Histomorphometric analysis of cutaneous remodeling in the early stage of the scleroderma model

INTRODUCTION/OBJECTIVES: Systemic sclerosis, or scleroderma, is a rheumatic disease characterized by autoimmunity, vasculopathy, and fibrosis of the skin and several internal organs. In the present study, our aim was to assess the skin alterations in animals with scleroderma during the first stages of disease induction. METHODS: To induce scleroderma, female New Zealand rabbits (n = 12) were subcutaneously immunized with 1 mg/ml of collagen V (Col V) in complete Freund's adjuvant, twice with a thirty-day interval. Fifteen days later, the animals received an intramuscular booster with type V collagen in incomplete Freund's adjuvant, twice with a fifteen-day interval. The control group was inoculated with 1 ml of 10 mM acetic acid solution diluted with an equal amount of Freund's adjuvant. Serial dorsal skin biopsies were performed at 7, 15, and 30 days and stained with H&E, Masson's trichrome and Picrosírius for morphological and morphometric analyses. RESULTS: Immunized rabbits presented a significant increase in collagen in skin collected seven days after the first immunization (p=0.05). CONCLUSION: The results from this experimental model may be very important to a better understanding of the pathogenic mechanisms involved in the beginning of human SSc. Therapeutic protocols to avoid early remodeling of the skin may lead to promising treatments for SSc in the future

Aerobic Exercise Attenuated Bleomycin-Induced Lung Fibrosis in Th2-Dominant Mice

Introduction The aim of this study was to investigate the effect of aerobic exercise (AE) in reducing bleomycin- induced fibrosis in mice of a Th2-dominant immune background (BALB/c). Methods BALB/c mice were distributed into: sedentary, control (CON), Exercise-only (EX), sedentary, bleomycin-treated (BLEO) and bleomycin-treated+exercised (BLEO+EX);(n = 8/group). Following treadmill adaptation, 15 days following a single, oro-tracheal administration of bleomycin (1.5U/kg), AE was performed 5 days/week, 60min/day for 4 weeks at moderate intensity (60% of maximum velocity reached during a physical test) and assessed for pulmonary inflammation and remodeling, and cytokine levels in bronchoalveolar lavage (BAL). Results At 45 days post injury, compared to BLEO, BLEO+EX demonstrated reduced collagen deposition in the airways (p<0.001) and also in the lung parenchyma (p<0.001). In BAL, a decreased number of total leukocytes (p<0.01), eosinophils (p<0.001), lymphocytes (p<0.01), macrophages (p<0.01), and neutrophils (p<0.01), as well as reduced pro-inflammatory cytokines (CXCL-1;p<0.01), (IL-1 beta;p<0.001), (IL-5;p<0.01), (IL-6;p<0.001), (IL-13;p<0.01) and pro-fibrotic growth factor IGF-1 (p<0.001) were observed. Anti-inflammatory cytokine IL-10 was increased (p<0.001). Conclusion AE attenuated bleomycin-induced collagen deposition, inflammation and cytokines accumulation in the lungs of mice with a predominately Th2-background suggesting that therapeutic AE (15-44 days post injury) attenuates the pro-inflammatory, Th2 immune response and fibrosis in the bleomycin model

Auto-imunidade e colágeno V Autoimmunity and collagen V

As proteínas da matriz extracelular (MEC) e seus componentes estão sendo amplamente estudados na literatura médica, assim como sua relação com o remodelamento tecidual presente nas doenças reumáticas. Neste artigo, mostramos a importância do estudo do colágeno do tipo V no entendimento da etiologia da esclerodermia, no que se refere ao desencadeamento da auto-imunidade nesta enfermidade. Estudos em nosso laboratório demonstram que a sensibilização com colágeno do tipo V em coelhos pode resultar em um modelo animal de esclerodermia. Diante destes fatos, sugerimos que pesquisas neste campo podem ser de grande valia no desenvolvimento de novas condutas terapêuticas.<br>The extracellular matrix (ECM) proteins and their components have been widely studied in medical literature, as well its relationship with the tecidual remodeling present in the rheumatic disease. In this paper we show the importance of understanding the role of type V collagen as an important trigger of rheumatic autoimmune diseases. Studies in our laboratory demonstrate that type V collagen sensibilization in rabbits, could result in an animal scleroderma model. In this way we suggested that researches in this field can be worthy in development of new therapeutic procedures

The remodeling of collagen fibers in rats ankles submitted to immobilization and muscle stretch protocol

