The action of subconjunctival cyclophosphamide in rabbits / Ação da ciclofosfamida subconjuntival em coelhos

ABSTRACT Objective: To observe clinically, in rabbits, the side effects of topical injection of subconjunctival cyclophosphamide, studying its role as an antifibrotic drug. Methods: Prospective study in 20 albino rabbits of New Zealand race. All rabbits were treated with cyclophosphamide, 10mg/ml in a volume of 0.3 ml, in the left eye through subconjunctival injection. They were evaluated for 1, 7, 30, and 60 days after the procedure. All the animals were examined for the detection of ocular reactions such as necrosis, hyperemia, chemosis, secretion, opacity, and iritis. Other side effects as changes in the behavior, in the feed, and the water consumption were also evaluated. Results: It was observed that from the 20 rabbits studied, three rabbits (15%) showed side effects only at the 24 hours analysis. One rabbit (5%) presented hyperemia, one rabbit (5%) had hyperemia associated with iritis, and one rabbit (5%) presented hyperemia associated with secretion. These reactions were not observed at 1, 7, 30, and 60 days. Conclusion: Cyclophosphamide subconjunctival injection induces minor side effects on the conjunctiva of rabbits such as hyperemia, associated with iritis and secretion.

RESUMO Objetivo: Observar clinicamente os efeitos colaterais de injeção subconjuntival de ciclofosfamida, pensando em sua ação como um agente antifibrótico. Métodos: Estudo prospectivo realizado com 20 coelhos albinos da raça Nova Zelândia. Todos os coelhos foram submetidos a 0,3 ml de injeção subconjuntival de ciclofosfamida 10mg/ml no olho esquerdo e foram avaliados de acordo com os efeitos locais no primeiro dia após a injeção, 7, 30 e 60 dias. Foram examinados para detecção de reações oculares como necrose, hiperemia, quemose, secreção, opacidade corneana, irite além de alterações comportamentais e variação no consumo de água e alimentação. Resultados: Dos 20 coelhos estudados, apenas 3 apresentaram reações oculares e somente na leitura de 24 horas. Um coelho (5%) apresentou hiperemia, 1 coelho (5%) apresentou hiperemia associada a presença de irite e 1 coelho (5%) apresentou hiperemia associada a presença de secreção. As reações não foram mais observadas durante os exames de 7, 30 e 60 dias. Conclusão: A ciclofosfamida subconjuntival causou poucos efeitos colaterais na conjuntiva dos coelhos. Os únicos efeitos encontrados foram hiperemia, irite e secreção.

Aliskiren attenuates cardiac dysfunction by modulation of the mTOR and apoptosis pathways

Aliskiren (ALS) is well known for its antihypertensive properties. However, the potential underlying the molecular mechanism and the anti-hypertrophic effect of ALS have not yet been fully elucidated. The aim of the present study was to investigate the role of ALS in mammalian target of rapamycin (mTOR) and apoptosis signaling using in vivo and in vitro models of cardiac hypertrophy. A rat model of cardiac hypertrophy was induced by isoproterenol treatment (5 mg·kg-1·day-1) for 4 weeks, with or without ALS treatment at 20 mg·kg-1·day-1. The expression of hypertrophic, fibrotic, and apoptotic markers was determined by RT-qPCR. The protein expression of apoptotic markers mTOR and p-mTOR was assessed by western blot analysis. The proliferation of H9C2 cells was monitored using the MTS assay. Cell apoptosis was analyzed using flow cytometry. In vivo, isoproterenol-treated rats exhibited worse cardiac function, whereas ALS treatment reversed these dysfunctions, which were associated with changes in p-mTOR, Bcl-2, Bax, and cleaved caspase-3 expression, as well as the number of apoptotic cells. In vitro, H9C2 cardiomyocyte viability was significantly inhibited and cardiac hypertrophy was induced by Ang II administration, but ALS reversed Ang II-induced H9C2 cardiomyocyte hypertrophy and death. Furthermore, Ang II triggered the activation of the mTOR and apoptosis pathways in hypertrophic cardiomyocytes that were inhibited by ALS treatment. These results indicated that ALS alleviated cardiac hypertrophy through inhibition of the mTOR and apoptosis pathways in cardiomyocytes.

