Identificação dos colágenos I, III, IV e a-SMA e participação dos miofibroblastos no processo fibrótico das endometroses equinas / Identification of collagens types I, III, IV and a-SMA and the participation of myofibroblasts in the fibrotic process in equine endometrosis

A endometrose é uma alteração degenerativa das glândulas uterinas e do estroma circundante, caracterizada pelo arranjo periglandular de miofibroblastos e pela deposição de matriz extracelular (ECM). O presente trabalho objetivou avaliar a expressão de colágenos tipos I, III e IV e α-actina de músculo liso (α-SMA) nas endometroses equinas, procurando esclarecer a participação dos miofibroblastos na progressão desses processos. Foram utilizadas 24 biópsias uterinas com diagnóstico de endometrose, recebidas pelo Serviço de Patologia Veterinária e de Reprodução Animal da FMVZ, Unesp, Botucatu, SP. Cortes histológicos foram submetidos às técnicas histoquímicas de tricrômico de Masson, picrosirius red sob luz polarizada e ácido periódico de Schiff (PAS) e imuno-histoquímicas para os três tipos de colágeno citados e α-SMA. Ainda, traçou-se um paralelo entre a técnica de picrosirius red e a imunomarcação dos colágenos tipos I e III. A análise histológica revelou que as fibras de colágeno denso correspondem ao colágeno tipo I, predominantes nas endometroses inativa e inativa destrutiva. As fibras de colágeno frouxo correspondem ao colágeno tipo III, predominantes nas endometroses ativas e ativas destrutivas. Nesses mesmos processos, a membrana basal revelou espessamento, aparentemente não relacionado ao colágeno tipo IV, e uma maior imunomarcação de miofibroblastos periglandulares em relação às endometroses inativa e inativa destrutiva. Dessa forma, nota-se que os miofibroblastos estão relacionados ao aumento na deposição de colágeno tipo III nos ninhos fibróticos ativos.(AU)

Endometriosis is a degenerative change of the uterine glands and surrounding stroma, characterized by periglandular arrangement of myofibroblasts and deposition of extracellular matrix (ECM). The aim of this study was to evaluate the expression of collagen type I, III and IV and α-smooth muscle actin (α-SMA) in equine endometriosis, and investigate the role of myofibroblasts in the progression of these processes. A parallel was made with histochemical techniques of Masson's trichrome, Picrosirius Red under polarized light and Periodic Acid-Schiff (PAS). Twenty four uterine biopsies received by the Veterinary Pathology Service and Animal Reproduction of FMVZ, UNESP, Botucatu, SP, were diagnosed with endometriosis. Histological analysis revealed that the orange dense collagen fibers correspond to type I collagen, being prevalent in inactive and inactive destructive endometriosis. The green loose collagen fibers correspond to type III collagen, and are predominant in active and active destructive endometriosis. In the same processes, a greater amount of periglandular myofibroblasts were observed in comparison to inactive and inactive destructive endometriosis. The presence of these cells in active processes are strongly related to an increased deposition of collagen type III in fibrotic nests. Regarding the basement membrane, the active destructive and active endometriosis shows thickening, apparently not related to an increase in expression of type IV collagen. The active destructive and inactive destructive endometriosis exhibited disruption areas in type IV collagen fibers. Thus, it is noted that the myofibroblasts are related to increased deposition of type III collagen in active fibrotic nests.(AU)

Identificação dos colágenos I, III, IV e α-SMA e participação dos miofibroblastos no processo fibrótico das endometroses equinas / Identification of collagens types I, III, IV and α-SMA and the participation of myofibroblasts in the fibrotic process in equine endometrosis

A endometrose é uma alteração degenerativa das glândulas uterinas e do estroma circundante, caracterizada pelo arranjo periglandular de miofibroblastos e pela deposição de matriz extracelular (ECM). O presente trabalho objetivou avaliar a expressão de colágenos tipos I, III e IV e α-actina de músculo liso (α-SMA) nas endometroses equinas, procurando esclarecer a participação dos miofibroblastos na progressão desses processos. Foram utilizadas 24 biópsias uterinas com diagnóstico de endometrose, recebidas pelo Serviço de Patologia Veterinária e de Reprodução Animal da FMVZ, Unesp, Botucatu, SP. Cortes histológicos foram submetidos às técnicas histoquímicas de tricrômico de Masson, picrosirius red sob luz polarizada e ácido periódico de Schiff (PAS) e imuno-histoquímicas para os três tipos de colágeno citados e α-SMA. Ainda, traçou-se um paralelo entre a técnica de picrosirius red e a imunomarcação dos colágenos tipos I e III. A análise histológica revelou que as fibras de colágeno denso correspondem ao colágeno tipo I, predominantes nas endometroses inativa e inativa destrutiva. As fibras de colágeno frouxo correspondem ao colágeno tipo III, predominantes nas endometroses ativas e ativas destrutivas. Nesses mesmos processos, a membrana basal revelou espessamento, aparentemente não relacionado ao colágeno tipo IV, e uma maior imunomarcação de miofibroblastos periglandulares em relação às endometroses inativa e inativa destrutiva. Dessa forma, nota-se que os miofibroblastos estão relacionados ao aumento na deposição de colágeno tipo III nos ninhos fibróticos ativos.(AU)

