 |
|
||||
|
|||||
| About | Latest Articles | Back-Issues | Articles
|
Search | Instructions | Online Submission | Subscribe | Etcetera | Contact |
| |||||||||||||||||||||
|
Arthritis, a complex connective and synovial joint destructive autoimmune disease: Animal models of arthritis with varied etiopathology and their significance SR Naik, SM WalaDepartment of Pharmacology, Sinhgad Institute of Pharmaceutical Sciences, Lonavala, Pune, Maharashtra, India
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0022-3859.138799
Animal models play a vital role in simplifying the complexity of pathogenesis and understanding the indefinable processes and diverse mechanisms involved in the progression of disease, and in providing new knowledge that may facilitate the drug development program. Selection of the animal models has to be carefully done, so that there is morphologic similarity to human arthritic conditions that may predict as well as augment the effective screening of novel antiarthritic agents. The review describes exclusively animal models of rheumatoid arthritis (RA) and osteoarthritis (OA). The development of RA has been vividly described using a wide variety of animal models with diverse insults (viz. collagen, Freund's adjuvant, proteoglycan, pristane, avridine, formaldehyde, etc.) that are able to simulate/trigger the cellular, biochemical, immunological, and histologic alterations, which perhaps mimic, to a great extent, the pathologic conditions of human RA. Similarly, numerous methods of inducing animal models with OA have also been described (such as spontaneous, surgical, chemical, and physical methods including genetically manipulated animals) which may give an insight into the events of alteration in connective tissues and their metabolism (synovial membrane/tissues along with cartilage) and bone erosion. The development of such arthritic animal models may throw light for better understanding of the etiopathogenic mechanisms of human arthritis and give new impetus for the drug development program on arthritis, a crippling disease. Keywords: Arthritis, osteoarthritis, rheumatoid arthritis
Arthritis is mainly characterized by inflammation of the synovial tissue/membrane and accompanying varying degrees of degeneration of cartilage and bone erosion. However, the correlation between inflammation and progressive destruction is not fully understood with rheumatoid arthritis (RA) or other types of arthritis. [1] Therefore, it is essential to explore the state-of-the-art of techniques/methods for assessing precisely the inflammation, cartilage damage, synovial joint destruction, and bone erosion which would find the possible solutions to define/explain the characteristic features of the inflammation that initiates progressive destruction. The major signs and symptoms observed are joint pain, joint enlargement or swelling, stiffness, warmth, and redness of the skin around joints. The different types of arthritis affecting various sites are peripheral arthritis, axial arthritis, ankylosing spondylitis, juvenile idiopathic arthritis, RA, fibromyalgia, systemic lupus erythematosus, osteoarthritis (OA), gout, and polymyalgia rheumatica. Considering the various arthritis disorders, we felt it is essential to focus on and project RA and OA due to their severity and irreversibility, which finally manifests as the colossal loss of human productivity and low quality of life, with a deep social and economic impact all over the world. Rheumatoid arthritis RA can be defined as a chronic inflammatory disease with systemic autoimmune component, and is mainly characterized by aggressive synovial hyperplasia, synovitis, progressive destruction of cartilage, and bone erosion with painful swelling of small joints, fatigue, prolonged stiffness, and fever caused by immune responses and specific innate inflammatory processes. RA causes premature mortality, deformity of joints, loss of function, loss of productivity, and low quality of life. The ethiopathology of RA is still not completely understood, and till date, there is no effective treatment to cure RA. However, phytochemicals, and biotechnologically and synthetically derived magic therapeutic bullets may be able to provide relief from its severity as well as the low quality of life, or cure the arthritic condition. [2],[3] Osteoarthritis OA can broadly be defined as a heterogeneous condition of transient and progressive structural changes in joint tissues, especially articular cartilage, subchondral bone, synovium, and synovial fluid, resulting in the development of bony spurs and cysts at the margins of the joints, and also affecting chondrocyte metabolism and the composition of extracellular matrix. [4],[5] OA affects knee joints, hips, small joints of hands, spine, and joints associated with mild synovitis, causing pain and stiffness. Some of the critical paradigms associated with OA are: articular cartilage edema, fibrillation and erosion with concomitant