Rhein alleviates renal interstitial fibrosis by inhibiting tubular cell apoptosis in rats

BACKGROUND: Ureteral obstruction causes injury of the renal tissues and can irreversibly progress to renal fibrosis, with atrophy and apoptosis of tubular cells. The goal of the current study was to examine the effects of rhein on the apoptosis o renal tubular cells as well as renal fibrosis using a rodent model of unilateral ureteral obstruction (UUO). METHODS: UUO was induced through ureteral ligation, then animals received treatments with rhein or vehicle. The control rats only received sham operation. The renal tissue was harvested 1 week after surgery for assessment of kidney fibrosis. RESULTS: The expressions of collagen I and α-smooth muscle actin (α-SMA), as well as the severity of renal tubular apoptosis and fibrosis were time-dependently increased following UUO. Treatments with rhein partially inhibited such responses. Renal interstitial fibrosis was associated with STAT3 (signal transducer and activator of transcription 3) phosphorylation as well as altered expressions of Bax and Bcl2, both apoptosis-related proteins. Treatment with rhein also partly blocked these responses. CONCLUSION: These findings demonstrated that rhein mitigated apoptosis of renal tubular cell as well as renal fibrosis in a UUO rodent model. This curative effect is likely mediated via suppression of STAT3 phosphorylation.

The effects of ankaferd blood stopper and microporous polysaccharide hemospheres on epidural fibrosis in rat laminectomy model

ABSTRACT PURPOSE: To investigate whether topically administered hemostatic agents ankaferd blood stopper and microporous polysaccharide hemospheres can decrease epidural fibrosis after laminectomy in rats. METHODS: Eighteen adult male Sprague-Dawley rats were equally and randomly divided into three groups. In the treatment groups, ankaferd blood stopper and microporous polysaccharide hemospheres topically administrated upon duramater surface after laminectomy. Fibroblast count, epidural fibrosis and arachnoidal involvement were evaluated and graded histopathologically. RESULTS: Our data revealed that the count of fibroblasts, the grading of epidural fibrosis and arachnoideal involvement in the rats treated with microporous polysaccharide hemospheres were significantly less than the control group. Although the arachnoideal involvement in ankaferd blood stopper group were significantly less than the control group, there were no statistical differences when comparing the grading of epidural fibrosis and the fibroblasts count between the treatment groups and the control group. CONCLUSION: The ankaferd blood stopper and microporous polysaccharide hemospheres reduced epidural fibrosis and arachnoideal involvement after laminectomy in rats.

Alterações iniciais da fibrose septal por Capillaria Hepatica (Bancroft, 1893) em ratos / Initial changes of septal fibrosis Capillaria hepatica (Bancroft, 1893) in rats

espaço-porta é o local de origem da fibrose em muitas doenças crônicas hepáticas. Essa área do fígado participa da drenagem linfática hepática e abriga diversos elementos celulares potencialmente fibrogênicos. Estudos sobre a fibrose hepática relacionados à infecção experimental de ratos pelo helminto Capillaria hepatica têm demonstrado que a fibrose começa em áreas portais com a distribuição de septos que sulcam o parênquima hepático se desenvolvendo em áreas próximas ao espaço de Disse. Entretanto, apesar de esta fibrose ocorrer de forma paralela aos sinusóides, estudos têm revelado que não apenas as células estreladas hepáticas participam da fibrose septal, mas também outros tipos celulares residentes nos espaços-porta. Diante destes aspectos, o presente estudo desenvolveu-se com o intuito de investigar a contribuição das células potencialmente fibrogênicas dos espaços-porta, nas fases iniciais da infecção, onde a fibrose se concentra. Para isso, foram utilizados fragmentos de fígado, em blocos parafinados, disponíveis nos arquivos do Laboratório de Patologia Experimental (CPqGM/Fiocruz) provenientes de ratos infectados com 800 ovos de Capillaria hepatica e foi possível observar que ocorreu a proliferação de colangiócitos e a concentração de miofibroblastos em áreas portais, além da ativação de células estreladas hepáticas, sendo todos os resultados vistos por meio da coloração de rotina HE, Picro-sírius vermelho e imunohistoquímica para α-actina de músculo liso, CD31 e GFAP.