To evaluate the remodeling of collagen fibers in the articular cartilage of rat ankles, with and without immobilization, after application of muscle stretching protocol. Twenty three Wistar rats were divided into four groups: immobilized (I), n = 6; immobilized and stretched (IS), n = 6; stretched (S), n = 6 and control (C), n = 5. The animals in groups I and IS were submitted to immobilization. After the period of immobilization, the animals in groups IS and S were submitted to a muscle stretching protocol. At the end of the experiment, the animals were euthanized and the joints removed, processed and stained with Picrosirius red. The analysis was carried out using a polarized light microscope. The density of collagen fibers were quantified according to the intensity of birefringence displayed. By way of statistical analyses, the right and left hind limbs of the different groups were compared based on the total density of collagen fibers, the density of thick collagen fibers and the density of thin collagen fibers. Immobilization promoted a reduction in density of the thin fibers and of total collagen. The muscle stretching protocol after immobilization promoted a reduction in density of the total collagen and of the thick fibers, but the density of the thin fibers showed the same values as control. The collagen fibers were remodeled by the different stimuli. Immobilization was harmful to the collagen fibers and the muscle stretching protocol only recovered the thin collagen fibers.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP

Meniscectomia parcial como modelo experimental de osteoartrite em coelhos e efeito protetor do difosfato de cloroquina Partial meniscectomy as an experimental model of osteoarthritis in rabbits and protector effect of chloroquine diphosphate

OBJETIVO: Estabelecer as alterações morfológicas e o remodelamento do tecido cartilaginoso na progressão da osteoartrite (OA) experimental para estudar o efeito do difosfato de cloroquina na cartilagem osteoartrítica. MÉTODOS: A osteoartrite experimental foi induzida em coelhos por meio de meniscectomia parcial. Para analisar a evolução da doença experimental foram estudados três grupos de dez animais, sacrificados a 3, 14 e 22 semanas de indução da doença. Para avaliar o efeito do difosfato de cloroquina um grupo de animais (n = 6) foi tratado preventivamente com 3 mg/kg ao dia, iniciados um mês antes da indução da osteoartrite, e mantidos até o sacrifício (22 semanas). Realizou-se análise histológica das articulações (H&E, tricrômico de Masson) e imunofluorescência para colágeno dos tipos I, II e XI. A intensidade da agressão articular foi quantificada pelo escore de Mankin. RESULTADOS: O modelo experimental reproduziu todas as alterações morfológicas observadas na osteoartrite humana. Animais que receberam difosfato de cloroquina não desenvolveram lesões histológicas observadas na OA. Neste grupo houve significante preservação da estrutura da cartilagem articular (p < 0,0001), conservação da celularidade (p < 0,0001), proteoglicanas, demonstrados pela coloração de azul de anilina (p < 0,005) e integridade da linha de crescimento (p < 0,001), além da inibição da formação de osteófitos, do bloqueio da neoformação óssea e do não-aparecimento de colágeno tipo I (tecido osteocartilaginoso). CONCLUSÃO: O modelo experimental de meniscectomia parcial reproduz de forma gradativa as alterações morfológicas encontradas na osteoartrite, e estudos preliminares com o difosfato de cloroquina sugerem tratar-se de medicamento barato e com grande potencial de emprego como droga condroprotetora.<br>OBJECTIVE: The aim of this study was to develop an experimental model of osteoarthritis (OA) that could reproduce morphologic alterations viewed in this disease and to study the effect of chloroquine diphosphate on cartilage remodeling. METHODS: osteoarthritis was induced in rabbits by performing partial meniscectomy. To establish the experimental disease evolution, three groups of ten animals were sacrificed at 3, 14, 22 weeks after disease induction. To evaluate the effect of chloroquine diphosfate in OA progression, a group of six animals was treated preventively with 3 mg/kg/day, started one month prior to osteoarthritis induction and kept until the day of sacrifice (22 weeks). Histopathological (Masson trychrome, H&E), biochemical and immunofluorescence analyses to types I, II and XI collagens were made in all animals. Mankin's score was employed to quantify the severity of articular damage. RESULTS: The experimental model reproduced all of the alterations observed in osteoarthritis. Animals treated with chloroquine diphosfate did not develop morphological changes found in OA. There was significant preservation of articular cartilage tissue (p < 0,0001), maintenance of cellular morphology (p < 0,0001), proteoglicans, as demonstrated by aniline blue coloration (p < 0,005) and tidemark protection (p < 0,001), besides inhibition of osteophytes formation and absence of type I collagen expression. CONCLUSION: The experimental model of partial meniscectomy reproduces gradually, all the cartilage morphologic changes found in human osteoarthritis. Preliminary study done with choroquine diphosfate indicates that it is a cheap and effective drug to act as condroprotective agent in OA

Butylated hydroxytoluene induces type-V collagen and overexpression of remodeling genes/proteins in experimental lung fibrosis