proliferation of chondrocytes, decreased staining of matrix proteoglycans (PGs), subchondral bone thickening, deformation of the articular surface, osteophyte formation, synovial intimal cell hyperplasia, and synovial fibrosis. Increased formation of cytokines activates matrix metalloproteinases (MMPs), important proteolytic enzymes responsible for the degradation of cartilage and bone. [6] Considering the aforementioned characteristic changes in the cartilage and supporting tissue, bone erosion, and molecular alterations, and to understand the existence of a complex relationship between the different disease mechanisms, many animal models were developed for the purpose of therapeutic evaluation of novel anti-osteoarthritic agents. [7] Animal Models of RA Considering the occurrence of several pathophysiological events in RA, different animal models are simulated for evaluation and determination of the therapeutic efficacy of new molecules. Animal model of human disease can be defined as a homogenous set of animals which have inherited, naturally acquired, or experimentally induced biological process, amenable to scientific investigation that in one or more respects might resemble the disease in humans, and is used to understand the indefinable processes and the diverse mechanisms involved. [8],[9] Collagen-induced arthritis Collagen type II (CII) is a major autoantigen in RA, and the prevalence of CII-specific antibodies and T cells in the early phase of RA suggests CII-specific immunity plays a major role in the induction of inflammation in articular joints and causing joint destruction. [10],[11] Hence, collagen-induced arthritis (CIA) models (mice, rats, and monkeys) are used widely because the model is found to be similar to that of human RA, mainly in its clinical and biochemical (synthesis of proinflammatory cytokines, prostaglandins, leukotrienes, and MMPs), immunological (activation of macrophages and inflammatory cells, overexpression of major histocompatibility complex II molecules, and enhanced cytokine production), and radiological and histologic aspects (inflammatory cell/leukocyte infiltration, inflammation of synovial joints, destruction of cartilage, bone erosion, and joint space narrowing). [12] Induction of arthritis The CII emulsion is prepared by mixing equal volumes of CII in 10 mM acetic acid and incomplete Freund's adjuvant. Then, 100 μg and 500 μg of CII emulsion are injected subcutaneously at the base of the tail of DBA/1J mice and Sprague-Dawley rats, respectively, on day 0 and day 7 for the induction of arthritis. All the symptoms of arthritis are observed between 12 and 14 days after CII emulsion administration. The various biological parameters, viz. paw volume, ankle diameter, grip strength, motility of joints, and pain threshold, are studied during arthritis development. The effects of test substances are assessed on 14-45 days by evaluating the various biological parameters mentioned above with visual (a score of 0-4) scoring system. The antiarthritic activity can be evaluated by prophylactic as well as therapeutic treatment. At the end of the experiment, radiographic analysis of normal and arthritic hind legs is performed using X-ray technique. The radiological criteria considered are: 0, no tissue swelling or bone damage; 1, tissue swelling and edema; 2, joint erosion; 3, bone erosion; and 4, osteophyte formation. The radiology scores for both hind paws are used to calculate the arthritic index. Various antioxidants (lipid peroxides, myeloperoxidase, superoxide dismutase, catalase, glutathione peroxidase, glutathione, nitric oxide), serum lysosomal enzymes (cathepsin D, lactate dehydrogenase, β-glucuronidase), metabolic products of the connective tissue (hydroxyproline, sialic acid, N-acetyl-β-d-glucosaminidase), intermediary energy metabolites (mitochondrial ATPase, succinic dehydrogenase), and polyamines and proinflammatory cytokines [interleukin (IL)-1β, tumor necrosis factor alpha (TNF-α), transforming growth factor beta (TGF-β)] in edema tissue and serum are the important biochemical indicators for antiarthritic drugs. [13],[14],[15],[16],[17],[18],[19] Freund's adjuvant-induced polyarthritis in rats Experimental arthritis is induced in rats by the method of Newbould. [20] In the plantar region of right hind paw of each rat is injected subcutaneously 0.1 ml (10 mg/ml of heat-killed Mycobacterium tuberculosis suspension prepared in mineral oil) of complete Freund's adjuvant (CFA). Polyarthritis syndrome is manifested by the development of:
X-rays of hind legs of rats are taken prior to sacrifice in order to study the effect of test drug on cartilage degeneration, bone erosion, and pannus formation. Rats are sacrificed either on 15 th or 29 th day for studying the biochemical changes in blood and inflamed tissues, including histologic alterations. Pristane-induced arthritis The non-antigenic chemical, pristane (2, 6, 10, 14- tetramethylpentadecane), is known to induce delayed onset of arthritis and unparalleled chronicity by involving CD4 + T cells, immunodominant environmental antigens, and heat shock protein 65 (hsp 65) and activating lymphocytes. [21] Intraperitoneal injection of pristane in the rodents induces chronic arthritis (onset from 60 to 200 days) that closely resembles RA. Alternatively, pristine-induced arthritis (PIA) can also be achieved in rats by intradermal injection of 150 μl at the base of the tail, which develops arthritis within 2-3 weeks and progresses with a relapsing course that persists for more than 6-10 weeks. The PIA is characterized clinically by joint swelling, pathologic alterations (polymorphonuclear cell infiltration and formation of pannus in affected joints), activated proinflammatory cytokines, and enhanced humoral/cellular responses to several putative joint autoantigens. [22],[23] Both PIA and RA are characterized by elevated levels of circulating rheumatoid factor, agalactosyl immunoglobulin G (IgG), cytokines [IL-1, IL-4, IL-6, TNF-α, and interferon gamma (IFN-γ)], glucose-6-phosphate isomerase, chondroitin sulfate B, collagen I, collagen II, aggrecan, and DNA of the connective tissue within 4-12 months post-pristane injection. [24] The development of arthritis is monitored by a macroscopic scoring system for four limbs with a scale ranging from 0 to 4, as mentioned earlier. PG-induced arthritis Aggrecan, a known cartilage-specific PG core protein, is used as an antigen to induce arthritis in rodents which exhibits a biological profile similar to that of collagen arthritis. PG aggrecan has also been reported to induce erosive polyarthritis and spondylitis in BALB/c mice, which is attributed to the G1 domain of the PG. [25],[26] PG-induced arthritis is directly related to the interaction between T cells (CD4 + T cells) and B cells, and these are found to be the major immune cells participating in the genesis of arthritis. [27],[28] Accumulation of PG-specific IgG in the cartilage and inflamed joints leads to loss of cartilage. Thus, the interaction between IgG containing immune complexes and FcγRs is the major factor in the progression of inflammation and development of arthritis. The arthritis in female BALB/c mice (12-14 weeks old) is induced by intraperitoneal injection of 150 μg PG (obtained from human spondylitis/arthritic cartilage patients) prepared in dimethyldioctadecylammonium bromide. Subsequently, BALB/c mice received a booster dose of 100 μg PG at an interval of 3 rd and 6 th week and were observed for inflammatory reactions/arthritic symptoms (such as varied degrees of edema, erythema with ankylosis) scored on the basis of intensity on a scale of 0-4 in a blind fashion for every 2 weeks up to a period of 18 weeks and the arthritic index is calculated. Histopathologic alterations observed are:
Cartilage oligomeric matrix protein-induced arthritis Cartilage oligomeric matrix protein (COMP) is a 524-kDa homopentameric extracellular matrix glycoprotein, belonging to thrombospondin family. COMP is synthesized largely by chondrocytes, and is localized extracellularly and present in cartilage, tendon, vitreous of the eye, and vascular and smooth muscle cells. In adults, COMP is most abundantly found in the inter-territorial matrix of articular cartilage, and is a sensitive indicator of the progression of arthritis. [30],[31],[32] RA is induced by immunization with COMP, which finally manifests as autoimmune arthritis due to cross-reactive immune responses to autologous mouse COMP. Further, COMP-induced arthritis largely relies on T cell recognition as well as major histocompatibility complex (MHC) molecules. COMP-immunized mice develop a strong and specific IgG response to COMP, increased B cells, CD4 + T cells, and absence of cytotoxic CD8 + T cells. This model develops symptoms analogous to human arthritic symptoms such as synovial hyperplasia, increased synovial volume, cellular infiltration, and the unique feature of a chronic relapsing disease phase observed mainly in females. [33] Induction of arthritis Mice are immunized initially by intradermal injection of 100 μg of COMP emulsified in 100 μl of CFA at the base of the tail. Subsequently (after 35 days), a booster injection of 50 μg of COMP in 50 μl of Freund's incomplete adjuvant is administered intradermally at the base of the tail. The mice are monitored three times a week for 160 days for the development of arthritis. Assessment of the number and degree of joints affected is performed by a macroscopic scoring system on a scale of 0-4 and the following are assessed:
Avridine-induced arthritis in rats Avridine [(N,N-dioctadecyl-N',N'-bis (2-hydroxyethyl) propanediamine], a potent synthetic non-immunogenic adjuvant, is used to induce arthritis in rats. Avridine-induced arthritis (AIA) is similar to collagen and Freund's adjuvant induced arthritis with respect to many pathophysiological symptoms. The development of arthritis is largely mediated by T cells and influenced by both MHC and non-MHC genes. [35] It is documented that LEW and DA rats are the most sensitive and develop severe arthritis with avridine. It is found that the infiltration of CD4 + T lymphocytes and MHC class II expression occurs prior to the development of arthritis. Induction of arthritis Avridine is solubilized in Freund's incomplete adjuvant and injected subcutaneously at a varied concentration, ranging from 3.75 to 7.5 mg/rat, either at the base of the tail or into the plantar region of hind paws of the rat. Development of arthritis was found to be 100% at 7.5 mg and 60-70% at 3.75 mg within 4 weeks period. Arthritis index is scored by visual scoring system on a scale of 0-4, observing the following parameters, respectively, in the four limbs:
Streptococcal cell wall-induced arthritis Streptococcal cell wall (SCW)-induced arthritis is a widely accepted rat model because it exhibits many critical symptoms of human RA, viz. infiltration of polymorphonuclear cells, CD4 + T cells, macrophages, hyperplasia of the synovial membrane, pannus formation, and significant erosion of cartilage and bone. In this model, synovitis can be induced within 24 h with maximum swelling by intraarticular injection of the antigen into an ankle joint. A characteristic feature of this model is development of spontaneous response, which allows the precise analysis of both cellular and biological mechanisms. [37] The active participation of TNF-α, IL-1α, IL-4, nitric oxide synthase (NOS), cyclooxygenase, P-selectin, vascular cell adhesion molecule-1, macrophage inflammatory protein (MIP)-2, MIP-1α, and monocyte chemoattractant protein (MCP)-1 in SCW-induced experimental arthritis has been demonstrated. [38],[39],[40],[41] Induction of SCW arthritis Streptococcus pyogenes T12 gram-positive organisms are cultured overnight, and the cell walls are harvested, centrifuged at 10,000 g (contain 11% muramic acid), and then used for the induction of arthritis. Unilateral arthritis is induced in mice by intraarticular injection of 25 μg SCW prepared in 6 μl phosphate buffered saline (PBS) into the right knee joint, and the non-injected left knee joint is used as a control. The same injection is repeated on different days (0, 7, 14, and 21) to induce chronic arthritis. The degree of severity of inflammation in the knee joints is assessed by macroscopic scoring system. Full fledged joint inflammation is manifested on 28 th day. Histologic examination of the total knee joints indicates changes in patella and femur/tibia regions, which are accompanied by bone erosion, exudate formation, and infiltration of granulocytes. [38] Formaldehyde-induced arthritis An acute rat model of formaldehyde-induced arthritis is used for screening antiarthritic agents. Development of arthritis is attributed to the release of histamine, serotonin, and prostaglandin at the site of injection. The rats are injected with formaldehyde (0.1 ml of 2% v/v) into the subplantar region of the hind paws on 1 st and 3 rd days. The edema of the hind paw is measured daily for 10 days by plethysmometer. The manifestation of arthritis is assessed by various parameters, viz. hind paw edema, diameter of the ankle joint, joint motility, pain threshold, and grip strength. Similarly, the degenerative changes of cartilages and bone erosion are also evaluated by X-ray studies of the hind limbs. The histopathologic studies are also performed to see the cellular changes, pannus formation, and other degenerative changes of cartilages and bone. [42],[43]
Naturally/spontaneous occurring OA models Virtually all the spontaneous animal models of OA are found to exhibit morphologic changes that resemble human OA conditions. Guinea pig, mouse, and nonhuman primate are considered to be the best spontaneous models of OA, and enable us to understand the slow progressive OA that may resemble human OA. The major advantage of such animal models is primarily due to their resemblesance to natural progression of the disease without any direct intervention or stimulation. The clinical symptoms and findings reported in spontaneous occurring OA models are outlined in [Table 1]. [44],[45],[46],[47],[48],[49]
Surgically induced OA Various types of injuries to cartilage lead to OA in humans, which have been simulated in animal models by manipulating anterior cruciate ligament transection (ACLT) and meniscectomy model, with special reference to knee OA [Table 2]. [50],[51],[52],[53],[54],[55],[56],[57],[58],[59],[60],[61] The disease progression is found to be more rapid in animal models than in humans, thus making the models less amenable to therapeutic intervention. However, cartilage damage observed in such models resembles human knee OA. Hence, these models have been used widely for evaluation of analgesics and structure-modifying agents. However, based on the observations, the use of rodents as the model is debatable as the disease is confined largely to knee joints.
