Simultaneous observation of collagen and elastin in normal and pathological tissues: analysis of Sirius-red-stained sections by fluorescence microscopy

In order to observe collagen and elastic fibers simultaneously, sections of human aorta, skin, lung, liver, and bladder were stained by Sirius red and analyzed by fluorescence microscopy. In all cases, the fibers of collagen presented the characteristic fluorescent red-orange color that results from the interaction of this extracellular protein with the dye, whereas elastic fibers showed strong green fluorescence (intrinsic fluorescence). This method efficiently detects collagen and elastic fibers when these two structures are present and could have valuable applications in processes that involves both fibers.

Evaluation of Laser Phototherapy in the Inflammatory Process of the Rat's TMJ Induced by Carrageenan

Aim: The aim of this study was to evaluate, by light microscopy, the effects of laser phototherapy (LPT) at 780nm or a combination of 660 and 790 nm, on the inflammatory process of the rat temporomandibular joint (TMJ) induced by carrageen. Background: Temporomandibular disorders (TMDs) are frequent in the population and generally present an inflammatory component. Previous studies have evidenced positive effects of laser phototherapy on TMDs. However, its mechanism of action on the inflammation of the TMJ is not known yet. Materials and Methods: Eighty-five Wistar rats were divided into 9 groups: G1, Saline; G2, Saline + LPT IR; G3, Saline + LPT IR + R; G4, Carrageenan; G5, Carrageenan + LPT IR; G6, Carrageenan + LPT IR + R; G7, previous LPT + Carrageenan; G8, previous LPT + carrageenan + LPT IR; and G9, previous LPT + carrageenan + LPT IR + R, and then subdivided in subgroups of 3 and 7 days. After animal death, specimens were taken, routinely cut and stained with HE, Sirius Red, and Toluidine Blue. Descriptive analysis of components of the TMJ was done. The synovial cell layers were counted. Results: Injection of saline did not produced inflammatory reaction and the irradiated groups did not present differences compared to non-irradiated ones. After carrageenan injection, intense inflammatory infiltration and synovial cell layers proliferation were observed. The infrared irradiated group presented less inflammation and less synovial cell layers number compared to other groups. Previous laser irradiation did not improve the results. Conclusion: It was concluded that the LPT presented positive effects on inflammatory infiltration reduction and accelerated the inflammation process, mainly with IR laser irradiation. The number of synovial cell layers was reduced on irradiated group.

Polarized Light (lambda 400-2000 nm) on Third-Degree Burns in Diabetic Rats: Immunohistochemical Study

Aim: The aim of this study was to evaluate with light microscopy the healing process of third-degree burns on diabetic rats treated with polarized light (lambda 400-2000 nm, 20 or 40 J/cm(2)/session, 40 mW/cm(2), 2.4 J/cm(2)/min, 5.5-cm beam diameter). Background: Uncontrolled diabetes mellitus causes severe disruption of the body's metabolism, including healing. Polarized light sources have been shown to be effective in improving healing in many situations. Animals and Methods: Diabetes mellitus was induced with streptozotocin (60 mg/kg) in 45 male Wistar albino rats, and a third-degree burn (1.5 by 1.5 cm) was created on the dorsum of each animal under general anesthesia. The animals were randomly distributed into three groups: control, 20 J/cm(2), and 40 J/cm(2). Each group was then divided into three subgroups based on time of death (7, 14, 21 d). Phototherapy (20 or 40 J/cm(2) per session) was carried out immediately after the burning and repeated daily until the day before death. Following animal death, specimens were removed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE) or Sirius Red or immunomarked with CK AE1/AE3 antibody. Qualitative and semiquantitative analyses were performed under light microscopy. The results were statistically analyzed. Results: The animals treated with 20 J/cm(2) showed significant differences with regard to revascularization and re-epithelialization. Although the 40 J/cm(2) group showed stimulation of fibroblastic proliferation as an isolated feature, no other difference from the control was observed. Conclusion: Our results suggest that the use of polarized light at 20 J/cm(2) effectively improves the healing of third-degree burns on diabetic animals at both early and late stages of repair.

Bile duct ligation in neonatal rats: Is it a valid experimental model for biliary atresia studies?

