Galanin in adjuvant arthritis in the rat

OBJECTIVE: To study the concentration changes of galanin in the ankles and spinal cord and to detect the distribution of galanin in different tissues in arthritic rats. METHODS: Adjuvant arthritis was induced by intradermal injection of Mycobacterium butyricum in Freund's incomplete adjuvant at the base of the tail. The concentrations of galanin were measured by radioimmunoassay (RIA) and the distributions of galanin were detected by immunoelectron microscopy (iEM). RESULTS: Measurements were taken on Day 28 after injection. RIA results showed that the concentration of galanin was significantly lower in the ankles and spinal cords of rats with adjuvant arthritis compared to controls. Our iEM results showed a heterogeneous distribution of galanin labelling in different cells and tissue compartments. In arthritic rats, we observed decreased galanin labelling in the sciatic nerve and in macrophage-like cells in the synovial membrane and increased labelling in monocyte lineage cells, polymorphonucleated lineage cells in the bone marrow, fibroblasts in the periosteum, osteoclasts and osteocytes, and lower labelling in osteoblasts compared to controls. CONCLUSION: Galanin is involved in the response to inflammation in adjuvant arthritis and might play a role in the regulation of inflammation and nociception. These findings are in accordance with a biological role of galanin in the development of inflammatory arthritis.     

Potential of indium-111 to measure inflammatory arthritis

Since an objective noninvasive method for measuring inflammation in arthritis is lacking, radioimaging experiments were conducted with 111indium-chloride (111InCl3) in the collagen model. A computer imaging index, reflecting uptake in the feet and ankles, was significantly greater in 16 rats immunized with collagen than in 6 nonimmunized rats (mean +/- SEM 1.18 +/- 0.04 vs 0.39 +/- 0.02, for the 2 groups, respectively, p less than 0.001). Additional analyses provided evidence that the 111InCl3 technique can be used to measure objectively progression of the arthritic response of rats of immunization with collagen, and pilot comparisons with conventional bone scintigraphy in 2 patients with rheumatoid arthritis suggested that the method is applicable to human arthritic disease.     

Inflammatory arthropathy in MRL hematopoietic chimeras undergoing Fas mediated graft-versus-host syndrome

OBJECTIVE: To determine whether overexpression of the Fas ligand (FasL) on activated lpr T lymphocytes could induce arthritic lesions when grafted into syngeneic wild-type MRL mice expressing normal Fas receptor levels. METHODS: Lethally irradiated MRL+/+ mice were reconstituted with congenic MRL/lpr bone marrow cells and splenocytes overexpressing FasL. Fas-mediated cytotoxic properties of repopulating lpr splenic lymphocytes were evaluated in vitro. Simultaneously, the hind paw ankles of the hematopoietic chimeras were histologically examined. RESULTS: The lpr lymphocytes repopulating the spleen overexpressed FasL and had in vitro Fas-mediated cytotoxic activity. Simultaneously, in vivo, articular (synovitis, pannus) and periarticular (periostitis) inflammation with bone resorption were observed. CONCLUSION: Arthritic lesions may be induced in Fas-expressing recipients by persistent engrafted syngeneic lymphocytes overexpressing FasL.     

IGF-I and IGF-I-binding proteins in rats with adjuvant-induced arthritis given recombinant human growth hormone

