Staining methods applied to glycol methacrylate embedded tissue sections

The use of glycol methacrylate (GMA) avoids some technical artifacts, which are usually observed in paraffin-embedded sections, providing good morphological resolution. on the other hand, weak staining have been mentioned during the use of different methods in plastic sections. In the present study, changes in the histological staining procedures have been assayed during the use of staining and histochemical methods in different GMA-embedded tissues.Samples of tongue, submandibular and sublingual glands, cartilage, portions of respiratory tract and nervous ganglion were fixed in 4% formaldehyde and embedded in glycol methacrylate. The sections of tongue and nervous ganglion were stained by H&E. Picrosirius, Toluidine Blue and Sudan Black B methods were applied, respectively, for identification of collagen fibers in submandibular gland, sulfated glycosaminoglycans in cartilage (metachromasia) and myelin lipids in nervous ganglion. Periodic Acid-Schiff (PAS) method was used for detection of glycoconjugates in submandibular gland and cartilage while AB/PAS combined methods were applied for detection of mucins in the respiratory tract. In addition, a combination of Alcian Blue (AB) and Picrosirius methods was also assayed in the sublingual gland sections.The GMA-embedded tissue sections showed an optimal morphological integrity and were favorable to the staining methods employed in the present study. In the sections of tongue and nervous ganglion, a good contrast of basophilic and acidophilic structures was obtained by H&E. An intense eosinophilia was observed either in the striated muscle fibers or in the myelin sheaths in which the lipids were preserved and revealed by Sudan Black B. In the cartilage matrix, a strong metachromasia was revealed by Toluidine Blue in the negatively-charged glycosaminoglycans. In the chondrocytes, glycogen granules were intensely positive to PAS method. Extracellular glycoproteins were also PAS positive in the basal membrane and in the region occupied by the lamina externa and reticular fibers surrounding each smooth muscle cells of the blood vessels. In the epithelial cells of the respiratory tract, acid and neutral mucins were histochemically detected by AB and PAS methods, respectively. Moreover, granules containing acid and neutral mucins were revealed in purple by AB and PAS concomitantly. In the sublingual gland sections, a distinct affinity of acid mucins by AB (in turquoise-blue) and collagen fibers by Picrosirius (in red) was obtained when these methods were combined. Although some routine dyes used in paraffin sections have showed a weak stain in historesin sections, our results showed that different dyes could be applied in GMA sections if modified staining procedures were assayed. Therefore, appropriate staining contrast and, thus, detection of one or different substances in a same section can be acquired in association to the good morphological resolution provided by GMA. (C) 2003 Elsevier Ltd. All rights reserved.

Low-power laser irradiation improves histomorphometrical parameters and bone matrix organization during tibia wound healing in rats

The influence of daily energy doses of 0.03, 0.3 and 0.9 J of He-Ne laser irradiation on the repair of surgically produced tibia damage was investigated in Wistar rats. Laser treatment was initiated 24 h after the trauma and continued daily for 7 or 14 days in two groups of nine rats (n=3 per laser dose and period). Two control groups (n=9 each) with injured tibiae were used. The course of healing was monitored using morphometrical analysis of the trabecular area. The organization of collagen fibers in the bone matrix and the histology of the tissue were evaluated using Picrosirius-polarization method and Masson's trichrome. After 7 days, there was a significant increase in the area of neoformed trabeculae in tibiae irradiated with 0.3 and 0.9 J compared to the controls. At a daily dose of 0.9 J (15 min of irradiation per day) the 7-day group showed a significant increase in trabecular bone growth compared to the 14-day group. However, the laser irradiation at the daily dose of 0.3 J produced no significant decrease in the trabecular area of the 14-day group compared to the 7-day group, but there was significant increase in the trabecular area of the 15-day controls compared to the 8-day controls. Irradiation increased the number of hypertrophic osteoclasts compared to non-irradiated injured tibiae (controls) on days 8 and 15. The Picrosirius-polarization method revealed bands of parallel collagen fibers (parallel-fibered bone) at the repair site of 14-day-irradiated tibiae, regardless of the dose. This organization improved when compared to 7-day-irradiated tibiae and control tibiae. These results show that low-level laser therapy stimulated the growth of the trabecular area and the concomitant invasion of osteoclasts during the first week, and hastened the organization of matrix collagen (parallel alignment of the fibers) in a second phase not seen in control, non-irradiated tibiae at the same period. The active osteoclasts that invaded the regenerating site were probably responsible for the decrease in trabecular area by the fourteenth day of irradiation. (C) 2003 Elsevier B.V. All rights reserved.