BA is the most important disease requiring liver transplantation in children. Common BDL in rats is a classic experimental model to study biliary obstruction. The response of the neonatal animal to BDL has yet to be completely understood and few reports have focused on the behavioral differences of the liver between neonatal and adult animals. Ninety newborn Wistar rats aged six days, weighing 8.0-13.9 g, and 90 adult Wistar rats weighing 199.7-357.0 g, were submitted to BDL. After surgery, they were randomly divided and killed on the 3rd, 5th, 7th, 14th, 21st and 28th day post-BDL. Hepatic biopsies were obtained and the following were measured: (i) semiquantification of the bile ductule proliferation and inflammatory infiltrate by HE stain, (ii) quanti. cation of portal and periportal fibrosis with the Sirius-red stain. Although the initial response of ductule proliferation and inflammatory infiltrate were less intense in the newborn animal, the portal and periportal fibrosis were higher when compared with adult animals (p < 0.0491). These findings may contribute to the understanding of the pathophysiology of BA.

Effect of ultrasound therapy on the repair of Gastrocnemius muscle injury in rats

The aim of this study was to evaluate the effect of the pulsed ultrasound therapy (PUT) in stimulating myoregeneration and collagen deposition in an experimental model of lacerative gastrocnemius muscle lesion in 30 Wistar rats. Fifteen rats were treated (TG) daily with 1 MHz pulsed ultrasound (50%) at 0.57 W/cm(2) for 5 min, and 15 were control animals (CG). Muscle samples were analyzed on postoperative days 4, 7 and 14 through H&E, Picrosirius-polarization and immunohistochemistry for desmin. The lesions presented similar inflammatory responses in both treated and control groups. The areal fraction of fibrillar collagen was larger in the TG at 4 days post-operatively (17.53 +/- 6.2% vs 6.79 +/- 1.3%, p = 0.0491), 7 days (31.07 +/- 7.45% vs 12.57 +/- 3.6%, p = 0.0021) and 14 days (30.39 +/- 7.3% vs 19.13 +/- 3.51%, p = 0.0118); the areal fraction of myoblasts and myotubes was larger in the TG at 14 days after surgery (41.66 +/- 2.97% vs 34.83 +/- 3.08%, p = 0.025). Our data suggest that the PUT increases the differentiation of muscular lineage cells, what would favor tissue regeneration. On the other hand, it is also suggested that there is a larger deposition of collagenous fibers, what could mean worse functional performance. However, the percentage of fibers seems to have stabilized at day 7 in TG and kept increasing in CG. Furthermore, the collagen supramolecular organization achieved by the TG is also significant according to the Sirius red staining results. (C) 2008 Elsevier B.V. All rights reserved.

Relationship between plasma cells and hepatic stellate cells in autoimmune hepatitis

Autoimmune hepatitis is an inflammatory chronic disease of the liver, which frequently results in cirrhosis. The present study aimed to verify the relationship between plasma cells and stellate cells in autoimmune hepatitis. Thirty-three pre-treatment, 11 post-treatment, and 10 normal liver biopsies were reviewed. Sirius Red staining (for semi-quantitative analysis of hepatic fibrosis) and immunohistochemistry were carried out: double staining for smooth muscle alpha-actin and plasma cell marker (for detection and localization of activated hepatic stellate cells and plasma cells, respectively); and single staining for glial fibrillary acid protein (for detection of hepatic stellate cells). We found an increase in the stellate cell population, mainly with an activated phenotype in autoimmune hepatitis, compared to the control group (liver specimens with no histological evidence of liver disease, obtained from patients undergoing hepatic resection for benign liver mass). A positive significant correlation was observed between stellate cells and scores of fibrosis (measured by Sirius Red) and the number of plasma cells. Additionally, there was a co-localization of plasma cells and activated stellate cells. We also observed a reduction in the number of plasma cells, hepatic stellate cells, and fibrosis in patients who had successfully been treated and had a second liver biopsy post-treatment. Our findings support that the number of plasma cells can be a surrogate marker for the severity of liver disease, reflecting the number of hepatic stellate cells and the amount of fibrosis. It remains to be seen if this is a result of a direct interaction between the plasma cells and hepatic stellate cells or the response to the same stimulus that affects both cellular types. (c) 2010 Elsevier GmbH. All rights reserved.