Portal space is the local of origin for fibrosis in many chronic liver diseases. This area is involved with lymph drainage and contains several cell types, potentially fibrogenic. Experimental studies related to hepatic fibrosis during Capillaria hepatica infection in rats have suggested that the septal fibrosis indeed takes origin from portal spaces, with the distribution of the septs in the parenchymal region in proximity areas of Disse space. However, despite this fibrosis occurs in parallel to sinusoids, studies have revealed that not only the hepatic stellate cells participate in septal fibrosis, but also other resident cell types in the portal spaces. In face these aspects, the goal of present study was investigate the contribution of the cells potentially fibrogenic in the portal space, in the early phases of the infection. For this, blocks in paraffin available of the liver of rats infected with 800 eggs of Capillaria hepatica archived in the Laboratory of Experimental Pathology (Research Center Gonçalo Moniz, Fiocruz - BA), were utilized and it was observed that proliferation of colangiocytes and concentration of myofibroblasts occurred portal areas, in addition to the activation of hepatic stellate cells. All results were analised by routine staining HE, Sirius red and immunohistochemistry for α-SMA, GFAP and CD31.

Adhesion prevention in reabsorbable polyethylene glycol hydrogel (Coseal®) coated polypropylene mesh in rabbits

PURPOSE: To evaluate of postoperative adhesion prevention and inflammatory response to polypropylene mesh, coated with reabsorbable hydrogel of polyethylene glycol (Coseal®) in contact with small bowel in an experimental model in rabbits. METHODS: Twenty female rabbits underwent laparotomy to implant two polypropylene meshes, 2x1cm, in the right and left flanks. The right mesh was protected with Coseal® spray (Group 1) and the left mesh received no treatment after implantation (Group 2). Thirty days after implantation, the rabbits underwent laparoscopy for adhesion analysis; the prosthesis were removed en bloc with the adjacent tissue for microscopic analysis of inflammation. Statistical analysis used the Mann-Whitney test. RESULTS: There was adhesion formation in five meshes (36%) from Group 1 and in 14 meshes (100%) from Group 2, with statistical significance (p<0.01). There were no differences in the inflammatory response, fibrosis, foreign body reaction, presence of collagen and type of inflammatory cells between the two groups. CONCLUSION: Polypropylene mesh coated with Coseal® showed a significantly lower rate of adhesion formation when compared with uncoated meshes, without interfering with inflammatory response.

El aumento de la expresión del ARNm de la enzima convertidora de angiotensina I homóloga (ECA-2) inducido por atorvastatina se asocia a menor fibrosis e hipertrofia ventricular izquierda en un modelo de cardiomiopatía diabética / Atorvastatin induced increase in homologous angiotensin i converting enzyme (ACE2) mRNA is associated to decreased fibrosis and decreased left ventricular hypertrophy in a rat model of diabetic cardiomyopathy

Objetivos. Evaluar el efecto de atorvastatina sobre la progresión del remodelado cardiaco y la expresión de ECA-2 en el miocardio de ratas diabéticas. Materiales y métodos. La diabetes fue inducida en ratas Holtzman con una inyección intraperitoneal de estreptozotocina. Los animales fueron divididos en tres grupos: (1) ratas control, (2) ratas diabéticas y (3) ratas diabéticas tratadas con atorvastatina (50 mg/kg/día). Después de ocho semanas de tratamiento, los corazones fueron extraídos para el análisis morfométrico, la cuantificación de colágeno y la determinación de los niveles de ARNm de ECA y ECA-2. Resultados. El índice de hipertrofia ventricular y el depósito de colágeno se incrementaron significativamente en las ratas diabéticas. La administración de atorvastatina previno estos cambios sin modificar los niveles de colesterol. La hiperglicemia produjo un incremento significativo en los niveles del ARNm de ECA y una marcada disminución en la expresión de ECA-2 en el miocardio de ratas diabéticas. La administración de atorvastatina indujo la expresión del ARNm de ECA-2 e inhibió la sobreexpresión del ARNm de ECA en el miocardio de las ratas diabéticas. Conclusiones. Nuestros resultados indican que la atorvastatina, independientemente de su capacidad para disminuir el colesterol, normaliza la relación de la expresión de ECA/ECA-2 y atenúa el desarrollo del remodelado adverso en el corazón diabético.