Adjuvant-induced arthritis in rats is associated with growth failure, hypermetabolism and accelerated protein breakdown. We have previously reported that adjuvant-induced arthritis in rats results in a decrease in body weight gain, pituitary GH mRNA, circulating GH and IGF-I together with an increase in serum IGF-binding proteins (IGFBPs). The aim of this study was to analyze the role of GH in the decrease in body weight and in the alterations in the IGF-I system observed in chronic inflammation. Male Wistar rats were injected with complete Freund's adjuvant and 16 days later arthritic rats were injected daily with recombinant human GH (rhGH) (3 IU/kg s.c.) for 8 days; control rats received 250 microl saline. Arthritis significantly decreased body weight gain and serum IGF-I. These decreases were not due to the reduced food intake, since in pair-fed rats they were not observed. Furthermore, administration of rhGH to arthritic rats increased body weight gain without modifying food intake. To further investigate the effect of GH administration, 14 days after adjuvant injection both control and arthritic rats were treated with 0, 1.5, 3 or 6 IU/kg of rhGH. GH treatment at the dose of 3 and 6 IU/kg significantly increased body weight gain in arthritic rats. GH administration, at the higher dose of 6 IU/kg, increased hepatic and serum concentrations of IGF-I in both control and arthritic rats. In control rats, rhGH at the three doses assayed increased circulating IGFBP-3. GH treatment in arthritic rats decreased IGFBP-1 and -2, and did not modify IGFBP-4. GH treatment at the dose of 3 IU/kg also decreased circulating IGFBP-3 in arthritic rats. These data suggest that GH treatment can ameliorate the catabolism observed in adjuvant-induced arthritis, an effect mediated, at least in part, by modifications in the circulating IGFBPs.     

Arthritis-induced increase in serum levels of IGF-binding protein-3 in rats is secondary to the decrease in its proteolytic activity

Adjuvant-induced arthritis is a chronic inflammatory illness that induces a catabolic state, with a decrease in pituitary GH and hepatic IGF-I synthesis. We have previously observed an increase in serum IGF-binding protein-3 (IGFBP-3) in arthritic rats, and found that GH administration prevents the increase in circulating IGFBP-3 in arthritic rats. The aim of this work was therefore to study IGFBP-3 synthesis in the liver as well as its proteolysis in serum as the two possible causes of the increased circulating IGFBP-3 in arthritic rats. The effect of recombinant human GH (rhGH) administration was also analysed. Adult male Wistar rats were injected with complete Freund's adjuvant or vehicle, and 14 days later they were injected s.c. daily until day 22 after adjuvant injection with rhGH (3 IU/kg) or saline. Three hours after the last GH injection, all rats were killed by decapitation. Arthritis increased serum IGFBP-3 levels (P<0.01). The increase in serum IGFBP-3 levels in arthritic rats seems to be due to decreased proteolysis (P<0.01) rather than to an increased synthesis, since liver IGFBP-3 mRNA content was not modified by arthritis. GH administration to control rats resulted in an increase in both hepatic IGFBP-3 mRNA content and in serum IGFBP-3 levels in spite of the increase in IGFBP-3 proteolysis in serum. In arthritic rats, GH treatment did not modify liver IGFBP-3 synthesis, but it increased serum proteolysis of IGFBP-3, leading to a serum concentration of IGFBP-3 similar to that of control rats. Furthermore, there was a negative correlation between circulating IGFBP-3 and its proteolytic activity in the serum of adjuvant-induced arthritic rats. These data suggest that in chronic arthritis the increase in IGFBP-3 serum concentration is secondary to a decrease in proteolytic activity, rather than to an increase in hepatic IGFBP-3 gene expression.     

Plasma and bone osteocalcin levels in rats with type ii collagen—induced arthritis

Osteocalcin levels in plasma and bone were measured by enzyme immunoassay in rats with arthritis induced by immunization with type II collagen and in untreated control rats. Compared with levels in control rats, the plasma levels of osteocalcin in arthritic rats were markedly decreased 1–3 weeks after immunization; during weeks 8–14, these levels were significantly increased. The osteocalcin content of tarsal bones changed in parallel with the plasma levels. These data suggest that osteocalcin levels in the plasma of arthritic rats reflect alterations in bone formation activity.     

Plasma and bone osteocalcin levels in rats with type II collagen-induced arthritis

Osteocalcin levels in plasma and bone were measured by enzyme immunoassay in rats with arthritis induced by immunization with type II collagen and in untreated control rats. Compared with levels in control rats, the plasma levels of osteocalcin in arthritic rats were markedly decreased 1-3 weeks after immunization; during weeks 8-14, these levels were significantly increased. The osteocalcin content of tarsal bones changed in parallel with the plasma levels. These data suggest that osteocalcin levels in the plasma of arthritic rats reflect alterations in bone formation activity.     