Metalloproteinase inhibition ameliorates hypertension and prevents vascular dysfunction and remodeling in renovascular hypertensive rats

Altered activity of matrix metalloproteinases (MMPs) is implicated in the vascular remodeling of hypertension. We examined whether increased MMP-2 expression/activity plays a role in the vascular remodeling and dysfunction found in the two-kidney, one-clip (2K-1C) hypertension. Sham operated or 2K-1C hypertension rats were treated with doxycycline 30 mg/(kg day) (or vehicle). Systolic blood pressure was monitored weekly. After 8 weeks of treatment, aortic rings were isolated to assess endothelium-dependent and independent relaxations. Quantitative morphometry of structural changes, collagen, and elastin contents in the aortic wall were studied in hematoxylin/eosin, Sirius Red, and Orceine stained aortic sections, respectively. Aortic MMP-2 levels were determined by gelatin zymography and aortic MMP-2 proteolytic activity was measured using DQ gelatin as the substrate after MMP-2 was captured by a specific antibody and immobilized on a microplate. Aortic MMP-2/tissue inhibitor of metalloprotemases (TIMP)-2 mRNA levels were determined by real time RT-PCR. Doxycycline attenuated 2K-1C hypertension (215 +/- 8 mmHg versus 167 +/- 13 mmHg in 2K-1C rats and 2K-1C + doxy rats, respectively; P < 0.01) and prevented the 35% reduction in endothelium-dependent vasorelaxation found in 2K-1C rats. Doxycycline prevented the increases in media thickness, and was associated with lower media/lumen and cross-sectional areas (all P<0.01). Doxycycline also prevented excessive collagen and elastin deposition in the vascular wall. Increased MMP-2 and Pro-MMP-2 levels and MMP-2 activity were found in the aortas of 2K-1C rats (all P<0.05). A 21-fold increase (P<0.001) in the ratio of MMP-2/TIMP-2 mRNA expression was found in the 2K-1C group, whereas this ratio remained unaltered in 2K-1C+doxy rats. Our results suggest that MMP-2 plays a role in 2K-1C hypertension and its structural and functional vascular changes, which were attenuated by doxycycline. (C) 2007 Elsevier Ireland Ltd. All rights reserved.

Inhibition of COX 1 and 2 prior to Renal Ischemia/Reperfusion Injury Decreases the Development of Fibrosis

Ischemia and reperfusion injury (IRI) contributes to the development of chronic interstitial fibrosis/tubular atrophy in renal allograft patients, Cyclooxygenase (COX) 1 and 2 actively participate in acute ischemic injury by activating endothelial cells and inducing oxidative stress. Furthermore, blockade of COX I and 2 has been associated with organ improvement after ischemic damage. The aim of this study was to evaluate the role of COX I and 2 in the development of fibrosis by performing a COX I and 2 blockade immediately before IRI We subjected C57BI/6 male mice to 60 min of unilateral renal pedicle occlusion, Prior to surgery mice were either treated with indomethacin (IMT) at days -1 and 0 or were untreated. Blood and kidney samples were collected 6 wks after IRI. Kidney samples were analyzed by real-time reverse transcription-poly me rase chain reaction for expression of transforming growth factor beta (TGF-beta), monocyte chemoattractant protein 1 (MCP-1), osteopontin (OPN), tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-1 beta, IL-10, heme oxygenose 1 (HO-1), vimentin, connective-tissue growth factor (CTGF), collagen 1, and bone morphogenic protein 7 (BMP-7), To assess tissue fibrosis we performed morphometric analyses and Sirius red staining. We also performed immunohistochemical analysis of anti-actin smooth muscle, Renal function did not significantly differ between groups. Animals pretreated with IMT showed significantly less interstitial fibrosis than nontreated animals. Gene transcript analyses showed decreased expression of TGF-beta, MCP-1,TNF-alpha, IL-1-beta, vimentin, collagen 1, CTGF and IL-10 mRNA (all P < 0.05), Moreover, HO-I mRNA was increased in animals pretreated with IMT (P < 0.05) Conversely, IMT treatment decreased osteopontin expression and enhanced BMP-7 expression, although these levels did rot reach statistical significance when compared with control expression levels, I he blockade of COX 1 and 2 resulted in less tissue fibrosis, which was associated with a decrease in proinflammatory cytokines and enhancement of the protective cellular response.