Endometriosis is a degenerative change of the uterine glands and surrounding stroma, characterized by periglandular arrangement of myofibroblasts and deposition of extracellular matrix (ECM). The aim of this study was to evaluate the expression of collagen type I, III and IV and α-smooth muscle actin (α-SMA) in equine endometriosis, and investigate the role of myofibroblasts in the progression of these processes. A parallel was made with histochemical techniques of Masson's trichrome, Picrosirius Red under polarized light and Periodic Acid-Schiff (PAS). Twenty four uterine biopsies received by the Veterinary Pathology Service and Animal Reproduction of FMVZ, UNESP, Botucatu, SP, were diagnosed with endometriosis. Histological analysis revealed that the orange dense collagen fibers correspond to type I collagen, being prevalent in inactive and inactive destructive endometriosis. The green loose collagen fibers correspond to type III collagen, and are predominant in active and active destructive endometriosis. In the same processes, a greater amount of periglandular myofibroblasts were observed in comparison to inactive and inactive destructive endometriosis. The presence of these cells in active processes are strongly related to an increased deposition of collagen type III in fibrotic nests. Regarding the basement membrane, the active destructive and active endometriosis shows thickening, apparently not related to an increase in expression of type IV collagen. The active destructive and inactive destructive endometriosis exhibited disruption areas in type IV collagen fibers. Thus, it is noted that the myofibroblasts are related to increased deposition of type III collagen in active fibrotic nests.(AU)

Avaliação da composição molecular da cápsula anterior da lente de cães idosos com catarata de alto risco / Molecular evaluation of the anterior lens capsule of older dogs suffering from high risk cataracts

Atualmente, a cápsula anterior e o epitélio da lente tem sido cada vez mais estudados, com o intuito de reduzir as possíveis complicações do pós-operatório da remoção da catarata, tal como a opacidade da cápsula posterior, alteração ocasionada principalmente pela diferenciação e migração das células do epitélio lenticular para a cápsula posterior da lente. O objetivo deste estudo foi analisar a composição molecular da cápsula anterior da lente pela técnica histoquímica de PAS (avaliação de proteoglicanos) e picrosirius red (avaliação de colágeno IV), em cães idosos com catarata diabética e não diabética do tipo hipermadura, submetidos ao uso ou não de azul de tripano a 0,1 % durante a facoemulsificação. Vinte e sete cães foram estudados, incluindo 21 fêmeas e 6 machos, de 8 a 12 anos de idade (média = 9,6 anos), de diversas raças e divididos em 2 grupos: GC (catarata hipermadura) e GCD (catarata diabética). Os resultados das análises realizadas mostraram que ambas as amostras, tanto as provenientes das cataratas hipermaduras, quanto das diabéticas, apresentam semelhante composição molecular de proteoglicanos e colágeno IV e isto independente da utilização de azul de tripano a 0,1 %. Conclui-se, portanto, que se os resultados obtidos forem decorrentes de alterações provocadas pelo rápido metabolismo da catarata diabética e pela cronicidade da catarata hipermadura sugere-se que o comprometimento da estrutura capsular seja de intensidade equivalente e, por consequência, que isto também possa prejudicar o metabolismo das células do epitélio anterior da lente, diminuindo assim a incidência da opacidade da cápsula posterior de cães com catarata diabética e hipermadura submetidos à facoemulsificação.(AU)

Nowadays, the anterior lens capsule and its epithelium have been being frequently studied aiming to reduce the incidence of posterior lens capsule opacity, a complication that frequently occurs after surgical removal of cataracts, due to epithelium cells differentiation and migration to the posterior pole. The objective of this study was to evaluate by histochemistry (PAS and picrosirius red) analysis two important molecular components of the anterior lens capsule (proteoglycans and type IV collagen) in older diabetic and non-diabetic dogs, with diabetic and hypermature cataracts, after phacoemulsification surgery utilizing 0.1% trypan blue or not. Twenty seven dogs, including 21 female and 6 male dogs, with ages varying from 8 to 12 years old (mean = 9.6 yo) of different breeds were studied. The animals were divided into 2 groups: GC (hypermature cataracts) and GCD (diabetic cataracts). Results showed that, besides their different pathophysiologies, both types of capsules studied (diabetic and hypermature ones) presented the same molecular composition of proteoglycans and type IV collagen, since no statistical significant differences were observed. In addition, 0.1% trypan blue was not capable to induce any other evident alteration for the samples. In conclusion, our findings suggest that, if the results consist in alteration induced by the aggressive metabolism of the diabetic cataract or the chronicity of the hypermature one, it is of the same intensity and independent of the use of 0.1% trypan blue. It is also possible to suggest that this alteration must be capable to compromise lens epithelium cell metabolism, which should probably favour future lens posterior capsule studies.(AU)

