Effects of ultrasound and stretching on skeletal muscle contusion in rats: immunohistochemistry analysis / Efectos de ultrasonido y estiramiento en la contusión muscular esqueletal en ratas: análisis inmunohistoquímico

SUMMARY: The aim of this study was to evaluate the effects of stretching and therapeutic ultrasound (TUS) on desmin and laminin contents of rat muscle after contusion. Male Wistar rats (n = 35, 8-9 weeks of age, 271 ± 14g body weight) were divided into five groups: Control group (CG) (n= 03); Injured group (IG) (n= 8); Injured + ultrasound group (IUSG) (n= 8); Injured+stretching group (ISG) (n= 8); Injured +ultrasound + stretching group (IUSSG) (n= 8). The application of ultrasound started 72 hours after the contusion, using the 50 % pulsed mode, 0.5 W/cm2, 5 min, once a day, for five consecutive days. Passive manual stretching was started on the tenth day after injury, with four repetitions of 30 s each and 30 s rest between repetitions, once a day, five times per week, for a total of ten applications. After 22 days, the rats were euthanazied and the gastrocnemius of both limbs removed for desmin and laminin immunohistochemistry morphometric measurement. Analysis was conducted using ANOVA one way post-hoc Tukey to parametric data and Kruskall-Wallis for non-parametric data. The IUSSG animals showed a larger area of desmin than ISG (p<0.05). It was found a decrease in laminin comparing IUSG to IG. However, laminin area was higher in ISG than all groups (p<0.05). UST isolated or in combination with stretching influenced gastrocnemius regeneration in different manners. While stretching applied isolated enhanced gastrocnemius regeneration noticed by the increase in laminin area, in combination with TUS strengthened the muscle healing rising desmin area.

RESUMEN: El objetivo de este estudio fue evaluar los efectos del estiramiento y la ecografía en los contenidos de desmina y laminina del músculo de rata después de la lesión. Ratas Wistar macho (n = 35, 8-9 semanas de edad, 271 ± 14 g de peso corporal) se dividieron en cinco grupos: grupo de control (CG) (n = 03); Grupo lesionado (GL) (n = 8); Lesionado + grupo de ultrasonido (LGU) (n= 8); Lesionado + grupo de estiramiento (LGE) (n = 8); Lesionado + ultrasonido + grupo de estiramiento (LUGE) (n = 8). La aplicación de ultrasonido comenzó 72 horas después de la lesión, usando el modo pulsado al 50 %, 0,5W / cm2, 5 min, una vez al día, durante cinco días consecutivos. El estiramiento manual pasivo se inició el décimo día después de la lesión, con cuatro repeticiones de 30 seg cada una y 30 seg de descanso entre repeticiones, una vez al día, cinco veces por semana, para un total de diez aplicaciones. Las ratas fueron sacrificadas después de 22 días, y se extrajo el músculo gastrocnemio de ambos miembros para la medición morfométrica de desmina y laminina a través de inmunohistoquímica. El análisis se realizó utilizando ANOVA unidireccional Tukey post-hoc para datos paramétricos y Kruskall-Wallis para datos no paramétricos. Los animales LUGE mostraron un área mayor de desmina que LGE (p <0,05). Se encontró una disminución en la laminina comparando LGU con GL. Sin embargo, el área de laminina fue mayor en LGE que en todos los grupos (p <0,05). El tratamiento con ultrasonido aislado o en combinación con estiramiento influyó en la regeneración del músculo gastrocnemio de diferentes maneras. Si bien el estiramiento aplicado, en combinación con tratamiento de ultrasonido, fortaleció el área de desmina, la regeneración del músculo gastrocnemio mejoró por el aumento en el área de laminina aumentando la curación muscular.