Zero-Time Kidney Histology Predicts Early Graft Function

Our purposes are to determine the impact of histological factors observed in zero-time biopsies on early post transplant kidney allograft function. We specifically want to compare the semi-quantitative Banff Classification of zero time biopsies with quantification of % cortical area fibrosis. Sixty three zero-time deceased donor allograft biopsies were retrospectively semiquantitatively scored using Banff classification. By adding the individual chronic parameters a Banff Chronic Sum (BCS) Score was generated. Percentage of cortical area Picro Sirius Red (%PSR) staining was assessed and calculated with a computer program. A negative linear regression between %PSR/ GFR at 3 year post-transplantation was established (Y=62.08 +-4.6412X; p=0.022). A significant negative correlation between arteriolar hyalinosis (rho=-0.375; p=0.005), chronic interstitial (rho=0.296; p=0.02) , chronic tubular ( rho=0.276; p=0.04) , chronic vascular (rho= -0.360;P=0.007), BCS (rho=-0.413; p=0.002) and GFR at 3 years were found. However, no correlation was found between % PSR, Ci, Ct or BCS. In multivariate linear regression the negative predictive factors of 3 years GFR were: BCS in histological model; donor kidney age, recipient age and black race in clinical model. The BCS seems a good and easy to perform tool, available to every pathologist, with significant predictive short-term value. The %PSR predicts short term kidney function in univariate study and involves extra-routine and expensive-time work. We think that %PSR must be regarded as a research instrument.

Contribution to the histology of Biomphalaria glabrata

A combination of histological techniques applied to the study of Biomphalaria glabrata yielded some interesting new data about the histology of this snail, a major intermediate host of Schistosoma mansoni in Brazil. Three kinds of pigments were identified: a dark pigment which bleached following oxidation with potassium permanganate; a lipofuchsin-like, diastase-resistant PAS-positive pigment and an iron-containing pigment, probably related to hemosiderin. Calcium was detected in small deposits within the connective tissue and forming a dense core inside the chitinous radular teeth. The presence of fibrils, staining with sirius-red and birefringence under polarized light strongly suggest primitive collagen tissue. The radular apparatus appeared as a storing site for glycogen, while abundant Alcian-blue positive material (proteoglycans) was extremely concentrated in the radular sac.

Morphological aspects of the myocarditis and myositis in Calomys callosus experimentally infected with Trypanosoma cruzi: fibrogenesis and spontaneous regression of fibrosis

Calomys callosus a wild rodent, is a natural host of Trypanosoma cruzi. Twelve C. callosus were infected with 10(5) trypomastigotes of the F strain (a myotropic strain) of T. cruzi. Parasitemia decreased on the 21 st day becoming negative around the 40th day of infection. All animals survived but had positive parasitological tests, until the end of the experiment. The infected animals developed severe inflammation in the myocardium and skeletal muscle. This process was pronounced from the 26 th to the 30th day and gradually subsided from the 50 th day becoming absent or residual on the 64 th day after infection. Collagen was identified by the picro Sirius red method. Fibrogenesis developed early, but regression of fibrosis occurred between the 50th and 64th day. Ultrastructural study disclosed a predominance of macrophages and fibroblasts in the inflammatory infiltrates, with small numbers of lymphocytes. Macrophages had active phagocytosis and showed points of contact with altered muscle cells. Different degrees of matrix expansion were present, with granular and fibrilar deposits and collagen bundles. These alterations subsided by the 64th days. Macrophages seem to be the main immune effector cell in the C. callosus model of infection with T. cruzi. The mechanisms involved in the rapid fibrogenesis and its regression deserve further investigation.