Objectives. This study has investigated the effect of atorvastatin on the progression of cardiac remodelling and ACE- 2 expression in diabetic myocardium in rats. Materials and Methods. Diabetes was induced in Holtzman rats with an intraperitoneal injection of streptozotocin. The animals were divided into 3 groups: (1) normal control rats, (2) diabetic rats and (3) diabetic rats treated orally with atorvastatin (50 mg/kg/day). After eight weeks of treatment, the hearts were removed for morphometric studies, collagen content assay and genetic expressions of ACE and ACE2 mRNA. Results. Myocardial hypertrophy index and collagen deposition were increased in diabetic rats, but not in the treated-diabetic rats, without producing changes in cholesterol levels. Myocardial ACE mRNA levels were increased while ACE2 mRNA levels were decreased in diabetic rats. Atorvastatin administration attenuated overexpression of ACE mRNA and overexpression of ACE-2 mRNA in diabetic rats. Conclusions. Our results indicate that atorvastatin, independently of its cholesterol-lowering capacity, lowers the ACE/ACE2 ratio to normal values and attenuates the development of adverse remodeling in the diabetic heart.

Antifibrotic effects of magnesium lithospermate B on hepatic stellate cells and thioacetamide-induced cirrhotic rats

Magnesium lithospermate B (MLB) is one of the major active components of Salvia miltiorrhizae. The anti-oxidative effects of Salvia miltiorrhizae have been previously reported. The aim of this study was to investigate the effect of purified MLB on hepatic fibrosis in rats and on the fibrogenic responses in hepatic stellate cells (HSCs). Hepatic fibrosis was induced in rats by intraperitoneal thioacetamide (TAA) injections over a period of 8 or 12 weeks. MLB was orally administered daily by gavage tube. Serum AST and ALT levels in TAA + MLB group were significantly lower than those in TAA only group at week 8. Hepatic fibrosis was significantly attenuated in TAA + MLB group than in TAA only group at week 8 or 12. Activation of HSCs was also decreased in TAA + MLB group as compared to TAA only group. Hepatic mRNA expression of alpha-smooth muscle actin (alpha-SMA), TGF-beta1, and collagen alpha1(I) was significantly decreased in TAA + MLB group as compared to TAA only group. Incubation with HSCs and MLB (> or =100 microM) for up to 48 h showed no cytotoxicity. MLB suppressed PDGF-induced HSC proliferation. MLB inhibited NF-kappaB transcriptional activation and monocyte chemotactic protein 1 (MCP-1) production in HSCs. MLB strongly suppressed H2O2-induced reactive oxygen species (ROS) generation in HSCs, and MLB inhibited type I collagen secretion in HSCs. We concluded that MLB has potent antifibrotic effect in TAA-treated cirrhotic rats, and inhibits fibrogenic responses in HSCs. These data suggest that MLB has potential as a novel therapy for hepatic fibrosis.

Effect of felodipine on myocardial and renal injury induced by aldosterone-high salt hypertension in uninephrectomized rats

It has been recently shown that calcium channel blockers might have a protective effect on cardiac fibrogenesis induced by aldosterone. The objective of this study was to evaluate the protective effect of felodipine, a dihydropyridine calcium channel blocker, against heart and kidney damage caused by aldosterone-high sodium intake in uninephrectomized rats. Wistar rats were divided into three groups: CNEP (uninephrectomized + 1 percent NaCl in the drinking water, N = 9); ALDO (same as CNEP group plus continuous infusion of 0.75 µg/h aldosterone, N = 12); ALDOF (same as ALDO group plus 30 mg·kg-1·day-1 felodipine in the drinking water, N = 10). All results were compared with those of age-matched, untreated rats (CTL group, N = 10). After 6 weeks, tail cuff blood pressure was recorded and the rats were killed for histological analysis. Blood pressure (mmHg) was significantly elevated (P < 0.05) in ALDO (180 ± 20) and ALDOF (168 ± 13) compared to CTL (123 ± 12) and CNEP (134 ± 13). Heart damage (lesion scores - median and interquartile range) was 7.0 (5.5-8.0) in ALDO and was fully prevented in ALDOF (1.5; 1.0-2.0). Also, left ventricular collagen volume fraction ( percent) in ALDOF (2.9 ± 0.5) was similar to CTL (2.9 ± 0.5) and CNEP (3.4 ± 0.4) and decreased compared to ALDO (5.1 ± 1.6). Felodipine partially prevented kidney injury since the damage score for ALDOF (2.0; 2.0-3.0) was significantly decreased compared to ALDO (7.5; 4.0-10.5), although higher than CTL (null score). Felodipine has a protective effect on the myocardium and kidney as evidenced by decreased perivascular inflammation, myocardial necrosis and fibrosis.