Demyelination arrest and remyelination induced by glatiramer acetate treatment of experimental autoimmune encephalomyelitis

The interplay between demyelination and remyelination is critical in the progress of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). In the present study, we explored the capacity of glatiramer acetate (GA, Copaxone) to affect the demyelination process and/or lead to remyelination in mice inflicted by chronic EAE, using both scanning electron microscopy and immunohistological methods. Spinal cords of untreated EAE mice revealed substantial demyelination accompanied by tissue destruction and axonal loss. In contrast, in spinal cords of GA-treated mice, in which treatment started concomitantly with disease induction (prevention), no pathology was observed. Moreover, when treatment was initiated after the appearance of clinical symptoms (suppression) or even in the chronic disease phase (delayed suppression) when substantial demyelination was already manifested, it resulted in a significant decrease in the pathological damage. Detection of oligodendrocyte progenitor cells (OPCs) expressing the NG2 or O4 markers via colocalization with the proliferation marker BrdU indicated their elevated levels in spinal cords of GA-treated mice. The mode of action of GA in this system is attributed to increased proliferation, differentiation, and survival of OPCs along the oligodendroglial maturation cascade and their recruitment into injury sites, thus enhancing repair processes in situ.     

Arthritic disease is more severe in older rats in a kaolin/carrageenan-induced arthritis model

This study examined in an arthritis animal model whether elderly onset rheumatoid arthritis (EORA) is a more severe disease than younger onset rheumatoid arthritis. Arthritis was induced by injecting 5% kaolin/carrageenan into the left tibiotarsal ankles of 18-month-old and 4-week-old rats. Various parameters were measured to evaluate the arthritic progression of kaolin/carrageenan-induced arthritis in the rats. Immunohistochemical staining of arthritic joints was performed to determine the degree of inflammation in old and young rats. Measurements of ankle volume and thickness, arthritic index, number of squeaks, and the paw pressure test showed the 18-month-old rats had more severe disease than the young rats in a kaolin/carrageenan-induced arthritis model. The degree of inflammation and MMP-1 expression of arthritic joints in old rats was significantly higher than that of young rats based on histological evaluation with hematoxylin and eosin (H&E) staining and immunochemistry. More severe disease symptoms were found in old rats with EORA, but the molecular mechanisms still remain to be elucidated. Understanding the molecular mechanisms will be helpful to develop clinical protocols to efficiently treat patients with EORA, which is difficult to control with current protocols.     

Axonal growth of embryonic stem cell-derived motoneurons in vitro and in motoneuron-injured adult rats

We generated spinal motoneurons from embryonic stem (ES) cells to determine the developmental potential of these cells in vitro and their capacity to replace motoneurons in the adult mammalian spinal cord. ES cell-derived motoneurons extended long axons, formed neuromuscular junctions, and induced muscle contraction when cocultured with myoblasts. We transplanted motoneuron-committed ES cells into the spinal cords of adult rats with motoneuron injury and found that approximately 3,000 ES cell-derived motoneurons (25% of input) survived for >1 month in the spinal cord of each animal. ES cell-derived axonal growth was inhibited by myelin, and this inhibition was overcome by administration of dibutyryl cAMP (dbcAMP) or a Rho kinase inhibitor in vitro and in vivo. In transplanted rats infused with dbcAMP, approximately 80 ES cell-derived motor axons were observed within the ventral roots of each animal, whereas none were observed in transplanted rats not treated with dbcAMP. Because these cells replicate many of the developmental and mature features of true motoneurons, they are an important biological tool to understand formation of motor units in vitro and a potential therapeutic tool to reconstitute neural circuits in vivo.     

