID) of 177Lu–micro liposomes in the synovial liquid for the entire observation period. Leakage of free 177Lu or 177Lu–liposomes from the synovial fluid has not been detected, indicating to a possible application of 177Lu–liposomes in radiosynoviorthesis (RSO) therapy.

Early Signs of Bone and Cartilage Changes Induced by Treadmill Exercise in Rats.

This study aims to investigate the earliest alterations of bone and cartilage tissues as a result of different exercise protocols in the knee joint of Wistar rats. We hypothesize that pretraining to a continuous intense running protocol would protect the animals from cartilage degeneration. Three groups of animals were used: (i) an adaptive (pretraining) running group that ran for 8 weeks with gradually increasing velocity and time of running followed by a constant running program (6 weeks of 1.12 km/hour running per day); (ii) a non-adaptive running (constant running) group that initially rested for 8 weeks followed by 6 weeks of constant running; and (iii) a non-running (control) group. At weeks 8, 14, and 20 bone and cartilage were analyzed. Both running groups developed mild symptoms of cartilage irregularities, such as chondrocyte hypertrophy and cell clustering in different cartilage zones, in particular after the adaptive running protocol. As a result of physical training in the adaptive running exercise a dynamic response of bone was detected at week 8, where bone growth was enhanced. Conversely, the thickness of epiphyseal trabecular and subchondral bone (at week 14) was reduced due to the constant running in the period between 8 and 14 weeks. Finally, the intermediate differences between the two running groups disappeared after both groups had a resting period (from 14 to 20 weeks). The adaptive running group showed an increase in aggrecan gene expression and reduction of MMP2 expression after the initial 8 weeks running. Thus, the running exercise models in this study showed mild bone and cartilage/chondrocyte alterations that can be considered as early-stage osteoarthritis. The pretraining adaptive protocol before constant intense running did not protect from mild cartilage degeneration. © 2017 The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Foamy virus‐adenovirus hybrid vectors for gene therapy of the arthritides

Genetic treatments of chronic arthritic conditions are essentially dependent on safe and efficient vector systems. To combine features of the efficient transduction of adenovirus vectors with the advantage of stable integration into the host cell genome of apathogenic prototype foamy virus vectors, hybrid vectors (FAD) have been established. In the present study, we have generated and investigated the use of safe FAD vectors for direct gene delivery to joints. We generated recombinant FAD encoding enhanced green fluorescent protein (EGFP) or human interleukin 1 receptor antagonist protein (IL1RA) cDNA, and explored their transgene expression profile, as well as the bioactivity of the IL1RA transgene in vitro. The feasibility of IL1RA gene delivery to articular tissues was investigated in a pilot study employing direct FAD injections to the knee joints of Wistar rats. FAD vectors efficiently transduced human or rat fibroblasts with EGFP or IL1RA transgene in vitro. Levels of IL1RA transgene expression were high, stable and functional in vitro. Transduced synovial fibroblasts and high levels of IL1RA protein (10-35 ng/ml) could be detected in vivo in the synovium of Wistar rats 3-5 days after injection of FAD vectors to the knee joints. Our results indicate that FAD vectors are capable of efficient in vivo gene transfer to synovium and merit further investigation as a means of providing efficient and long-term intra-articular transgene expression for treatment of the arthritides.

Bioevaluation of 90Y-labeled particles in animal model of arthritis

The aim of the present study was to evaluate the behaviour of (90)Y-labeled particles when injected into an arthritic knee joint of Wistar rats with severe inflammation induced using Complete Freund's Adjuvant (CFA). (90)Y-ferric hydroxide macroaggregates ((90)Y-FHMA), (90)Y-hydroxyapatite ((90)Y-HA) and (90)Y-phosphate particles ((90)YPO(4)) were prepared, subjected to quality control analyses and in vitro stability studies. Biodistribution studies were carried out by intra-articular injection into knee joints of Wistar rats induced with chronic inflammatory arthritis using CFA and by monitoring the radioactivity for retention and leakage. All the three preparations exhibited ~99% localization in knee joints even at 24 h p.i. with very small amounts observed in the liver and lungs, possibly due to leakage of the radiolabeled particles from the inflamed knee joint. Absence of any radioactivity in the femur indicated the in vivo stability of the particle preparations. The biodistribution patterns were very similar in the normal and arthritic rats and were associated with negligible leakage (up to 24 h) from the knee joint indicating the potential of all the (90)Y-radiolabeled preparations reported here, for use in radiation synoviorthesis.