Testicular interstitial cells, and steroidogenic detection in the protogynous fish, Synbranchus marmoratus (Teleostei, Synbranchidae)

The swamp eel, Synbranchus marmoratus, is a freshwater protogynic diandric species. Primary males develop directly as males while secondary males arise from the sex reversal of females. Fishes from Argentine and Brazil inland waters were collected, examined and compared for this study. In order to characterize the interstitial testicular compartment, light and electron microscopy techniques and an enzyme histochemical examination for steroidogenic cells detection were used. The interstitial compartment of S. marmoratus is composed of Leydig and myoid cells, collagen fibers, blood cells, macrophages,and amyelinic nerves. At the ultrastructural level, no differences were observed in the interstitial tissue, either between specimens from the different sampling sites or between primary and secondary males. Leydig cells are present in all testes examined throughout the year. A cytoplasmatic reaction of 3beta-HSD was detected only in Leydig cells during sex reversal and in both type of males, mainly during the regressed and early maturation classes (autumn and winter). Leydig cells possess the typical fine structural characteristics associated with steroidogenesis. Furthermore, in both type of males, during sex reversal and after the spawning period, the number of granulocytes and macrophages present in the testes increased, suggesting that they could be involved in phagocytosis and resorption of damaged cells. (C) 2004 Elsevier Ltd. All rights reserved.

Prostate carcinogenesis induced by N-methyl-N-nitrosourea (mnu) in gerbils: Histopathological diagnosis and potential invasiveness mediated by extracellular matrix components

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Histology, histochemistry and stereology of the adipose fin of Prochilodus lineatus

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Morphological analysis of colon goblet cells and submucosa in type I diabetic rats submitted to physical training

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Structure, histochemistry, and ultrastructure of the epithelium and stroma in the gerbil (Meriones unguiculatus) female prostate

The female prostate has aroused scientific interest because it is subjected to the same diseases compromising the male prostate during aging. The objective of this work was to characterize structurally, cytochemically, and ultrastructurally the tissue compartments of the normal adult female prostate of Meriones unguiculatus gerbils. The morphological analyses showed that the gerbil's female prostate is constituted of a cluster of glands and ducts inserted in a musculofibrous stroma. The alveolar epithelium is differentiated and consisted of basal proliferating cells, intermediary cells, and secretory cells. The secretory cells are the most numerous cell type and continuously secrete glycoproteins. The basal cells are the source of the secretory cells and they are then responsible for the alveolus renovation. The prostatic stroma is abundant and rich in elastic and collagen fibers, which are closely associated with smooth muscle cells and fibroblasts. The results showed that the gerbil's female prostate shows morphological and ultrastructural homology to the human female prostate (Skene's gland), and despite being a small organ, it is a mature and physiologically active gland. (C) 2003 Elsevier Ltd. All rights reserved.

Short-term stromal alterations in the rat ventral prostate following alloxan-induced diabetes and the influence of insulin replacement

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Fluoxetine Inhibits Inflammatory Response and Bone Loss in a Rat Model of Ligature-Induced Periodontitis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of Chamomilla recutita (L.) on oral wound healing in rats

Objectives. The purpose of this study was to evaluate the effect of Chamomilla recutita on the healing of ulcers in rats.Study Design. A 5-mm wound was inflicted on the tongue of 36 rats. Treatment group animals were treated topically with 0.04 mL/day of chamomile ointment, whereas control group animals were not treated. Animals were sacrificed after 3, 7 or 10 days. Semi-quantitative analysis of the degree of inflammation, fibroblast count and wound size was performed, as well as histometric analysis of re-epithelialization and percentage of collagen fibers of the lesion.Results. Animals treated with chamomile showed the best results regarding epithelialization and percentage of collagen fibers after 10 days. As expected, time had a statistically significant effect (p<0.05) on fibroblast count, epithelialization, inflammation and wound size; animals sacrificed at 3 days showed the worst results.Conclusions. Chamomile stimulated re-epithelialization and the formation of collagen fibers after 10 days of treatment; it did not, however, influence inflammation or fibroblast count.