123I-antileukoproteinase scintigraphy reveals microscopic cartilage alterations in the contralateral knee joint of rats with "monarticular" antigen-induced arthritis

OBJECTIVE: To assess the involvement of the contralateral knee joint in monarticular antigen-induced arthritis (AIA) by scintigraphy with the cationic (pI >10), 123I-labeled, serine proteinase inhibitor antileukoproteinase (123I-ALP) and to compare the scintigraphic findings with those of radiography and high-resolution ex vivo magnetic resonance imaging (MRI). METHODS: Lewis rats with chronic AIA were examined 2.5 months following arthritis induction (injection of 500 microg of methylated bovine serum albumin/saline into the ipsilateral [arthritic] knee joint and injection of phosphate buffered saline into the contralateral knee joint following systemic immunization). 123I-ALP was injected intravenously into normal rats (n = 4) or rats with AIA (n = 6). The ipsilateral and contralateral knee joints and both ankles were examined by scintigraphy and radiography. Joint cartilage was examined by high-resolution ex vivo MRI, histopathology, and measurement of tissue radioactivity. RESULTS: ALP accumulation (typically observed in normal articular cartilage) was lost in both the ipsilateral and the contralateral knee joints, but not in the clinically unaffected ankles of rats with AIA. In both knee joints, 123I-ALP target:background ratios and cartilage radioactivity correlated negatively with the loss of toluidine blue staining in cartilage, which documents the depletion of charged matrix molecules. Findings of histopathology confirmed mild alterations in the ipsilateral knee joint and even milder alterations in the contralateral knee joint, while the ankles were normal. Radiography and high-resolution ex vivo MRI failed to detect abnormalities in the contralateral knee joint. CONCLUSION: Loss of ALP accumulation appears to document proteoglycan depletion, even in the microscopically altered cartilage of the contralateral knee joint in AIA. These findings underscore the high sensitivity of 123I-ALP for in vivo detection of biochemical cartilage alterations in arthritis, and furthermore, question the use of the contralateral knee joint as a normal control in AIA.     

Methotrexate suppresses passive adjuvant arthritis: studies on the metabolism of methotrexate in mononuclear cells derived from normal and adjuvant arthritic rats

Passive transfer of adjuvant arthritis by spleen cells is suppressed by methotrexate. Mononuclear cells derived from spleens of normal and adjuvant arthritic Lewis rats were incubated with [3H]-methotrexate and harvested at various periods of time. The amount of methotrexate and its various polyglutamates were quantitated. The results of these studies indicate that the kinetics of uptake of methotrexate by mononuclear cells from normal and adjuvant arthritic rats are similar. However, the amount of methotrexate polyglutamates accumulating in the mononuclear cells of adjuvant arthritic rats was significantly lower than that observed in mononuclear cells derived from normal rats.     

The role of cartilage minor collagens in inducing arthritis

Arthritogenic properties of native types IX, XI, and II collagen were investigated in female Wistar rats. Immunization with native type-XI or -II collagen led to the arthritic reaction in 60% of investigated rats. After boosting, a distinct increase in anticollagen antibodies was observed followed by temperature rise (swelling and redness of affected joints). At the end of the experiment (after 7 weeks) subchondral bone destruction was detectable upon x-ray examination. Histological observation of the affected knee and ankle joints showed progressive destruction of the articular cartilage and subchondral bone accompanied by proliferative synovitis with extensive formation of fibrous tissue found in joints of rats immunized with native type-XI collagen. Immune response to collagen types (determined by ELISA) reached its peak between the 14th and the 21st day. From the acute stage to the chronic stage a decrease of serum anticollagen antibodies was observed to remain constant. Rats immunized with native type IX and denatured type XI collagen did not develop arthritis.     

Expression of osteoclast differentiation factor at sites of bone erosion in collagen-induced arthritis