Decellularization of placentas: establishing a protocol

Biological biomaterials for tissue engineering purposes can be produced through tissue and/or organ decellularization. The remaining extracellular matrix (ECM) must be acellular and preserve its proteins and physical features. Placentas are organs of great interest because they are discarded after birth and present large amounts of ECM. Protocols for decellularization are tissue-specific and have not been established for canine placentas yet. This study aimed at analyzing a favorable method for decellularization of maternal and fetal portions of canine placentas. Canine placentas were subjected to ten preliminary tests to analyze the efficacy of parameters such as the type of detergents, freezing temperatures and perfusion. Two protocols were chosen for further analyses using histology, scanning electron microscopy, immunofluorescence and DNA quantification. Sodium dodecyl sulfate (SDS) was the most effective detergent for cell removal. Freezing placentas before decellularization required longer periods of incubation in different detergents. Both perfusion and immersion methods were capable of removing cells. Placentas decellularized using Protocol I (1% SDS, 5 mM EDTA, 50 mM TRIS, and 0.5% antibiotic) preserved the ECM structure better, but Protocol I was less efficient to remove cells and DNA content from the ECM than Protocol II (1% SDS, 5 mM EDTA, 0.05% trypsin, and 0.5% antibiotic).

Imunoproteção de ilhotas pancreáticas microencapsuladas em biomateriais inovadores e seu potencial terapêutico no diabetes mellitus tipo 1 / Immunoprotection of pancreatic islets microencapsulated in inovative biomaterials and its therapeutic potential in type 1 Diabetes Mellitus

O transplante de ilhotas microencapsuladas constitui uma alternativa terapêutica interessante para o Diabetes Mellitus tipo 1, permitindo um melhor controle glicêmico e eliminando a necessidade de imunossupressão. Entretanto, a manutenção a longo prazo da viabilidade das células-ß ainda é um desafio. No isolamento, a perda da matriz extracelular e as condições hipóxicas subsequentes afetam decisivamente a sobrevivência e funcionalidade das ilhotas. Objetivo Para diminuir o estresse sobre o enxerto, levando a um sucesso prolongado do transplante, propôs-se a adição de perfluorocarbono (PFC) ou laminina (LN), moléculas associadas respectivamente à oxigenação e interações célula-célula, ao biomaterial baseado em alginato, Biodritina, adequado ao encapsulamento celular. Metodologia Para testar a estabilidade das formulações PFC-Biodritina e LN-Biodritina, microcápsulas foram submetidas a diferentes estresses (rotacional, osmótico, temperatura e cultura) por 7 e 30 dias. A pureza do biomaterial foi avaliada pela coincubação com macrófagos murinos RAW264.7, por 3, 9 e 24h, quando a ativação dos macrófagos foi observada pela expressão gênica de IL- 1ß e TNFα. Microcápsulas implantadas i.p. em camundongos foram recuperadas após 7 ou 30 dias, para análises de biocompatibilidade. A expressão de níveis de mRNA (bax, bad, bcl-2, bcl-XL, xiap, caspase 3, mcp1/ccl2, hsp70, ldh, insulina 1 e 2), proteínas (Bax, Bcl-XL e Xiap) e a atividade de Caspase3 foram avaliadas em ilhotas microencapsuladas com PFC- e LN-Biodritina, após cultura de 48h em condições de normóxia e hipóxia (<2% O2). Camundongos diabéticos foram transplantados com ilhotas encapsuladas nas diferentes formulações e os animais foram monitorados pelas variações de massa corporal, glicêmicas e pela funcionalidade do enxerto (TOTGs). As ilhotas foram recuperadas de animais normo ou hiperglicêmicos e uma análise de biocompatibilidade das cápsulas foi realizada, assim como a avaliação funcional das células-ß. Após o explante, a glicemia dos animais normoglicêmicos foi monitorada para se atestar a eficiência das ilhotas transplantadas. Resultados Microcápsulas de PFC- e LN-Biodritina são tão estáveis e biocompatíveis quanto as de Biodritina. Para ilhotas encapsuladas em ambos os materiais, em normóxia ou hipóxia, observou-se uma modulação gênica que sugere proteção contra apoptose. Adicionalmente, encontrou-se uma diminuição na expressão de genes indicadores de estresse (mcp1, hsp70). Uma diminuição nos níveis de mRNA de ldh foi vista para PFC-Biodritina, mas o oposto foi encontrado para LN-Biodritina. As diferenças encontradas na expressão proteica sugerem o mesmo padrão anti-apoptótico. Caspase3 não foi modulada por nenhum biomaterial. Nos experimentos de transplante, apenas LN-Biodritina levou reversão prolongada do diabetes, com 60% dos animais normoglicêmicos, 198 dias pós-cirurgia, comparado a 9% do grupo Biodritina. O TOTG demonstrou que camundongos transplantados com ilhotas encapsuladas secretaram mais insulina do que controles, 60 (LN-Biodritina) ou 100 (PFC- e LN-Biodritina) dias pós-cirurgia. O explante restabeleceu a hiperglicemia nos camundongos. Microcápsulas recuperadas de animais hiperglicêmicos apresentavam uma extensa adesão celular. Testes de secreção de insulina in vitro demonstraram que somente ilhotas do grupo normoglicêmico responderam às variações da concentração de glicose. Conclusão A adição de moléculas bioativas à Biodritina é capaz de diminuir o estresse em ilhotas isoladas e tem o potencial de melhorar a terapia pelo transplante de ilhotas