Preparation and preliminary biological evaluation of 177Lu-labelled hydroxyapatite as a promising agent for radiation synovectomy of small joints

Lutetium-(177)Lu) is considered to be a promising radionuclide for use in radiation synovectomy of small-sized joints owing to its favourable decay characteristics [t(1/2)=6.73 days, E(beta)(max)=0.49 MeV, E(gamma)=113 keV (6.4%), 208 keV (11%)] and feasible and cost-effective production route. Hydroxyapatite particles are regarded as one of the most suitable carriers for applications in radiation synovectomy, and labelling with (177)Lu has been envisaged. The present work describes the preparation and preliminary biological evaluation of (177)Lu-labelled hydroxyapatite particles. (177)Lu-labelled hydroxyapatite particles were prepared using (177)Lu produced by thermal neutron irradiation of a natural (2.6% (177)Lu) Lu(2)O(3) target and hydroxyapatite particles (particle size, 2-10 microm) prepared in-house. The biological efficacy of the radiolabelled preparation was tested by recording serial gamma scintigraphic images after injecting the agent in both normal and arthritic knee joints of Wistar rats. (177)Lu-hydroxyapatite was prepared with high yield and high radiochemical purity (approximately 99%) and the radiolabelled particles showed excellent in-vitro stability at room temperature. Serial scintigraphic images of normal and arthritic Wistar rats showed complete retention of activity within the synovial cavity, with no measurable activity leaching out from the joint until 168 h post-injection. Studies with (177)Lu-hydroxyapatite indicate its potential for use as an agent for radiation synovectomy of digital joints, as a viable alternative to (169)Er-based agents. The results also demonstrate the possibility of preparing a large number of patient doses of (177)Lu-hydroxyapatite from indigenously produced (177)Lu using a natural target.

Sodium Iodoacetate-Induced Experimental Osteoarthritis and Associated Pain Model in Rats

Degenerative lesions were induced in the knee joint of Wistar rats by intraarticular injection of chondrocyte metabolism inhibitor mono-iodoacetate (MIA) at doses of 0, 0.3 or 3 mg/joint. Histopathological examination and the measurement of hind paw weight ratio as an index of joint pain by incapacitance tester were performed. Histological findings that are similar to those observed in human osteoarthritis (OA), such as disorganization of chondrocytes, erosion and fibrillation of cartilage surface, and subchondral bone exposure etc., were observed in a MIA-dose-dependent manner. Saflanin-O fast green staining revealed that marked diffuse reduction of proteoglycan in cartilage tissue of rats treated with MIA. The clinical scores of the joint pain were closely correlated to the grade of histological findings. We conclude that the present experimental model in combination with a novel dual channel weight averager would be very useful for the study of human OA, and could be applied for estimation of therapeutic effect of new anti-OA drugs.

The role of IL-4 in the control of mechanical stress-induced inflammatory mediators by rat chondrocytes