Efeito do resfriamento sobre a textura post-mortem da carne do peixe matrinxã Brycon cephalus

Os mecanismos que causam o amolecimento e a perda na textura post-mortem da carne de matrinxã foram determinados por meio das mudanças na microestrutura do músculo, imediatamente após a morte e depois de 12 horas de estocagem a -3°C. As observações na microestrutura, realizadas com microscópio eletrônico de transmissão, foram semelhantes aos resultados obtidos na força de ruptura do músculo medidos com o texturômetro. Os valores da força da ruptura foram menores para a carne após o resfriamento. Observou-se que as fibras do colágeno do tecido conectivo pericelular se desintegraram e que as do colágeno do tecido conectivo do miocommata conservaram sua arquitetura e integridade. Houve pouca degradação da linha Z. Isso sugere que o amolecimento post-mortem da carne de mantrinxã, durante a estocagem a -3°C, é causado pela degradação do tecido conectivo pericelular.

Eletroacupuntura no reparo do tendão calcanear comum em coelhos após enxertia com peritônio bovino

Avaliou-se o efeito da eletroacupuntura (EA) sobre os aspectos macroscópicos e microscópicos em 36 coelhos que tiveram o tendão calcanear comum seccionado e que, após 30 dias, receberam enxerto de peritônio bovino conservado em solução saturada de sal. Após a cirurgia, os animais foram distribuídos em três grupos de igual número: os do grupo E10 foram estimulados pela EA do segundo ao 11º dia de pós-operatório, os do grupo E20 do 11º ao 31º dia de pós-operatório e os do grupo C não foram estimulados. Posteriormente, os animais foram redistribuídos em três subgrupos de igual número, os quais foram observados no pós-operatório por 11, 31 e 91 dias. Nos coelhos do E10 e E20, ocorreu diminuição de aderência da área enxertada aos tecidos circunvizinhos. Aos 31 dias de pós-operatório, observou-se nos do E10 tecido conjuntivo. Verificou-se intensa neovascularização no local enxertado em todos os animais que receberam estímulo pela EA. Nos coelhos do E10, aos 91 dias, verificou-se intensa quantidade de tecido conjuntivo, com fibras colágenas organizadas e paralelas ao eixo do tendão. O estímulo pela EA na fase precoce do pós-operatório estimulou a maturação cicatricial com mínima reação inflamatória.

Histologia da pele da carpa prateada (Hypophtalmichthys molitrix) e testes de resistência do couro

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Análise histológica e morfométrica da área cruenta tratada com membrana de silicone: estudo em ratos

Background. Thin study evaluated the effects of a silicone membrane on the treatment of the raw flesh area in wounds.METHODS. The experimental study was carried out with 30 male Wistar rats divided into three groups: in the G(G) group, the raw area was treated with gauze, in the G(H) group with homograft, and in the G(S) group with a silicone membrane. Animals were sacrificed at fourteen days of postoperative. The studied attributes were body mass variation, histological study quantifed by morphometric analysis evaluating the number of neovessels, fibroblasts, collagen fibers, leucocytes, monocytes; and using a micrometric ruler measurement of the raw area thickness. Data were then submitted to statistical analysis.RESULTS. There was no significant difference between the animals' mass (p=0.0685). Predominance of neovessels (p<0.01), fibroblasts (p<0.001) and thickness of the raw flesh were observed in G(S) animals. There was predominance of leucocytes (p<0.021) and monocytes (p<0.0001) in G(H) animals. Also, no significant difference between the groups as for collagen fibers (p=0.0536) was observed.CONCLUSION. The silicone membrane promoted granulation tissue with a large number of neovessels, fibroblasts and greater thickness.

Epithelial-stromal transition of MMP-7 immunolocalization in the rat ventral prostate following bilateral orchiectorny

Epithelial cells from involuting rat ventral prostate (VP) express Matrilysin (MMP-7) mRNA. Herein, we investigated by immunohistochemistry the NIMP-7 protein location and its association with tissue changes following castration in the VP. Normal and castrated adult male Wistar rats were sacrificed at different times after surgery. VP was examined by immunocytochemistry and immunoprecipitation. Castration promoted a shrinking of prostate ducts with an extensive stromal remodeling. In the VP from normal rats, MMP-7 immunoreactivity was found in epithelial secretory granules. Three days after castration, immunostaining for MMP-7 was found in both the epithelial secretory granules and in the stroma just below the epithelium, mainly at the distal ductal tips. At seven and 21 days after castration, the immunostaining for MMP-7 was found only in the stromal space. Immunoprecipitation confirmed the specificity of the primary antibody by rescuing a pro-enzyme form (28 kDa) in the prostate extracts. The present results suggest that MMP-7 participates in the epithelial-stromal interface remodeling of the ventral prostate during the involution achieved by castration, probably in the degradation of components of the epithelial basement membrane. (c) 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.