Anomalous histoarchitecture with increased levels of type-V collagen (Col V) in lungs of human idiopathic pulmonary fibrosis (IPF) and bleomycin (BLM) airway-centered interstitial fibrosis suggest that this collagen can be a possible trigger involved in the pathogenesis of these diseases. Butylated hydroxytoluene (BHT) injury model revealed a distal involvement of lung parenchyma with significant endothelial injury and fibrotic response, contrasting with the BLM airway-centered insult. We undertook this study to analyze whether BHT alters distal airway/alveolar epithelial cells (AECs) and extracellular matrix (ECM) signaling involved in the initiation and progression of pulmonary fibrosis in a different pathway concerning overexpression of Col V. Female mice C57BL/6 (n=6) were instilled intraperitoneally with 400mg/kg of BHT dissolved in 1 mL of corn oil and euthanized at day 14 or 21 after BHT administration. Morphometry, immunohistochemistry and transmission electron microscopy were performed to characterize microscopic and submicroscopic changes of AECs and endothelial cells through transforming growth factor beta (TGF-β) basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) expression. Immunofluorescence and immunogold electron microscopy were performed to characterize Col V. Quantitative polymerase chain reaction (qPCR) was used to confirm differential levels of RNA messenger. BHT lungs showed marked fibrotic areas and hyperplastic AECs. The alveolar damage caused destruction of elastic fibers and a critical increase of Col V in ECM of distal lung parenchyma. Fibrogenesis-promoting markers TGF-β, bFGF and VEGF were also overexpressed in situ, coinciding with up-regulation in remodeling enzymes, growth factors, cytokines, transduction and transcription genes. BHT alters distal lung parenchyma signaling involved in pulmonary fibrosis highlighted similarities to human IPF in a pathway involving Col V arising as a promissory model to identify effective therapeutic targets

Radiofrequency preserves histoarchitecture and enhances collagen synthesis in experimental tendon injury

We investigated the action of radiofrequency (RF) on the healing process after inducing experimental lesions of the Achilles tendon in rats. Wistar rats were surgically subjected to bilateral partial transverse sectioning of the Achilles tendon. The right tendon was treated with radiofrequency (RFT), whereas the left tendon served as a control (CT). On the third postoperative day, the rats were divided into three experimental groups consisting of ten rats each, which were treated with monopolar radiofrequency (Tonederm™) until they were sacrificed on the 7th, 14th or 28th days. The histological specimens were studied for inflammatory cell content, collagen types I and III, immunostaining and morphometry. Total collagen were biochemically analyzed and to evalute fibroblast and myofibroblast proliferation by vimentin and α-actin smooth muscle immunohistochemistry methods. Statistical analysis was performed using the Student's ttest, the sign test and the Kruskal-Wallis test to compare tendons treated with radiofrequency with the non-treated tendons (α=5%; α=10%). Larger amounts of collagen I with hydroxyproline content and myofibroblast cells were clearly evident within 7 days (p<0.05). No difference was observed in the inflammatory cell content between the groups. We found better collagen arrangement with RF administration across the entire time studied. Radiofrequency administration preserves histoarchitecture and enhances collagen synthesis during the initial phases of cicatrization, suggesting that the treatment can provide improved stiffness during the most vulnerable phases of tendon healing. Clinical studies may include RF among the therapeutic tools in tendinous lesion management

Collagen V and vascular injury promote lung architectural changes in systemic sclerosis

Background: Systemic sclerosis (SSc) is a multisystem disorder characterized by inflammation, fibrosis and vascular damage. The aim of this study was to evaluate the interactions between basement membrane disruption, endothelial injury and collagen V deposition on the vascular wall, as well as their association with pulmonary function tests in patients with SSc. Method: The endothelial apoptosis was assessed by TUNEL and electron microscopy, and quantified through the point-counting technique. To evaluate basement membrane integrity, laminin immunostaining and electron microscopy were used. Immunofluorescence and morphometric analysis were used to determine the amount of collagen V in the vascular walls in 23 open lung biopsies of patients with SSc without pulmonary hypertension. Normal lung tissue was obtained from five individuals who had died of traumatic injuries. Results: The apoptosis index in SSc was higher in the endothelial cells (13.83 +/- 6.83) when compared with the control (2.51 +/- 2.06) group (P < 0.001) and confirmed by electron microscopy. We observed an important disruption of the basement membrane on the vascular wall shown by discontinuous laminin immunostaining and electron microscopy. An increase in collagen V on the vascular wall of the SSc group was observed (45.28 +/- 13.21), when compared with control group (22.90 +/- 4.13, P < 0.001), and this difference was statistically significant. An inverse correlation was found between vital capacity, forced vital capacity, forced expiratory volume in 1 s, vascular collagen V and endothelial apoptosis (P < 0.05). Conclusions: We conclude that the endothelial apoptosis and vascular collagen V interaction reinforce the vascular pathway in the SSc pathogenesis. Further studies are needed to determine whether this relationship is causal or consequential. Please cite this paper as: Parra ER, Aguiar AC Jr, Teodoro WR, de Souza R, Yoshinari NH and Capelozzi VL. Collagen V and vascular injury promote lung architectural changes in systemic sclerosis. The Clinical Respiratory Journal 2009; 3: 135-142.Brazilian agencies: the National Council for Scientific and Technological Development (CNPq)Foundation for the Support of Research of the State of Sao Paulo (FAPESP)[2006/14566-9]Universidade de São Paulo - Laboratories for Medical Research[LIM 05] - FM/USPUniversidade de São Paulo - Laboratories for Medical Research[LIM17] - FM/USPHospital das Clinicas, Faculdade de Medicina da Universidade de Sao Paulo HC-FM_US