Transplantation of microencapsulated islets represents an attractive therapeutical approach to treat type 1 Diabetes Mellitus, accounting for an improved glycemic control and the abolishment of immunosuppressive therapies. However, maintenance of long-term ß-cell viability remains a major problem. During islet isolation, the loss of extracellular matrix interactions and the hypoxic conditions thereafter dramatically affect ß-cell survival and function. Objective To lessen the burden of islet stress and achieve a better outcome in islet transplantation we tested the addition of perfluorocarbon (PFC) or laminin (LN), molecules associated respectively with oxygenation and cell-cell interaction, to Biodritin, an alginate-based material suitable for cell microencapsulation. Methodology To test the stability of PFC-Biodritin and LN-Biodritin composites, microcapsules were subjected to different stresses (rotational, osmotic, temperature and culture) for 7 and 30 days. To assess biomaterial purity microcapsules were co-incubated with RAW264.7 murine macrophage cell line for 3, 9 and 24h and macrophage activation was detected through mRNA levels of IL-1ß and TNFα. Microcapsules were implanted i.p. in mice and retrieved after 7 or 30 days, for biocompatibility analyses. Gene expression at mRNA (bax, bad, bcl-2, bcl-XL, xiap, caspase 3, mcp1/ccl2, hsp70, ldh, insulin 1 and 2) and protein (Bax, Bcl-XL and Xiap) levels, together with Caspase3 activity, were evaluated in islets microencapsulated in PFC- or LN-Biodritin, upon culturing for 48h in normoxic or hypoxic (<2% O2) conditions. Diabetic mice were transplanted with PFC- or LN-Biodritin microencapsulated islets, followed by assessments of body weight, glycemia and graft function by oral glucose tolerance tests (OGTTs). Microencapsulated islets were retrieved from normoglycemic or hyperglycemic mice and biocompatibility analyses of the beads together with a functional assessment of the graft followed. After graft removal, normoglycemic animals had their glycemias monitored to attest the efficacy of the transplanted islets. Results PFC- and LN-Biodritin microcapsules were as stable and biocompatible as Biodritin. For both biomaterials in normoxia and hypoxia a modulation in gene expression was observed in islets associated with a protection against apoptosis. Also, a decreased expression of stress-related genes (mcp1, hsp70) was evidenced. ldh mRNA levels were down-regulated in PFC-Biodritin microencapsulated islets but upregulated in the presence of LN. Increased levels of insulin mRNA were observed. The differences seen in protein expression indicated the same anti-apoptotic pattern. Caspase3 activity was not different between groups. Concerning diabetes reversal experiments, only mice transplanted with LN-Biodritin microencapsulated islets presented a better outcome, with 60% remaining euglycemic at 198 days post-surgery, compared with 9% for the Biodritin group. OGTT showed that mice transplanted with encapsulated islets secreted more insulin than normal mice, 60 (LN-Biodritin) or 100 days (PFC- and LN-Biodritina) posttransplant. Hyperglycemia was achieved after the retrieval of microcapsules showing graft efficacy. Retrieved microcapsules revealed an extensive overgrowth in most beads from hyperglycemic mice. A static glucose stimulated insulin secretion test revealed that only islets from normoglycemic subjects were able to secrete insulin according to glucose concentration. Conclusion- The addition of bioactive molecules to Biodritin may lessen the stress of isolated islets and have the potential to improve islet transplantation therapy.