Avaliação da composição molecular da cápsula anterior da lente de cães idosos com catarata de alto risco / Molecular evaluation of the anterior lens capsule of older dogs suffering from high risk cataracts

Atualmente, a cápsula anterior e o epitélio da lente tem sido cada vez mais estudados, com o intuito de reduzir as possíveis complicações do pós-operatório da remoção da catarata, tal como a opacidade da cápsula posterior, alteração ocasionada principalmente pela diferenciação e migração das células do epitélio lenticular para a cápsula posterior da lente. O objetivo deste estudo foi analisar a composição molecular da cápsula anterior da lente pela técnica histoquímica de PAS (avaliação de proteoglicanos) e picrosirius red (avaliação de colágeno IV), em cães idosos com catarata diabética e não diabética do tipo hipermadura, submetidos ao uso ou não de azul de tripano a 0,1 % durante a facoemulsificação. Vinte e sete cães foram estudados, incluindo 21 fêmeas e 6 machos, de 8 a 12 anos de idade (média = 9,6 anos), de diversas raças e divididos em 2 grupos: GC (catarata hipermadura) e GCD (catarata diabética). Os resultados das análises realizadas mostraram que ambas as amostras, tanto as provenientes das cataratas hipermaduras, quanto das diabéticas, apresentam semelhante composição molecular de proteoglicanos e colágeno IV e isto independente da utilização de azul de tripano a 0,1 %. Conclui-se, portanto, que se os resultados obtidos forem decorrentes de alterações provocadas pelo rápido metabolismo da catarata diabética e pela cronicidade da catarata hipermadura sugere-se que o comprometimento da estrutura capsular seja de intensidade equivalente e, por consequência, que isto também possa prejudicar o metabolismo das células do epitélio anterior da lente, diminuindo assim a incidência da opacidade da cápsula posterior de cães com catarata diabética e hipermadura submetidos à facoemulsificação.(AU)

Nowadays, the anterior lens capsule and its epithelium have been being frequently studied aiming to reduce the incidence of posterior lens capsule opacity, a complication that frequently occurs after surgical removal of cataracts, due to epithelium cells differentiation and migration to the posterior pole. The objective of this study was to evaluate by histochemistry (PAS and picrosirius red) analysis two important molecular components of the anterior lens capsule (proteoglycans and type IV collagen) in older diabetic and non-diabetic dogs, with diabetic and hypermature cataracts, after phacoemulsification surgery utilizing 0.1% trypan blue or not. Twenty seven dogs, including 21 female and 6 male dogs, with ages varying from 8 to 12 years old (mean = 9.6 yo) of different breeds were studied. The animals were divided into 2 groups: GC (hypermature cataracts) and GCD (diabetic cataracts). Results showed that, besides their different pathophysiologies, both types of capsules studied (diabetic and hypermature ones) presented the same molecular composition of proteoglycans and type IV collagen, since no statistical significant differences were observed. In addition, 0.1% trypan blue was not capable to induce any other evident alteration for the samples. In conclusion, our findings suggest that, if the results consist in alteration induced by the aggressive metabolism of the diabetic cataract or the chronicity of the hypermature one, it is of the same intensity and independent of the use of 0.1% trypan blue. It is also possible to suggest that this alteration must be capable to compromise lens epithelium cell metabolism, which should probably favour future lens posterior capsule studies.(AU)

Avaliação da composição molecular da cápsula anterior da lente de cães idosos com catarata de alto risco / Molecular evaluation of the anterior lens capsule of older dogs suffering from high risk cataracts

Atualmente, a cápsula anterior e o epitélio da lente tem sido cada vez mais estudados, com o intuito de reduzir as possíveis complicações do pós-operatório da remoção da catarata, tal como a opacidade da cápsula posterior, alteração ocasionada principalmente pela diferenciação e migração das células do epitélio lenticular para a cápsula posterior da lente. O objetivo deste estudo foi analisar a composição molecular da cápsula anterior da lente pela técnica histoquímica de PAS (avaliação de proteoglicanos) e picrosirius red (avaliação de colágeno IV), em cães idosos com catarata diabética e não diabética do tipo hipermadura, submetidos ao uso ou não de azul de tripano a 0,1 % durante a facoemulsificação. Vinte e sete cães foram estudados, incluindo 21 fêmeas e 6 machos, de 8 a 12 anos de idade (média = 9,6 anos), de diversas raças e divididos em 2 grupos: GC (catarata hipermadura) e GCD (catarata diabética). Os resultados das análises realizadas mostraram que ambas as amostras, tanto as provenientes das cataratas hipermaduras, quanto das diabéticas, apresentam semelhante composição molecular de proteoglicanos e colágeno IV e isto independente da utilização de azul de tripano a 0,1 %. Conclui-se, portanto, que se os resultados obtidos forem decorrentes de alterações provocadas pelo rápido metabolismo da catarata diabética e pela cronicidade da catarata hipermadura sugere-se que o comprometimento da estrutura capsular seja de intensidade equivalente e, por consequência, que isto também possa prejudicar o metabolismo das células do epitélio anterior da lente, diminuindo assim a incidência da opacidade da cápsula posterior de cães com catarata diabética e hipermadura submetidos à facoemulsificação.