Avaliação da cicatrização de feridas em dorso de ratos tratados com mitomicina C tópica e posterior reforço com injetável / Study of wound healing in rats treated with topical and injected mitomycin-C

Estudo experimental em animais. A mitomicina C vem sendo usada como inibidor de fibroblastos, acarretando, com isso, diminuição do processo cicatricial em feridas cirúrgicas. OBJETIVO: Este trabalho visa avaliar o uso de Mitomicina C para diminuir o processo cicatricial, através de seu uso tópico com reforços posteriores injetáveis. MATERIAL E MÉTODOS: Foi usado um modelo de feridas em dorso de ratos, com retirada circular da pele e cicatrização por segunda intenção. Foram usados 18 ratos, divididos em três grupos: controle; com uso tópico; e com reforço de mitomicina C injetável, mensalmente e por 2 meses. Após 3 meses os animais foram sacrificados e as cicatrizes retiradas cirurgicamente e submetidas a estudo histológico. RESULTADOS: Notou-se sob vários critérios que a cicatrização com o uso tópico é menos intensa, mas ao se usar o reforço injetável os parâmetros voltam a ser comparados ao do grupo controle. DISCUSSÃO: Acreditamos que a administração injetável de mitomicina C nas cicatrizes, pela sua elevada característica tóxica, acarreta destruição tecidual e neoformação cicatricial. CONCLUSÕES: A mitomicina C diminui o processo cicatricial quando usada topicamente, mas acarreta aumento da cicatrização quando nestas feridas são feitos reforços injetáveis.

Experimental study in animals. Introduction: Mitomycin C has been used as a fibroblasts inhibitor, thus reducing the scaring process in surgical wounds. AIM: This paper aims at assessing the use of Mitomycin C to reduce the scaring process through its topical use and later injected reinforcements. MATERIALS AND METHODS: We used a model of a creating a wound in the dorsum of rats, removing a circular piece of skin and letting it heal by itself. We used 18 rats, divided in three groups. The first group - Control, the second with topical use, and a third group with injected mitomycin C reinforcement, monthly for 2 months. After 3 months the animals were slaughtered and the scars were surgically removed and sent for histology study. RESULTS: We noticed, under different criteria, that healing with topical mitomycin is less intense; however, when it was injected, the parameters were again comparable to those from the control group. DISCUSSION: We believe that injected mitomycin C in the scar, since it is highly toxic, it destroys tissue and brings about scar neoformation. CONCLUSIONS: Mitomycin C reduces the scaring process when used topically; however, it increases scar tissue formation when injected in these wounds.

Estudo de membrana biológica em ratos na prevenção de fibrose pós laminectomia / Inhibition of peridural fibrosis after laminectomy using biological sheet in rat model

Os autores investigaram a aplicação de uma membrana biológica, constituída de cortical óssea bovina descalcificada, em cirurgias de coluna vertebral de ratos, com o intuito de tratar o defeito ósseo minimizando ou impedindo a herniação do tecido muscular para dentro do canal raquidiano pós laminectomia, inibindo a formação de fibrose pós-operatória e avaliando a biocompatibilidade do material. O estudo foi feito utilizando-se de ratos Wistar-EPM, que após intervalos de 8, 16 e 24 semanas, foram sacrificados, sendo removidas as peças cirúrgicas para análise anatomopatológica. A membrana biológica evitou a herniação do tecido muscular para o canal raquidiano, sendo totalmente reabsorvida em todas as peças analisadas, demonstrando sua biocompatibilidade e favorecendo a neoformação óssea e evitando aderências.