Os ácidos graxos de cadeia curta na cicatrização de anastomoses colônicas: estudo experimental em ratos

OBJETIVO: Avaliar a ação dos ácidos graxos de cadeia curta (AGCC) na cicatrização de anastomoses colônicas em ratos. MÉTODO: Utilizaram-se 50 ratos divididos em 4 grupos. Dois desses grupos, vinte e seis animais, foram submetidos à operação de Hartmann; metade destes (grupo HF) recebeu infusões retais diárias, no pós-operatório, de solução fisiológica a 0,9% ; a outra metade (grupo HA) recebeu solução de butirato de sódio à 80mmol/l (AGCC) (HA). Os dois grupos restantes, vinte e quatro animais foram submetidos à anastomose colônica término-terminal a 4 cm da borda anal. Destes, 12 animais (grupo AF) receberam infusões retais de soro fisiológico a 0,9% e 12 animais (grupo AA) receberam a solução com AGCC. Os animais foram avaliados no 7º e 14º dia do pós-operatório. Foi realizada análise histológica da densitometria do colágeno pela coloração do sirius red, utilizando-se a microscopia de polarização com método computadorizado. RESULTADOS: Comparando-se os grupos submetidos à cirurgia de Hartmann, portanto na ausência do trânsito intestinal, observou-se concentração significativamente maior de colágeno I e total no grupo que recebeu AGCC (p=0,001). Comparando-se os grupos submetidos à anastomose término-terminal, com interferência do trânsito intestinal, não se encontrou diferença significante da concentração de colágeno I e total (p=0,056 e p=0,397, respectivamente). A ação isolada do trânsito intestinal promoveu também aumento da produção de colágeno, quando comparado ao grupo sem atuação do trânsito intestinal. CONCLUSÃO: A administração via retal de AGCC, na ausência do trânsito intestinal mostrou-se de grande valia, promovendo aumento da síntese do colágeno. Independentemente da interferência do trânsito intestinal, o principal efeito do AGCC esteve relacionado com o aumento da maturação do colágeno.

Enxerto homólogo congelado de diafragma na correção dos defeitos da parede abdominal de ratos

OBJETIVOS: Análise morfológica e biomecânica do enxerto homólogo congelado de diafragma para correção de defeito da parede abdominal em ratos. MÉTODOS: Os animais foram distribuidos em controle (20 ratos Wistar) e experimento (30 ratos Wistar). Os do grupo controle foram submetidos à laparotomia mediana e sutura da parede abdominal; já os do grupo experimento, à ressecção da parede abdominal e reconstrução com enxerto homólogo congelado de diafragma. Os animais foram submetidos à eutanásia no 3° e 6° mês de pós-operatório e avaliados quanto à presença de complicações pós-operatórias, integridade do enxerto, presença de aderências, avaliação tensiométrica, avaliação histopatológica com H/E e com sirius-red (colágeno tipo I e III). RESULTADOS: Houve integração do enxerto em todos os animais sem complicações. Aderências foram semehantes entre os grupos controle e experimento após três e seis meses. Observaram-se maior força máxima, força de ruptura e tensão nos animais do grupo experimento aos três meses de pós-operatório (p=0,001; p=0,012 e p=0,001, respectivamente). Na correlação entre as diferentes variáveis estudadas houve correlação estatisticamente significante entre força máxima e tensão nos grupos controle e experimento (p=0,001 e p= 0,001), e nos subgrupos três e seis meses (p=0,002 e p=0,001). Correlacionaram-se força máxima e colágeno tipo I (p=0,04) e Índice de Maturação do Colágeno (IMaC) e força máxima (p=0,03) ambos somente no grupo controle, mas nos subgrupos 3 e 6 meses (p=0,045 e p=0,038). O número de monomorfonucleares e força máxima também apresentou significância estatística tanto para o grupo controle quanto para o experimento (p=0,005 e p=0,004, respectivamente). CONCLUSÃO: O enxerto homólogo congelado de diafragma mostrou ser boa alternativa no reparo de grandes defeitos da parede abdominal em ratos.

Efeito dos Estrógenos Conjugados e da Medroxiprogesterona sobre a Mama: Estudo Experimental