Nowadays, the anterior lens capsule and its epithelium have been being frequently studied aiming to reduce the incidence of posterior lens capsule opacity, a complication that frequently occurs after surgical removal of cataracts, due to epithelium cells differentiation and migration to the posterior pole. The objective of this study was to evaluate by histochemistry (PAS and picrosirius red) analysis two important molecular components of the anterior lens capsule (proteoglycans and type IV collagen) in older diabetic and non-diabetic dogs, with diabetic and hypermature cataracts, after phacoemulsification surgery utilizing 0.1% trypan blue or not. Twenty seven dogs, including 21 female and 6 male dogs, with ages varying from 8 to 12 years old (mean = 9.6 yo) of different breeds were studied. The animals were divided into 2 groups: GC (hypermature cataracts) and GCD (diabetic cataracts). Results showed that, besides their different pathophysiologies, both types of capsules studied (diabetic and hypermature ones) presented the same molecular composition of proteoglycans and type IV collagen, since no statistical significant differences were observed. In addition, 0.1% trypan blue was not capable to induce any other evident alteration for the samples. In conclusion, our findings suggest that, if the results consist in alteration induced by the aggressive metabolism of the diabetic cataract or the chronicity of the hypermature one, it is of the same intensity and independent of the use of 0.1% trypan blue. It is also possible to suggest that this alteration must be capable to compromise lens epithelium cell metabolism, which should probably favour future lens posterior capsule studies.

Three days of intermittent stretching after muscle disuse alters the proteins involved in force transmission in muscle fibers in weanling rats.

The aim of this study was to determine the effects of intermittent passive manual stretching on various proteins involved in force transmission in skeletal muscle. Female Wistar weanling rats were randomly assigned to 5 groups: 2 control groups containing 21- and 30-day-old rats that received neither immobilization nor stretching, and 3 test groups that received 1) passive stretching over 3 days, 2) immobilization for 7 days and then passive stretching over 3 days, or 3) immobilization for 7 days. Maximal plantar flexion in the right hind limb was imposed, and the stretching protocol of 10 repetitions of 30 s stretches was applied. The soleus muscles were harvested and processed for HE and picrosirius staining; immunohistochemical analysis of collagen types I, III, IV, desmin, and vimentin; and immunofluorescence labeling of dystrophin and CD68. The numbers of desmin- and vimentin-positive cells were significantly decreased compared with those in the control following immobilization, regardless of whether stretching was applied (P<0.05). In addition, the semi-quantitative analysis showed that collagen type I was increased and type IV was decreased in the immobilized animals, regardless of whether the stretching protocol was applied. In conclusion, the largest changes in response to stretching were observed in muscles that had been previously immobilized, and the stretching protocol applied here did not mitigate the immobilization-induced muscle changes. Muscle disuse adversely affected several proteins involved in the transmission of forces between the intracellular and extracellular compartments. Thus, the 3-day rehabilitation period tested here did not provide sufficient time for the muscles to recover from the disuse maladaptations in animals undergoing postnatal development.

Three days of intermittent stretching after muscle disuse alters the proteins involved in force transmission in muscle fibers in weanling rats

The aim of this study was to determine the effects of intermittent passive manual stretching on various proteins involved in force transmission in skeletal muscle. Female Wistar weanling rats were randomly assigned to 5 groups: 2 control groups containing 21- and 30-day-old rats that received neither immobilization nor stretching, and 3 test groups that received 1) passive stretching over 3 days, 2) immobilization for 7 days and then passive stretching over 3 days, or 3) immobilization for 7 days. Maximal plantar flexion in the right hind limb was imposed, and the stretching protocol of 10 repetitions of 30 s stretches was applied. The soleus muscles were harvested and processed for HE and picrosirius staining; immunohistochemical analysis of collagen types I, III, IV, desmin, and vimentin; and immunofluorescence labeling of dystrophin and CD68. The numbers of desmin- and vimentin-positive cells were significantly decreased compared with those in the control following immobilization, regardless of whether stretching was applied (P<0.05). In addition, the semi-quantitative analysis showed that collagen type I was increased and type IV was decreased in the immobilized animals, regardless of whether the stretching protocol was applied. In conclusion, the largest changes in response to stretching were observed in muscles that had been previously immobilized, and the stretching protocol applied here did not mitigate the immobilization-induced muscle changes. Muscle disuse adversely affected several proteins involved in the transmission of forces between the intracellular and extracellular compartments. Thus, the 3-day rehabilitation period tested here did not provide sufficient time for the muscles to recover from the disuse maladaptations in animals undergoing postnatal development.