Chemically induced OA Intraarticular injections of diverse chemical agents (physiological saline, corticosteroid, estrogen, papain, collagenase, monosodium iodoacetate, quinolone antibiotics, etc.) induced acute inflammation with cartilage degeneration and degradation of extracellular matrix, which are found to be somewhat similar to those in human OA [Table 3]. [62],[63],[64],[65],[66],[67],[68],[69] In addition, the oxidative stress induces marked changes in enzymes that are closely participating in the progression of OA. This model is particularly beneficial for rapidly developing synovitis associated with pain and decreased motility of joints. In such models, no severe changes are observed at early stage of OA and the changes are also found to be reversible. Thus, apparently, such models seem to have specific advantage in the evaluation of nonsteroidal and steroidal anti-inflammatory drugs. Nevertheless, experimental findings of such animal models suggest the altered pathology does not fully represent human OA conditions.
Physical models of OA Physical/biomechanical models of OA are the routinely used models to reproduce mainly sport traumatism and accidental traumas in humans and veterinary orthopedic research, respectively. Surgical interventions (desmotomy, meniscectomy, deliberate patellar contusion or luxation, continuous immobilization, non-physiological overload) are the techniques used for induction of mechanical stress on articular cartilage and impaired congruency, motility, and joint instability. Such models induce rapid and severe cartilage degeneration than the spontaneous models. The deterioration due to physical alterations adversely affects the metabolism of chondrocytes of subchondral bone, leading to degeneration and damages. [70] The Pond-Nuki model The tearing and stretching of ligaments often result in the rupture of ligaments, leading to OA. Canine stifle surgical cranial cruciate ligament (CCL) resection proposed by Pond and Nuki [56] is a widely accepted model of OA. The method involves the mechanical instability that triggers pathophysiological alterations leading to OA in dog, which in many respects resembles human OA. However, clinical investigations on CCL of the knee failed to explain the probable cause for its spontaneous rupture without any trauma. Even though the clinical opinion differs, the onset of symptoms of OA is normally observed within 3-5 weeks after CCL rupture. [71] The resection can also be performed by percutaneous stab incision, open arthrotomy, and arthroscopically guided transection. [72] The physio-anatomical changes in bone, cartilage, and synovial membrane in dogs occur within a period of 16 weeks, which is akin to natural OA. [73] Onset of inflammation in synovial fluid is observed within 13 weeks of subjecting subchondral bone to focal alteration stress. [74] It causes significant changes in mineral density within 3-12 weeks, and is known to induce the activity of metalloproteinases. Immobilization model Immobilization is the most common orthopedic treatment practised clinically for musculoskeletal injury, deformity, and inflammation. Prolonged immobilization results in joint contracture and atrophy of muscle and bone. Experimental immobilization is used as a model for OA which triggers morphologic degeneration and biochemical changes in joint cartilage. Immobilization of hamsters for 3 months decreased the content of PG and synovial fluid volume in the stifle joints. [75] Torelli et al. have demonstrated that splint immobilization of the stifle joint in rabbits (12 weeks) developed OA, which is closely related to the morphologic and biochemical changes observed in cartilage and bone structure. [76] Canine groove model Canine model of OA is developed by damaging the articular cartilage of the weight-bearing areas of the femoral condyles in one knee, without damaging the subchondral bone, and without affecting joint stability. To trigger OA, the affected joints are loaded by fixing the contralateral control limb to the trunk of the dog temporarily. In this model, it is reported that within 10 weeks a characteristic change occurs in collagen metabolism, turnover of PG, MMP activity, and histoarchitecture alterations, viz. moderate cartilage destruction, fibrillation of the articular surface, and chondrocyte clustering. The degenerative changes represent the naturally occurring