A complex environment controls the metabolism of chondrocytes in the inflammatory joint. Although mechanical stimuli are essential for the chondrocyte metabolism, excessive mechanical force contributes to the release of inflammatory mediators leading the cartilage destruction under the arthritic condition. IL-4 is a chondroprotective cytokine, which is also involved in the integrin-mediated chondrocyte mechano-transduction. We examined the effects of cyclic mechanical stress on the gene expression profile of rat chondrocytes, and tested the in vitro effects of recombinant IL-4. Chondrocytes were obtained from the knee joints of 7-day-old Wister rats. Cyclic mechanical stress was applied on the cultured chondrocytes for 24 hours using Flexercell strain unit (Flexcell International Co., Hillsboro, NC, USA) with 0.5 Hz, 7% elongation, with or without treatment by recombinant IL-4. The cDNA microarray analysis revealed that, of 1080 genes, 37 genes were upregulated and 46 genes were downregulated after mechanical stress. RT-PCR and real-time PCR analysis confirmed that treatment by IL-4 resulted in the significant downregulation of mRNA expression of cysteine proteinase cathepsin B, and the inducible form of nitric oxide synthase. Next, an osteoarthritis model was created in the knee joints of Wistar rats (200 g) with anterior cruciate ligament and medial collateral ligament transection. The rats were killed 2, 4, or 6 weeks after the surgery, and the gross morphology and histology of the knee joint cartilage were examined. The preliminary results showed that intra-articular administration of IL-4 exerted a protective effect on the development of osteophytes, and cartilage lesions in this model of osteoarthritis. These results suggested the possible role of IL-4 in the control of mechanical stress-induced inflammatory mediators in vitro and in vivo.

Development of Osteoarthritis in the Knee Joints of Wistar Rats After Strenuous Running Exercise in a Running Wheel by Intracranial Self-Stimulation

The influence of excessive running load on the development of knee osteoarthritis (OA) was investigated in male Wistar rats. Running exercises were performed in a running wheel using intracranial self-stimulation to motivate Wistar rats to run daily distances of 500 m at 5 days/week. Hereby, ten rats ran a distance of 15 km within three weeks while a further ten rats run a total of 30 km within six weeks. Thirteen Wistar rats without running exercises served as controls. Complete knee joint sections of all rats were evaluated histologically using MANKINs grading system with categorization of the findings into non, mild moderate, and severe osteoarthritis. In addition, immunoreactivity of the chondrocytes to MMP-3 as an important cartilage degrading enzyme in OA was assessed by immunostaining with monoclonal MMP-3 IgG antibodies. Histological assessment of the knee joint sections revealed a significant increase in osteoarthritic changes with higher running load. While in rats with 15 km running all but two knee joints showed mild OA, moderate OA was the predominant finding in rats with 30 km running. In contrast, no OA was found in the controls. Immunostaining for MMP-3 revealed a significant increase in immunoreactivity of the chondrocytes to MMP-3 with higher running load, indicating a running load-depending production of this cartilage-degrading enzyme in the course of increasing OA. Compared to 47.4% immunoreactive chondrocytes to MMP-3 in the controls, this ratio rose to 70.4% in rats with 15 km running and even up to 89.9% in rats with 30 km running. In conclusion, in Wistar rats, excessive running load leads to marked, running distance-depending osteoarthritic changes which are caused, at least in part, by an increase in MMP-3 production rising with greater running distance. Within this exercise model of OA, intracranial self-stimulation is an effective method to motivate Wistar rats to extremely excessive running in a running wheel. This model offers a wide range of further approaches to studying different processes of the development of OA.

Chondrocytes synthesize type I collagen and accumulate the protein in the matrix during development of rat tibial articular cartilage.

The present study was designed to investigate whether or not chondrocytes in articular cartilage express type I collagen in vivo under physiological conditions. Expressions of the gene and the phenotype of type I collagen were examined in rat tibial articular cartilage in the knee joint during development. Knee joints of Wistar rats at 1, 5, and 11 weeks postnatal were fixed in 4% paraformaldehyde with or without 0.5% glutaraldehyde and decalcified in 10% EDTA. After the specimens were embedded in paraffin and serial sections made, adjacent sections were processed for immunohistochemistry and in situ hybridization for type I collagen. The epiphysis of the tibia was composed of cartilage in week- 1 rats. Formation of articular cartilage was in progress in week 5 as endochondral ossification proceeded and was completed in week 11. Anti-type I collagen antibody stained only the superficial area of the epiphysis in week 1, but the immunoreactivity was expanded into the deeper region of the articular cartilage with development in weeks 5 and 11. Hybridization signals for pro-alpha 1 (I) collagen were seen in some of chondrocytes in the epiphysis of the week-1 tibia. The most intense signals were identified in chondrocytes in week 5 and the signals appeared weaker in week 11. The present study demonstrated that chondrocytes synthesize type I collagen and accumulate the protein in the matrix during development of the articular cartilage.