Analysis of the frequency and nature of hyaline ring granulomas in inflammatory odontogenic cysts.

AIM: To determine the prevalence of hyaline ring granulomas (HRGs) in a large case series of inflammatory odontogenic cysts, and to investigate the nature of these structures. METHODOLOGY: All records from the patients diagnosed with inflammatory odontogenic cysts between January 1970 and April 2009 were reviewed. Histologic sections were evaluated by light microscopy and cases with HRGs for which sufficient biological material was available were submitted to histochemical analysis (Masson's trichrome) and immunohistochemistry (CD34, CD68 and collagen IV). RESULTS: Twenty-two (3.3%) of the 661 cases of inflammatory odontogenic cysts diagnosed during the study period presented HRGs. The relative frequency of HRGs was higher amongst residual radicular cysts (6.1%), followed by paradental cysts (5.6%) and radicular cysts (3.0%). HRGs appeared as roughly circular homogeneous/fibrillar masses in 14 (63.6%) cases and as round structures enclosing amorphous material in 3 (13.6%) cases. Most (77.8%) roughly circular homogeneous/fibrillar masses were positive for collagen, whereas all (100.0%) round structures enclosing amorphous material were negative for this protein. Immunohistochemistry showed that most mononucleated cells and all multinucleated giant cells were positive for CD68, but negative for CD34, in all cases. In addition, collagen IV immunostaining was negative in amorphous structures and weakly positive in homogeneous/fibrillar masses. CONCLUSIONS: The present results suggest a very low frequency of HRGs in inflammatory odontogenic cysts and support the hypothesis that these structures arise from the implantation of foreign material, most likely food particles of plant or vegetable origin. The diverse microscopic features of HRG possibly represent different developmental stages of this structure.

Immunohistochemical analysis of collagen content and types in the rectus abdominis muscle of cadavers of different ages.

PURPOSE: To assess the collagen content and types in the rectus abdominis muscle of cadavers of different ages. METHODS: Forty fresh adult male cadavers, at room temperature, were obtained from the Institute of Legal Medicine of Franca and dissected within 24 hours of death. The cadavers were divided into two groups: Group A (n=20), 18 to 30 years of age, and Group B (n=20), 31 to 60 years of age. Bilateral incisions were made in the middle portion of anterior rectus sheath 3 cm superiorly and 2 cm inferiorly to the umbilicus and four fragments of the rectus abdominis muscle were dissected. The samples were fixed in 10% buffered formalin and sent for immunohistochemical analysis to determine collagen content and types. RESULTS: Immunohistochemical results revealed higher amounts of type I and type III collagen in Group A. However, no difference in the amount of type IV collagen was found between the groups. CONCLUSION: The amount of type I and type III collagen was higher in group A.

Comparative analysis of the immunohistochemical expression of collagen IV, MMP-9, and TIMP-2 in odontogenic cysts and tumors.

OBJECTIVE: The aim of this study was to evaluate the immunohistochemical expression of collagen IV, matrix metalloproteinase (MMP) 9 and tissue inhibitor of MMP (TIMP) 2 in dentigerous cysts (DCs), radicular cysts (RCs), keratocystic odontogenic tumors (KOTs), and ameloblastomas. STUDY DESIGN: Twenty cases of DCs, 20 RCs, 20 KOTs, and 20 ameloblastomas were selected and analyzed by immunohistochemistry. RESULTS: Most DCs and RCs showed continuous and >50% staining for collagen IV in the basement membrane of the epithelium, whereas predominantly discontinuous thin and ≤ 50% staining was observed in KOTs and ameloblastomas, with a significant difference in staining percentage (P < .001). MMP-9 was diffusely distributed and localized in both epithelial and mesenchymal cells of all of the lesions analyzed. The staining percentage was higher in the epithelium (P = .058) and mesenchyme (P = .005) of KOTs and ameloblastomas. Moreover, the distribution pattern, location, and percentage of expression of TIMP-2 were similar in the lesions studied, except for ameloblastoma, with a significant difference in staining percentage (P < .001). CONCLUSION: These results demonstrate that the interaction between collagen IV, MMP-9, and TIMP-2 is an important factor for the establishment of differences in the biologic behavior of the odontogenic cysts and tumors studied.

Supraorganized collagen enhances Schwann cell reactivity and organization in vitro.