OBJECTIVE: To investigate the cellular mechanism of bone destruction in collagen-induced arthritis (CIA). METHODS: After induction of CIA in DA rats, a histologic study of the advanced arthritic lesion was carried out on whole, decalcified joints from the hindpaws of affected animals. To conclusively identify osteoclasts, joint tissue sections were stained for tartrate-resistant acid phosphatase (TRAP) enzyme activity, and calcitonin receptors (CTR) were identified using a specific rabbit polyclonal antibody. The expression of messenger RNA (mRNA) for the osteoclast differentiation factor (also known as receptor activator of nuclear factor kappaB ligand [RANKL]) was investigated using in situ hybridization with a specific riboprobe. RESULTS: TRAP-positive and CTR-positive multinucleated cells were invariably detected in arthritic lesions that were characterized by bone destruction. Osteoclasts were identified at the pannus-bone and pannus-subchondral bone junctions of arthritic joints, where they formed erosive pits in the bone. TRAP-positive multinucleated cells were detected within synovium and at the bone erosive front; however, CTR-positive multinucleated cells were present only at sites adjacent to bone. RANKL mRNA was highly expressed in the synovial cell infiltrate in arthritic joints, as well as by osteoclasts at sites of bone erosion. CONCLUSION: Focal bone erosion in CIA is attributed to cells expressing definitive features of osteoclasts, including CTR. The expression of RANKL by cells within inflamed synovium suggests a mechanism for osteoclast differentiation and activation at sites of bone erosion. Inhibitors of RANKL may represent a novel approach to treatment of bone loss in rheumatoid arthritis.     

Measurement of radiographic changes in adjuvant-induced arthritis in rats by quantitative image analysis

Objective. To apply quantitative analytical methods to the evaluation of radiographic images in experimental arthritis. Methods. Adjuvant was used to induce arthritis in rats. Arthritis progression was followed by conventional methods. In addition, digitized images of radiographs of the calcaneus were examined for changes in the mean and in the distribution pattern of gray values. Periosteal new bone formation was measured as an increase in image area of the calcaneus. Results. Significant changes in the gray value profile and increases in periosteal bone formation occurred in arthritic rats. More extensive changes occurred in Lewis rats than in Sprague-Dawley rats. Analysis of serial radiographs revealed an initial decrease in the density of juxtaarticular bone, followed by progressive increases in gray value variation due to concurrent bone loss and bone formation. Eventually, bone formation in arthritic rats resulted in increased gray values above those in nonarthritic rats. Conclusion. Image analysis represents a sensitive, quantitative method for detecting radiographic changes in experimental arthritis.     

Innervation of bone from healthy and arthritic rats by substance P and calcitonin gene related peptide containing sensory fibers.

Mechanical stress causes remodelling of bone, a transformation of bone structure by physical forces through an unknown mechanism. Inflammation also affects bone structure, through altered use and the production of various inflammatory mediators. The peripheral nervous system may play both a sensory and an efferent role in the mechanical and inflammatory influences on bone structure. We studied the occurrence of substance P and calcitonin gene related peptide (CGRP) containing nerves in periosteal tissue, bone marrow, diaphysis and epiphysis of the ankle and knee joints of healthy and adjuvant arthritic rats. In arthritic animals, only ankle joints were affected by the inflammation. The periosteum was richly innervated both in healthy and arthritic animals. In arthritic rats few nerve fibers penetrated the woven, callous bone underlying the periosteum. Also bone marrow contained substance P and CGRP immunoreactive nerves in normal bone, whereas the hypercellular bone marrow of arthritic rats showed a decrease in the density of substance P and CGRP containing fibers. Epiphysis had a dense innervation compared to diaphysis. In contrast to large erosions, small peripheral erosions contained some CGRP immunoreactive fibers, perhaps as a sign of attempts of reactive repair. Our results suggest a local delivery system of potent peptide regulatory factors in bone, a system also affected by the pathophysiology of arthritis.     

Influence of chronic drug-induced achlorhydria by substituted benzimidazoles on the endocrine stomach in rats

The release of gastric somatostatinlike immunoreactivity and gastrin was studied in rats with chronic achlorhydria induced by the substituted benzimidazole BY 308. In vitro, stimulation of gastrin release by acetylcholine was slightly enhanced after 1 day of treatment but no further effects were observed compared to placebo controls. Four weeks of treatment evoked marked gastrin hypersecretion, which was atropine-resistant. Stimulation of gastrin release was inversely correlated to enhancement of basal gastrin levels. Chronic achlorhydria distinctly reduced somatostatin responses to isoproterenol, whereas potent stimulation was observed in controls. Treatment with BY 308 for 1 wk was associated with fully developed gastrin hypersecretion but isoproterenol-stimulated somatostatin release was still unaffected. Hypergastrinemia accompanied by increased antral gastrin and reduced antral and fundic somatostatin concentrations was also found in vivo after 4 wk of treatment with BY 308. It is concluded that chronic achlorhydria not only enhances storage and secretion of gastrin but also diminishes the secretion and tissue stores of somatostatin; adaptive changes of the somatostatin cell occur, however, with a much longer delay.     