Expressão da laminina na membrana basal em carcinoma escamocelular oral / Expression of basement membrane laminin in oral squamous cell carcinomas

A membrana basal é uma estrutura dinâmica que sofre modificações qualitativas e quantitativas durante a progressão do carcinoma escamocelular, e este processo é de fundamental importância na invasão neoplásica e metástase. OBJETIVO: O objetivo deste estudo foi investigar o comportamento da membrana basal em carcinomas escamocelulares de boca com diferentes graus de malignidade histológica através da expressão imuno-histoquímica da laminina. FORMA DE ESTUDO: Coorte histórica com corte transversal. MATERIAL E MÉTODO: Trinta e um casos de carcinoma escamocelular de boca foram submetidos à graduação histológica para tumores malignos. Através do método da streptavidina-biotina, foi verificada a expressão imuno-histoquímica da laminina em relação à sua intensidade e integridade nas lesões com diferentes graus de malignidade. RESULTADOS: Foram observadas diferenças estatisticamente significativas dos valores médios da intensidade e da continuidade de marcação da laminina em relação aos diferentes graus de malignidade. CONCLUSÃO: Ficou demonstrado que a laminina, uma glicoproteína presente na membrana basal, sofreu modificações diferenciadas em carcinomas escamocelulares de boca nos diferentes graus de malignidade histológica.

The basement membrane is a dynamic structure that undergoes quantitative and qualitative changes during the progression of squamous cell carcinoma, which is essencially important in tumoral invasion and metastasis. AIM: This study is aimed at investigating the behavior of the basement membrane in oral squamous cell carcinomas with different malignancy scores, which were obtained through the immunohistochemical expression of the laminin, a glycoprotein present in the basement membrane. STUDY DESING: History cross-sectional cohort. MATERIAL AND METHOD: Thirty-one cases of oral squamous cell carcinoma were subjected to histological grading of malignant tumors. The immunohistochemical expression of the laminin in lesions bearing different scores of malignancy was evaluated according to intensity and integrity, using the Streptavidin-Biotin complex method. RESULTS: We noticed significant differences in the media between intensity and continuity laminin expression in relation to different grades of malignancy. CONCLUSION: Different expressions of laminin, a glycoprotein present in basement membranes were evident in oral cell carcinomas within different grades of histological malignancy.

Merosin-positive congenital muscular dystrophy: neuroimaging findings

Congenital muscle dystrophy (CMD) is a heterogeneous group of autosomal recessive myopathies. It is known that CMD may affect the central nervous system (CNS). Some authors have shown that merosin-negative CMD patients may have encephalic metabolic disturbances. In order to study metabolic changes within the brain, the authors performed a magnetic resonance spectroscopy (MRS) study in a 1-year-old girl with merosin-positive CMD (MP-CMD). MRS of brain demonstrated that NAA/Cr ratio was decreased (1.52), while Cho/Cr ratio was increased (1.78). These findings suggest that metabolic changes in CNS can also be found in patients with MP-CMD.

A distrofia muscular congênita (DMC) é um grupo heterogêneo de miopatias autossômicas recessivas que também podem afetar o sistema nervoso central (SNC). Alguns autores mostraram previamente que pacientes com DMC por deficiência da merosina podem apresentar alterações metabólicas no encéfalo. Com o objetivo de estudar as possíveis alterações metabólicas no SNC, os autores realizaram um estudo por ressonância magnética com espectroscopia em uma paciente de 1 ano com DMC sem deficiência da merosina. A razão NAA/Cr estava reduzida (1,52), enquanto que a razão Cho/Cr estava aumentada (1,78). Estes achados sugerem que alterações metabólicas no SNC também podem ser encontradas em pacientes com DMC merosina-positiva.