We investigated the reactivity and expression of basal lamina collagen by Schwann cells (SCs) cultivated on a supraorganized bovine-derived collagen substrate. SC cultures were obtained from sciatic nerves of neonatal Sprague-Dawley rats and seeded on 24-well culture plates containing collagen substrate. The homogeneity of the cultures was evaluated with an SC marker antibody (anti-S-100). After 1 week, the cultures were fixed and processed for immunocytochemistry by using antibodies against type IV collagen, S-100 and p75NTR (pan neurotrophin receptor) and for scanning electron microscopy (SEM). Positive labeling with antibodies to the cited molecules was observed, indicating that the collagen substrate stimulates SC alignment and adhesion (collagen IV labeling - organized collagen substrate: 706.33 ± 370.86, non-organized collagen substrate: 744.00 ± 262.09; S-100 labeling - organized collagen: 3809.00 ± 120.28, non-organized collagen: 3026.00 ± 144.63, P < 0.05) and reactivity (p75NTR labeling - organized collagen: 2156.33 ± 561.78, non-organized collagen: 1424.00 ± 405.90, P < 0.05; means ± standard error of the mean in absorbance units). Cell alignment and adhesion to the substrate were confirmed by SEM analysis. The present results indicate that the collagen substrate with an aligned suprastructure, as seen by polarized light microscopy, provides an adequate scaffold for SCs, which in turn may increase the efficiency of the nerve regenerative process after in vivo repair.

Supraorganized collagen enhances Schwann cell reactivity and organization in vitro

We investigated the reactivity and expression of basal lamina collagen by Schwann cells (SCs) cultivated on a supraorganized bovine-derived collagen substrate. SC cultures were obtained from sciatic nerves of neonatal Sprague-Dawley rats and seeded on 24-well culture plates containing collagen substrate. The homogeneity of the cultures was evaluated with an SC marker antibody (anti-S-100). After 1 week, the cultures were fixed and processed for immunocytochemistry by using antibodies against type IV collagen, S-100 and p75NTR (pan neurotrophin receptor) and for scanning electron microscopy (SEM). Positive labeling with antibodies to the cited molecules was observed, indicating that the collagen substrate stimulates SC alignment and adhesion (collagen IV labeling - organized collagen substrate: 706.33 ± 370.86, non-organized collagen substrate: 744.00 ± 262.09; S-100 labeling - organized collagen: 3809.00 ± 120.28, non-organized collagen: 3026.00 ± 144.63, P < 0.05) and reactivity (p75NTR labeling - organized collagen: 2156.33 ± 561.78, non-organized collagen: 1424.00 ± 405.90, P < 0.05; means ± standard error of the mean in absorbance units). Cell alignment and adhesion to the substrate were confirmed by SEM analysis. The present results indicate that the collagen substrate with an aligned suprastructure, as seen by polarized light microscopy, provides an adequate scaffold for SCs, which in turn may increase the efficiency of the nerve regenerative process after in vivo repair.

Sequence variants in COL4A1 and COL4A2 genes in Ecuadorian families with keratoconus.

PURPOSE: Keratoconus (KTCN) is a non-inflammatory, usually bilateral disorder of the eye which results in the conical shape and the progressive thinning of the cornea. Several studies have suggested that genetic factors play a role in the etiology of the disease. Several loci were previously described as possible candidate regions for familial KTCN; however, no causative mutations in any genes have been identified for any of these loci. The purpose of this study was to evaluate role of the collagen genes collagen type IV, alpha-1 (COL4A1) and collagen type IV, alpha-2 (COL4A2) in KTCN in Ecuadorian families. METHODS: COL4A1 and COL4A2 in 15 Ecuadorian KTCN families were examined with polymerase chain reaction amplification, and direct sequencing of all exons, promoter and intron-exon junctions was performed. RESULTS: Screening of COL4A1 and COL4A2 revealed numerous alterations in coding and non-coding regions of both genes. We detected three missense substitutions in COL4A1: c.19G>C (Val7Leu), c.1663A>C (Thr555Pro), and c.4002A>C (Gln1334His). Five non-synonymous variants were identified in COL4A2: c.574G>T (Val192Phe), c.1550G>A (Arg517Lys), c.2048G>C (Gly683Ala), c.2102A>G (Lys701Arg), and c.2152C>T (Pro718Ser). None of the identified sequence variants completely segregated with the affected phenotype. The Gln1334His variant was possibly damaging to protein function and structure. CONCLUSIONS: This is the first mutation screening of COL4A1 and COL4A2 genes in families with KTCN and linkage to a locus close to these genes. Analysis of COL4A1 and COL4A2 revealed no mutations indicating that other genes are involved in KTCN causation in Ecuadorian families.

Vocal polyps: clinical, morphological, and immunohistochemical aspects.