Pirfenidone Suppressed the Development of Glomerulosclerosis in the FGS/Kist mouse

Pirfenidone(PFD) is a newly developed anti-fibrotic agent. We evaluated the effect of PFD for the prevention of renal fibrosis using a spontaneous progressive glomerulosclerosis animal model, FGS/Kist mice. Male and female FGS/Kist mice were fed a diet containing 0.5% PFD or the same control diet (CD) without PFD, for 1, 2, or 3-month periods. Body weight was monitored for the general effect of PFD on the mice. Proteinuria and glomerular filtration rate (GFR) were evaluated for renal function. The sclerosis index was examined for the morphological changes. There were no significant changes in body weight between the PFD and control groups in both sexes. Proteinuria levels were low in all the PFD groups compared to the corresponding CD groups. The sclerosis scores were also reduced in both sexes of the 3-month PFD groups (p<0.05), and glomerular filtration rates were increased in both sexes of the 3-month PFD groups compared to the CD groups. The treatment of PFD for 1 or 2-month periods did not have statistic significances but the treatment for 3 months had statistic significances in sclerosis and GFR compared to CD groups. These results suggested that long-term administration of PFD sup-pressed the progression of glomerulosclerosis and improved renal function of the renal function of the FGS/Kist mice.

Ultraestructura del hepatocito en diversas hepatopatías / Hepatocyte ultrastructure in liver diseases

Se estudiaron 14 biopsias hepáticas obtenidas de ocho pacientes con hepatitis viral aguda y series con hepatopatías crónicas, y se halló buena correlación de los datos clínicos con las alteraciones morfológicas por lo que parece recomendable hacer una clasificación etiológica para evitar que los pacientes progresen a cirrosis y desarrollen carcinoma hepatocelular. El estudio de la hepatitis viral mediante los microscopios de luz y electrónico de transmisión, correlacionado con la clínica y completado con estudios de histoquímica y virología, sirven para aclarar interrogantes en relación a la identificación del virus

Coroiditis multifocal: presentación de un caso / Multifocal choroiditis. A case report

Presentamos la evolución clínica de una paciente de 11 años de edad con panuveítis bilateral recurrente, caracterizada por la presencia de múltiples focos de coroiditis, atrofia peripapilar y fibrosis subretiniana. Se hacen consideraciones acerca del diagnóstico diferencial y tratamiento.

Enxerto de gordura nas laminectomias / Fat graft in laminectomies

O autor analisa as tomografias computadorizadas de 47 laminectomias realizadas em 32 pacientes. Os diagnósticos iniciais foram: hérnia discal, 29 casos; estenose em moléstia de Paget, um; cisto sinovial da articulaçäo interfacetária, um; e um caso de espondilolistese. O enxerto de gordura foi utilizado em 32 cirurgias. Conclui que o enxerto de gordura forma um plano de deslizamento, uma barreira, impedindo a penetraçäo da fibrose dentro do canal, ao contrário do que foi observado nas tomografias computadorizadas das laminectomias que näo receberam enxerto

Bloqueo ductal pancreático : importancia de la cantidad de sustancia oclusiva empleada / Pancreatic ductal blockade : importance of quantity occlusive substance used

Serie de 16 perros que luego de provocarles pancreatitis aguda necrohemorrágica con ClCa al 20% en lóbulo izquierdo, se les ocluyó el sistema ductal con 2 cm.3 de adhesivo tisular(Tissucol). Dentro de las 24 hs. murieron 2 perros, los restantes evolucionaron satisfactoriamente y se los sacrificó a los 30, 40 y 120 días. La sobrevida(87,5%) demuestra que rellenando sólo el continente canalicular, se interrumpe el proceso necrohemorrágico, descartando la posible influencia terapéutica del excedente que pasa al intersticio. Estudios histopatológicos revelaron que con 2cm3 en lugar de 4 a 7 cm3 disminuye la secuela fibrótica.