Objetivo: avaliar no tecido mamário a influência de drogas empregadas na terapia de reposição hormonal sobre a proliferação celular, a quantidade de colágeno e de fibras elásticas e as alterações histológicas gerais do parênquima. Método: utilizaram-se 61 ratas Wistar-UFPR, adultas, divididas em 5 grupos. O grupo padrão (n=12) representou o perfil hormonal ovariano normal. As 49 ratas restantes foram ooforectomizadas e a partir do 96º dia P.O. receberam a medicação designada para cada grupo durante 30 dias. O grupo EEC (n=13) recebeu 50 mg/dia de estrógenos eqüinos conjugados; o grupo MPA (n=12), 2,0 mg/dia de acetato de medroxiprogesterona; o grupo EEC + MPA (n=12), ambos, e o grupo AD (n=12), água destilada. No 31º dia de medicação todos os animais foram sacrificados e as mamas inguinais foram retiradas para análise histológica. A avaliação da proliferação celular nos ductos e ácinos foi realizada por método imuno-histoquímico utilizando-se anticorpo anti-PCNA. Utilizando-se a coloração de Sirius-Red quantificou-se o colágeno maduro (tipo I) e imaturo (tipo III). A coloração de Weigert avaliou a formação de fibras elásticas. A análise anatomopatológica foi realizada em coloração de hematoxilina-eosina, determinando-se o número de ácinos por ducto terminal, número de ductos por campo, presença de secreção intraductal e a intensidade de vacuolização intracitoplasmática. Resultados: o grupo EEC + MPA apresentou menor porcentagem de células ductais em proliferação (46,1%) (p<0,0001). Também mostrou maior taxa de proliferação das células acinares (66,3%), sendo semelhante ao grupo MPA (p=0,075) mas diferente dos demais grupos (p<0,004). No grupo EEC encontrou-se maior quantidade de colágeno imaturo (33,6%) (p<0,01) e o grupo MPA apresentou mais elevada concentração de fibras elásticas (11,7%) (p<0,0001). Os grupos EEC + MPA e MPA apresentaram hiperplasia acinar secretora, sendo intensa (91,7%) no grupo EEC + MPA e discreta (41,7%) ou moderada (58,3%) no grupo MPA, mas ambos diferentes dos demais grupos (p<0,097). Conclusões: os estrógenos eqüinos conjugados associados a acetato de medroxiprogesterona inibem a proliferação celular ductal e estimulam a proliferação celular acinar promovendo hiperplasia acinar secretora; os estrógenos eqüinos conjugados intensificam a formação de colágeno imaturo (tipo III) e o acetato de medroxiprogesterona aumenta a formação de fibras elásticas.

Avaliação das fibras colágenas de meniscos frescos e preservados em glicerina: estudo experimental em coelhos (Oryctolagus cuniculus)

No presente estudo foi avaliado o efeito da glicerina 98% sobre as fibras colágenas, arquitetura tecidual e o tamanho de meniscos mediais de coelhos da raça Nova Zelândia. Os meniscos foram separados em três grupos: (1) Grupo MF com meniscos frescos (grupo controle), (2) Grupo MG com meniscos preservados em glicerina 98% por 30 dias, e (3) Grupo MR com meniscos preservados em glicerina 98% por 30 dias e reidratados em NaCl 0,9%, por 12 horas. Os cortes histológicos foram corados com sirius red para identificação dos tipos de colágenos e observados em microscópio de luz polarizada, avaliando-se a concentração total de colágeno Tipo I e III e a disposição das fibras. Os meniscos frescos apresentaram significativamente maior concentração de fibras colágenas Tipo I e menor concentração de fibras colágenas Tipo III que os meniscos preservados (MG e MR); isto ocorreu devido à perda de água e conseqüente redução do tamanho dos meniscos e retração das fibras colágenas dos meniscos dos Grupos MG e MR; isto pode ter feito com que as fibras Tipo I, mais espessas e em maior quantidade, se tornassem mais evidentes do que as fibras colágenas Tipo III, que são mais delgadas e frágeis (fibrilas). Nos três grupos estudados, as fibras colágenas apresentaram-se de forma circunferencial, interpostas por fibras orientadas radialmente. Entretanto, nos grupos tratados (MG e MR) foi observado, em pequenas áreas, leve desorganização das fibras colágenas, o que correspondeu a 42,8% e 14,3% dos meniscos, respectivamente. O grupo de meniscos em glicerina apresentou redução significativa (p<0,05) no tamanho em relação ao Grupo MF. No Grupo MR, 85,7% dos meniscos retornaram ao seu tamanho inicial após a reidratação. A glicerina 98% é eficaz na preservação de meniscos, mantendo o tamanho, a arquitetura estrutural, a integridade e percentagem do colágeno dos meniscos preservados semelhante à de meniscos frescos, desde que sejam reidratados em NaCl 0,9% após preservação.