OBJECTIVES: To study the clinical, morphological, and immunohistochemical characteristics of vocal polyps. STUDY DESIGN: Prospective and retrospective. METHODS: Clinical study: 76 medical charts from patients with polyps were reviewed. Histology study: in 42 slides from surgical specimens, the following were analyzed: epithelium, basal membrane, and lamina propria. In the transmission and scanning electron microscopy (TEM and SEM) studies, eight new cases of polyps were included. An immunohistochemical study was carried out in the 42 specimens, using antibody antifibronectin, antilaminin, and anticollagen IV. RESULTS: Genders--43% males and 57% females; age range--between 21 and 40 years (36.85%); and between 41 and 60 years of age (51.31%); smoking and drinking-reported by 39 and 15 patients, respectively; associated symptoms-vocal abuse (61%), gastroesophageal (47%), and nasosinusal symptoms (32%); occupation--teachers (24.0%) and maids (18.0%). Histology--epithelial hyperplasia (31.71%), hyperkeratosis, (14.28%), edema (100%), vessel proliferation (92.86%), and congestion (83.33%). SEM--reduction in mucous lacing and increase in desquamating cells. TEM--hyperplastic epithelium, enlargement of the intercellular junctions, dense subepithelial network of collagen and basal membrane with adhesion loss. Immunohistochemistry--greater immunoexpression of fibronectin, laminin, and collagen IV around the vessels. CONCLUSIONS: In vocal polyps, the morphological analyses show lamina propria with edema, vessel proliferation and inflammation, basement membrane with adhesion loss in some areas and dense network of subepithelial collagen. Immunohistochemistry techniques identify pigmentation of the antibodies anti-fibronectin, anti-laminin, and anti-collagen IV in the endothelium of blood vessels.

Influence of laser photobiomodulation on collagen IV during skeletal muscle tissue remodeling after injury in rats.

OBJECTIVE: The aim of the present study was to determine the effect of GaAlAs low-level laser therapy (LLLT) on collagen IV remodeling of the tibialis anterior (TA) muscle in rats after cryolesion. BACKGROUND: Considerable interest exists in skeletal muscle regeneration in situations such as repair after exercise-induced muscle injury, after muscle transplantation, in muscular dystrophy, exercise-induced muscle injury, and the recovery of strength after atrophy due to disuse. A number of studies have demonstrated the potential of LLLT in facilitating the muscle-healing process; however, no consensus is found in the literature regarding the best laser-irradiation parameters. METHODS: Adult male Wistar rats (n = 45) were used and randomly divided into three groups: control (n = 5); nontreated cryolesioned group (n = 20), and LLLT-cryolesioned group (n = 20). The cryolesioned groups were analyzed at 1, 7, 14, and 21 days after the injury procedure. Laser irradiation was performed 3 times per week on the injured region by using the GaAlAs laser (660 nm; beam spot of 0.04 cm(2), output power of 20 mW, power density of 500 mW/cm(2), and energy density of 5 J/cm(2), for 10 sec). The muscles were removed, frozen, cryosectioned, and then stained with hematoxylin-eosin for the visualization of general morphology or used for immunohistochemical analysis of collagen IV. RESULTS: It was demonstrated that LLLT promotes an increase in collagen IV immunolabeling in skeletal muscle in the first 7 days after acute trauma caused by cryoinjury, but does not modify the duration of the tissue-repair process. Even with LLLT, the injured muscle tissue needs ∼21 days to achieve the same state of organization as that in the noninjured muscle. CONCLUSION: The collagen IV content is modulated in regenerating skeletal muscle under LLLT, which might be associated with better tissue outcome, although the histologic analysis did not detect tissue improvement in the LLLT group.

Immunohistochemical analysis of collagen content and types in the rectus abdominis muscle of cadavers of different ages / Imunohistoquímica do colágeno no músculo reto do abdome de cadáveres de diferentes grupos etários

PURPOSE: To assess the collagen content and types in the rectus abdominis muscle of cadavers of different ages. METHODS: Forty fresh adult male cadavers, at room temperature, were obtained from the Institute of Legal Medicine of Franca and dissected within 24 hours of death. The cadavers were divided into two groups: Group A (n=20), 18 to 30 years of age, and Group B (n=20), 31 to 60 years of age. Bilateral incisions were made in the middle portion of anterior rectus sheath 3 cm superiorly and 2 cm inferiorly to the umbilicus and four fragments of the rectus abdominis muscle were dissected. The samples were fixed in 10 percent buffered formalin and sent for immunohistochemical analysis to determine collagen content and types. RESULTS: Immunohistochemical results revealed higher amounts of type I and type III collagen in Group A. However, no difference in the amount of type IV collagen was found between the groups. CONCLUSION: The amount of type I and type III collagen was higher in group A.