Alterations in proteins of bone marrow extracellular matrix in undernourished mice

The objective of the present study was to determine the effect of protein malnutrition on the glycoprotein content of bone marrow extracellular matrix (ECM). Two-month-old male Swiss mice were submitted to protein malnutrition with a low-protein diet containing 4 percent casein as compared to 20 percent casein in the control diet. When the experimental group had attained a 20 percent loss of their original body weight, we extracted the ECM proteins from bone marrow with PBS buffer, and analyzed ECM samples by SDS-PAGE (7.5 percent) and ECL Western blotting. Quantitative differences were observed between control and experimental groups. Bone marrow ECM from undernourished mice had greater amounts of extractable fibronectin (1.6-fold increase) and laminin (4.8-fold increase) when compared to the control group. These results suggest an association between fluctuations in the composition of the hematopoietic microenvironment and altered hematopoiesis observed in undernourished mice

Rigid spine syndrome: case report

We describe a patient who had difficulty in walking since toddling stage and presented proximal upper and lower member weakness which have evolved to a progressive limitation of neck and trunk flexure, compatible with rigid spine syndrome. The serum muscle enzymes were somewhat elevated and the electromyography showed a myopatic change. The muscle biopsy demonstrated an active and chronic myopathy. The DNA analysis through PCR did not display any abnormality for dystrophin gene. The dystrophin by immnofluorescence was present in all fibers, but some interruptions were found in the plasma membrane giving it the appearance of a rosary. The test for merosin was normal.

Expressäo de laminina no germe dental do primeiro molar de ratos em desenvolvimento / Expression of laminin in the germ of the first molar tooth of developing rats

A regulaçäo da diferenciaçäo e do desenvolvimento dos vários tecidos que formam o germe dental ainda näo está bem estabelecida. Tem-se atribuído à membrana basal e a seus componentes esse papel. O objetivo deste trabalho foi investigar a distribuiçäo de laminina no germe dental do primeiro molar de ratos, utilizando um anticorpo policlonal antilaminina. A análise imunohistoquímica mostrou que a lamina é expressa de forma contínua nas membranas basais dos epitélios interno e externo do orgäo do esmalte, nos pequenos vasos sangüíneos e nas fibrilas nervosas localizadas na papila e no folículo dentário de ratos recém-nascidos. Nas regiöes em que ocorreu diferenciaçäo de células mesenquimais em odontoblastos e de células do epitélio interno em ameloblastos, a expressäo de laminina näo foi mais observada. Essas mudanças sugerem que a expressäo de laminina na membrana basal está relacionada com a diferenciaçäo celular e com a secreçäo de matriz orgânica

role of laminin immunolabelling in the management of basal cell carcinoma

The aim of the present study was to assess laminin production and distribution in basal cell carcinomas in relation to clinical presentation and behaviour. Of 30 cases with B.C.C., 12 expressed a peripheral linear pattern of laminin immunostaining [type I] whereas 18 cases expressed short discontinuous intratumoral labelling in relation to cell membranes [type II]. Type I labelling was recorded more frequently in primary cases, while type II labelling was more frequently recorded in recurrent and extensive cases. Based on correlation with clinical data, it was concluded that type II tumors with type II laminin immunolabelling are more aggressive and should be subjected to prompt surgical excision

Hepatic structural glycoprotein metabolism in liver cirrhosis and its correlation with different disease parameters

This study was conducted on 39 patients diagnosed as liver cirrhosis and 20 age and sex matched controls. Twenty-six patients had clinical and/or sonographic evidence of ascites, but only 22 could be subjected to sampling of their ascites. Serum laminin [LPI] was estimated in all patients and controls, while ascitic fluid LPI was assessed only in 22 samples. Mean value +/- SD of serum LPI in patients [2.36 +/- 0.8] was significantly higher than control group [1.47 +/- 0.25]. Ascitic fluid laminin showed a significant correlation to serum laminin. A ratio of ascitic fluid LPI/serum LPI was calculated with a mean +/- SD of 0.944 +/- 0.45. Mean value of +/- SD of serum LPI in patients with history of gastrointestinal [GI] bleeding [2.69 +/- 1.13] was significantly higher than in patients with no such history [2 +/- 0.92]. Serum LPI was also significantly correlated to the quantitative reduction of hepatic right and left robe spans as measured by ultrasonography. The results suggested that serum LPI values were correlated with the degree of hepatic fibrosis. The study provided a potential new risk factor for GI bleeding from esophageal varices. The study also provided, for the first time, the ratio of ascitic fluid LPI/serum LPI in patients with liver cirrhosis

Avances en la estructura y función de la membrana basal / Advances in basement membrane structure and function