erosion in OA development with age, characterized by slow but steady progression of degenerative events with domination over the repair potential of the cartilage matrix. [77] Mechanical overload model Mechanical factors such as chronic overload and higher degree of sudden stress play a major role in the pathogenesis of the OA. The model is simulated by mechanical overloading on the hind limb of dogs to produce subchondral microfractures of the femoral condyle to initiate a characteristic degeneration of the cartilage. Overloading is found to be a risk factor for the wear and tear of joints both in animals and humans. In general, this method can be an intermediate between naturally occurring and surgically induced OA models. [78] Hypermotility model The objective of the model is to simulate a physical wear and tear of cartilage, which is generally much above its normal capacity for physiological movements and repair. The benefit of this model is to provoke articular cartilage alterations that resemble human OA. Pap et al. demonstrated eloquently the destructive effect of strenuous physical exercise on the structural alterations of joints in rats. Intracranial self-stimulation causes rats to run distances of approximately 15 and 30 km within 3 and 6 weeks in a revolving wheel. By the end of the trial period, a significant elevation of metalloproteinases along with concomitant loss of chondrocyte from the superficial cartilage layer is reported. [79] Models of genetic modification Transgenic or knockout mice models with altered expression of transcription factors, viz. MMP, angiogenic factors, or extracellular proteins, adequately provide information pertaining to the mechanism(s) that control or modulate cartilage development and pathogenesis of OA. To simulate OA lesion swiftly, surgical methods are used in transgenic mice. Transgenic mice are a useful tool to understand the specific role of biomolecules and their pathways in the genesis of OA. It is difficult to accept that single gene defects are able to simulate OA in humans. Therefore, use of such animal models for evaluation of the effects of therapeutic agents on OA is questionable. The modifications of genes of growth factor signaling pathway (viz. Bmpr1a, TGF-β RII, Smad3, Ank, Npp1, α1 integrin, Runx2, Hif-2α, and miR-104) are involved in the development of OA with well-defined characteristic features such as cartilage degeneration/degradation, increased chondrocyte hypertrophy and progression, skeletal degeneration, cartilage erosion, crystal deposition, and increased osteophyte formation. Genetic manipulation of genes of extracellular matrix (viz. Col2a1, Col9a1, Col9a1, Col11a1, aggrecan, fibromodulin, fibromodulin/biglycan, and matrilin-3) may result in degenerative changes in cartilage, mild chondrodysplasia, tendon mineralization, and early-onset OA in adult mice. Similar genetic manipulation of the genes of proteinases (Mmp9, Mmp13, Adamts5, Timp3) and cytokines (IL-1β, IL-6, ICE, MK2, NOS2) are also reported to participate in the development of OA similar to that of humans. [80],[81],[82]
The simulation of animal models of arthritis is the major approach for understanding the cellular, molecular, biochemical, and pathologic events of the disorder. Among the arthritic disorders, RA and OA are the most complex destructive and immune origin crippling diseases. Till date, there are no effective therapeutic interventions for preventing as well as curing such diseases, largely due to the inability to simulate etiologically valid, identical animal models of human arthritis. However, in the present review, we have attempted to enumerate diverse animal models of RA and OA of a variety of species in order to gain a wider and perspective knowledge concerning the pathogenesis of arthritis, so that appropriate models can be adopted judiciously for efficient evaluation of novel therapeutic agents. Ostensibly, with the advantage of knowledge-based science, especially in molecular biology and biotechnology, we may perhaps be able to clinch success in identifying the complex events of arthritis and may provide impetus to develop more specific animal models of RA and OA. Such breakthrough might open up a new corridor for the development of effective and safe new-generation antiarthritic agents.
[Table 1], [Table 2], [Table 3]
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|||||||