Chronic hyponatremia exacerbates multiple manifestations of senescence in male rats

The syndrome of inappropriate antidiuretic hormone secretion (SIADH) is frequently responsible for chronic hyponatremia in the elderly due to age-related disruption of the inhibitory component of brain osmoregulatory mechanisms. Recent research has indicated that chronic hyponatremia is associated with gait disturbances, increased falls, and bone fragility in humans, and we have found that chronic hyponatremia causes increased bone resorption and reduced bone mineral density in young rats. In this study, we used a model of SIADH to study multi-organ consequences of chronic hyponatremia in aged rats. Sustained hyponatremia for 18 weeks caused progressive reduction of bone mineral density by DXA and decreased bone ash calcium, phosphate and sodium contents at the tibia and lumbar vertebrae. Administration of 10-fold higher vitamin D during the last 8 weeks of the study compensated for the reduction in bone formation and halted bone loss. Hyponatremic rats developed hypogonadism, as indicated by slightly lower serum testosterone and higher serum FSH and LH concentrations, markedly decreased testicular weight, and abnormal testicular histology. Aged hyponatremic rats also manifested decreased body fat, skeletal muscle sarcopenia by densitometry, and cardiomyopathy manifested as increased heart weight and perivascular and interstitial fibrosis by histology. These findings are consistent with recent results in cultured osteoclastic cells, indicating that low extracellular sodium concentrations increased oxidative stress, thereby potentially exacerbating multiple manifestations of senescence. Future prospective studies in patients with SIADH may indicate whether these multi-organ age-related comorbidities may potentially contribute to the observed increased incidence of fractures and mortality in this population.     

Lymphoid abnormalities in rats with adjuvant-induced arthritis. I. Mitogen responsiveness and lymphokine synthesis.

Lewis rats injected in the hind paw with Mycobacterium butyricum develop a severe polyarthritis which shares certain features in common with rheumatoid arthritis in man. Spleen and peripheral blood mononuclear cells from rats with this form of arthritic disease proliferate poorly in vitro in response to concanavalin A (con A), phytohaemagglutinin (PHA), and pokeweed mitogen (PWM). The splenic hyporesponsiveness appears within four days of M. butyricum injection (three to five days prior to the development of detectable arthritis), reaches a peak 16-22 days following injection, and persists for at least 40 days. Buffalo strain rats injected with M. butyricum do not develop arthritis, and their spleen cells respond normally to con A, PHA, and PWM. In response to lipopolysaccharide (LPS) the synthesis of interleukin 1 (IL-1) by spleen or peritoneal macrophages from arthritic Lewis rats equalled or exceeded that of macrophages from normal rats. In contrast splenic T cells from arthritic rats produced reduced amounts of interleukin 2 (IL-2; T cell growth factor) in response to stimulation with PHA or con A. Moreover, con-A-activated spleen cells from arthritic rats failed to bind IL-2 and to respond to this growth factor with increased 3H-TdR uptake as did normal spleen cells. In-vitro treatment of 'arthritic' cells with 10(-5) M indomethacin did not restore to normal their reduced mitogen responsiveness, and spleen cells from normal and arthritic rats were equally sensitive to the inhibitory effects of prostaglandin E2 on con-A-induced proliferative responses. These results indicate that peripheral lymphoid function is compromised in rats with adjuvant-induced arthritis and that this functional deficit is mediated by aberrant synthesis of and response to IL-2 by T cells of arthritic animals.