La zona de membrana basal es una estructura compleja. Está constituída por la membrana plasmática de la célula basal, lámina lúcida, lámina densa y región de anclaje; cada una de ellas posee diversos componentes moleculares. Sus principales funciones biológicas son actuar como soporte y barrera mecánica, permite la fijación dermoepidérmica, regular la filtración y permeabilidad y guiar la regeneración tisular

Serum laminin in hepatosplenic human schistosomiasis

Serum laminin level was measured in chronic schistosomiasis. A significant increase in the mean serum laminin levels was observed in patients with hepatosplenic (HS) schistosomiasis (2,57 ñ 0,83U/ml), as compared to those in patients with the hepatointestinal (HI) form of the disease (1,38 ñ 0,45-U/ml) and in the control group (1,15 ñ 0,31 U/ml). In the HS patients there was a significant direct relatiom between serum laminin and percutaneous splenic pulp pressure (r = 0,68). These findigs are compatible with an increased production of lamin in hepatosplenic schistosomiasis with may be related to the observed enlarged liver and spleen basement membranes in such disease

Inmunolocalización de la fibronectina, laminina y macrófagos en membranas vitreorretinianas de la retinopatia diabética proliferativa / Fibronectin, laminin and macrophages in proliferative diabetic retinopathy

A retinopatia diabética proliferativa (RDP) se caracteriza por uma proliferaçäo fibrovascular intraocular com a formaçäo de membranas prerretinianas que predispöem a desprendimentos de retina tracionais e hemorragias no vítreo. Ainda sem consideraçöes os estudos morfológicos acerca destas membranas, sua patogênese é apenas conhecida. Recentemente tem-se descrito a possível participaçäo de fatores tumoraes e de matriz extracelular na angiogênesis e em sua formaçäo. Com o objetivo de identificar a distribuiçäo de alguns destes possíveis fatores, temos realizado um estudo imunocitoquímico para a detecçäo da Laminina (LN) e da Fibronectina (FN), em uma série de 18 membranas fibrovasculares, obtidas de olhos portadores de RDP e submetidos à vitrectomia. Ambas glucoproteínas apresentam uma localizaçäo vascular; a FN em disposiçäo luminal, na LN em disposiçäo basal com respeito às células endoteliais dos capilares. A imunolocalizaçäo também tem confirmado a presença de macrófagos, sugerindo que o processo proliferativo intraocular pode ser considerado como uma forma modificada de formaçäo cicatricial

Fibronectin and laminin expression in breast cancer and lymph node metastases: lack of correlation with fibronectin plasmatic levels

Ya que las alteraciones de las membranas basales (BMs) se asocian a procesos de invasión y metástasis nos hemos propuesto estudiar si dos componentes de dichas membranas, la laminina (LA) y la fibronectina (FN) se ebcuentran alterados en carcinomas de mama humano y en sus respectivas metástasis ganglionares. Estos datos se analizaron en función del estadío clínico, grado histológico de malignidad y número de metástasis ganglionares. Se ha estudiado con el método de preoxidasa-antiperoxidasa la presencia y distribución de FN y LA en una serie de 25 tumores primários de mama, 22 nódulos linfáticos metastáscios y 10 biopsias de tejido mamario no tumoral. La tinción de las BMs par FN fue entre moderada y extensa en17/25 carcinomas, aunque éstas mostraron siempre discontinuidades. La tinción para LA de las BMs fue negativa o muy pobre e irregular en la mayoría de los casos. Se observó que 6/9 pacientes agrupados en estadio III (TNM) presentaron tinción pobre o negativa para FN en las BMs de los tumores primarios mientras que solo 1/15 de los pacientes agrupados en estadio I y II fue negativo para la misma glicoproteína. La desaparición de LA de las BMs se correlacionaría con el grado histológico de malignidad. En 19/22 metástasis ganglionares se destacó una marcada tinción intracelular par FN. La expresión de FN y LA en las BMs de metástasis ganglionares y los tumores primários correspondientes fue similar. No se hallo correlación entre los niveles de FN plasmática de las 19 pacientes estudiadas y la expresión de FN tisular. Este estudio demuestra alteraciones cuali y cuantitativas en la expresión de FN y LA en el tumor primario de mama y sugiere que los niveles de FN plasmáticos serían independientes de la expresión de la FN tisular tumoral