OBJETIVO: Avaliar o colágeno no músculo reto do abdome em cadáveres de diferentes faixas etárias. MÉTODOS: Foram dissecados 40 cadáveres adultos masculinos, não fixados, com tempo de óbito de até 24 horas, em temperatura ambiente, provenientes do Instituto Médico-Legal de Franca (SP - Brasil). Os cadáveres foram distribuídos em dois grupos: GRUPO A (n=20) - 18 a 30 anos e GRUPO B (n=20) -31 a 60 anos. Realizou-se incisão na porção mediana da lâmina anterior da bainha do músculo reto a 3 cm superiormente e 2 cm inferiormente ao umbigo em ambos os lados, sendo retirados quatro fragmentos de músculo reto do abdome. Esse material foi conservado em formalina tamponado a 10 por cento e enviado para imuno-histoquímica para determinação do tipo de colágeno. RESULTADOS: Na Imunihistoquímica os colágenos I e III foram estatisticamente maiores no grupo A, porém não houve diferença entre os grupos em relação ao colágeno IV. CONCLUSÃO: A quantidade de colágeno tipo I e III foi maior no grupo A.

Vocal fold nodules: morphological and immunohistochemical investigations.

The objective of this study was to investigate the morphological and immunohistochemical characteristics of vocal fold nodules. The study design was prospective and retrospective. For the histological study, we reviewed 15 slides from the surgical cases of vocal fold nodules, in which we analyzed epithelium, basal membrane (bm), and lamina propria. For the transmission and scanning electron microscopy (TEM, SEM) studies, five new cases on vocal fold nodules were included. Immunohistochemistry study was carried out in the 15 specimens, using antifibronectin, antilaminin, and anticollagen IV antibodies. The main histological alterations were epithelial hyperplasia (73.33%), basement membrane thickening (86.66%), edema, and fibrosis (93.33%). SEM--reduction in mucous lacing and increase in the desquamating cells, without epithelial erosion. TEM--hyperplasia of the epithelium, enlargement of the intercellular junctions, which was filled by fluid, subepithelial thickening of the lamina reticularis, and break points in the basal membrane. Immunohistochemistry--we identified greater immunoexpression of fibronectin on the basal membrane, on the lamina propria, and around the vessels. Antilaminin and anticollagen IV antibodies showed higher pigmentation on the endothelium of the vessels than that on the basal membrane. In vocal fold nodules, combined assessment using light microscopy, electron microscopy, and immunohistochemistry can reveal important morphological details useful in characterizing these lesions.

Alport syndrome: significance of gingival biopsy in the initial diagnosis and periodontal evaluation after renal transplantation.

Alport Syndrome (AS) is an important hereditary disorder affecting the glomerular basement membrane. Diagnosis of AS is based on the presence of hematuric nephropathy, renal failure, hearing loss, ocular abnormalities and changes in the glomerular basement membrane of the lamina densa. The aims of this case report were to show the changes in the gingival tissues in a patient with AS under therapy with cyclosporin-A after renal transplantation and to discuss the possible role of type IV collagen in gingival basal lamina as an alternative approach for the diagnosis of AS. A 20-year-old male patient with AS underwent periodontal therapy including a series of gingivectomy surgeries. Gingival samples obtained during the second surgery were examined histopathologically and by transmission electron microscopy for further pathological examination. Gingivectomy procedures have been performed every 6 months over the last 4 years. The excessive and fibrous gingival enlargements resulted in migration of the anterior teeth, but no alveolar bone loss occurred. This is the first report to demonstrate the possible changes in the gingival tissues caused by AS. It is suggested that gingival biopsy can be an initial diagnostic tool instead of renal or skin biopsies. Proper dental and periodontal care and regular visits to the dentist could provide limited gingival hyperplasia to patients with AS.

Alport syndrome: significance of gingival biopsy in the initial diagnosis and periodontal evaluation after renal transplantation

Alport Syndrome (AS) is an important hereditary disorder affecting the glomerular basement membrane. Diagnosis of AS is based on the presence of hematuric nephropathy, renal failure, hearing loss, ocular abnormalities and changes in the glomerular basement membrane of the lamina densa. The aims of this case report were to show the changes in the gingival tissues in a patient with AS under therapy with cyclosporin-A after renal transplantation and to discuss the possible role of type IV collagen in gingival basal lamina as an alternative approach for the diagnosis of AS. A 20-year-old male patient with AS underwent periodontal therapy including a series of gingivectomy surgeries. Gingival samples obtained during the second surgery were examined histopathologically and by transmission electron microscopy for further pathological examination. Gingivectomy procedures have been performed every 6 months over the last 4 years. The excessive and fibrous gingival enlargements resulted in migration of the anterior teeth, but no alveolar bone loss occurred. This is the first report to demonstrate the possible changes in the gingival tissues caused by AS. It is suggested that gingival biopsy can be an initial diagnostic tool instead of renal or skin biopsies. Proper dental and periodontal care and regular visits to the dentist could provide limited gingival hyperplasia to patients with AS.