Histomorphometry and detection of glycosaminoglycans in the endocervical epithelium of pregnant rats after local administration of hyaluronidase.

OBJECTIVE: The aim of the study was to detect the presence of glycosaminoglycans and to investigate histomorphometric aspects of the endocervical epithelium in pregnant rats after local administration of hyaluronidase. MATERIALS AND METHODS: Ten pregnant rats were randomly distributed into two groups. On day 18 of pregnancy 1 mL of distilled water and 0.02 mL of hyaluronidase were administered to the control group (CG) and the study group (SG), respectively On day 20 the rats were sacrificed, followed by dissection and removal of the uterine cervix, which was prepared for histomorphometry (endocervical epithelium thickness and leucocyte infiltration) and for immunohistochemistry with alcian blue reaction and its respective blockers. The paired Student t test was used to compare the groups. RESULTS: The SG was characterized by reduced epithelial thickness (mean: 291.01 +/- 71.1 vs. 764.30 +/- 50.94; p < 0.0001) and a larger number of eosinophils (mean: 3.72 +/- 1.60 vs. 0.54 +/- 0.70; p < 0.0001). Alcian blue staining (pH 0.5) indicated a very strong reaction (3+) for the CG. With pH 2.5, the staining was also very intense (4+) in the CG. With methylation, both groups showed negative reactions after alcian blue staining (pH 2.5). With the methylation reaction followed by saponification and with enzymatic digestion of the lamina, staining showed a weak reaction (1+) in both groups. CONCLUSION: The SG presented with significant alterations related to the reduction of epithelial thickness and an increase in leucocyte infiltration. Furthermore, the use of hyaluronidase resulted in a significant decrease of the sulfated glycosaminoglycans.

Alterações histoquímicas das glicosaminoglicanas na cérvice uterina no final da prenhez da rata albina após ministração local de hialuronidase / Histochemical changes of the glycosaminoglycans in the uterine cervix of pregnant rats after local injection of hyaluronidase

OBJETIVO: estudar as alterações histoquímicas relacionadas às glicosaminoglicanas da cérvice uterina da rata albina, após ministração local de hialuronidase no final da prenhez. MÉTODOS: dez ratas com teste de prenhez positivo foram distribuídas aleatoriamente em dois grupos, numericamente iguais. O Grupo Controle (Gc) foi constituído pelas ratas que receberam 1 mL de água destilada, dose única, no 18° dia da prenhez, sob anestesia, ministrado na cérvice uterina. O Grupo Experimental (Gex) constou de ratas que receberam, sob as mesmas condições do Gc, 0,02 mL de hialuronidase, diluído em 0,98 mL de água destilada (total de 1 mL). No 20° dia de prenhez, as ratas foram novamente anestesiadas e submetidas à dissecção, preparando-se a cérvice uterina para estudo histoquímico com coloração de alcian blue e seus bloqueios (pH=0,5, pH=2,5, metilação e saponificação). RESULTADOS: verificou-se na lâmina própria no Gc, reação fortemente positiva (+3) e, no Gex, reação negativa, na coloração de alcian blue no pH=0,5. Em pH=2,5 a coloração também se apresentou fortemente positiva (+4) no Gc e fracamente positiva (+1) no Gex. Após metilação, tanto o Gc quanto o Gex mostraram reação negativa após coloração de alcian blue no pH=2,5. Com a reação de metilação seguida de saponificação e na digestão enzimática em lâmina, a coloração da lâmina própria se mostrou negativa em ambos os grupos. CONCLUSÕES: há uma nítida predominância de glicosaminoglicanas sulfatadas no Gc em relação ao Gex e uma tênue quantidade de glicosaminoglicanas carboxiladas identificadas no Gex. As modificações evidenciadas na matriz extracelular sugerem que a hialuronidase injetada localmente na cérvix uterina promoveu alterações bioquímicas compatíveis com maturação cervical.

PURPOSE: to study the histochemical changes related to the uterine cervix glycosaminoglycan of the albino female rat, after local ministration of hyaluronidases at the end of pregnancy. METHODS: ten female rats with positive pregnancy tests were randomly distributed in two numerically equal groups. The control group (Cg) was built up with rats that received a single dose of 1 mL of distilled water in the uterine cervix, under anesthesia, at the 18th pregnancy day. In the experimental group (Exg), the rats received 0.02 mL of hyaluronidase, diluted in 0.98 mL of distilled water (1 mL as a total), under the same conditions as the Cg. At the 20th pregnancy day, the rats were anesthetized once again and submitted to dissection, and the cervix prepared for histochemical study with alcian blue dye and its blockades (pH=0.5, pH=2.5, methylation and saponification). RESULTS: strongly positive reaction in the lamina propria (+3) has been seen in the Cg, and negative reaction in the Exg, with pH=0.5 alcian blue staining. With pH=2.5, staining has also been strongly positive (+4) in the Cg, and weakly positive (+1) in the Exg slide. After methylation, both groups have shown negative reaction, with pH=2.5 alcian blue staining. The lamina propria staining became negative after methylation in both groups, followed by saponification and enzymatic digestion on slide. CONCLUSIONS: there is clear predominance of sulphated glycosaminoglycans in the Cg as compared to the Exg and a small amount of identified carboxylated glycosaminoglycans in the Exg. The changes evidenced in the extracellular matrix have suggested that the hyaluronidase injected in the uterine cervix has promoted biochemical changes compatible with cervix maturation.

Alteração da matriz extracelular da cérvice uterina durante a gestação / Changes in the extracellular matrix of the uterine cervix during pregnancy

A matriz extracelular do colo uterino sofre importantes mudanças em sua composição no decorrer da gestação e principalmente no período peripartal. Tais mudanças são necessárias para transformar o colo uterino imaturo, uma estrutrura fibrosa, em um órgão complacente o bastante para a passagem do feto no trabalho de parto. Conhecer melhor todo o processo de amadurecimento cervical é de fundamental importância para obterem-se melhores resultados na indução do parto.

[Histochemical changes of the glycosaminoglycans in the uterine cervix of pregnant rats after local injection of hyaluronidase]. / Alterações histoquímicas das glicosaminoglicanas na cérvice uterina no final da prenhez da rata albina após ministração local de hialuronidase.

PURPOSE: to study the histochemical changes related to the uterine cervix glycosaminoglycan of the albino female rat, after local administration of hyaluronidases at the end of pregnancy. METHODS: ten female rats with positive pregnancy tests were randomly distributed in two numerically equal groups. The control group (Cg) was built up with rats that received a single dose of 1 mL of distilled water in the uterine cervix, under anesthesia, at the 18th pregnancy day. In the experimental group (Exg), the rats received 0.02 mL of hyaluronidase, diluted in 0.98 mL of distilled water (1 mL as a total), under the same conditions as the Cg. At the 20th pregnancy day, the rats were anesthetized once again and submitted to dissection, and the cervix prepared for histochemical study with alcian blue dye and its blockades (pH=0.5, pH=2.5, methylation and saponification). RESULTS: strongly positive reaction in the lamina propria (+3) has been seen in the Cg, and negative reaction in the Exg, with pH=0.5 alcian blue staining. With pH=2.5, staining has also been strongly positive (+4) in the Cg, and weakly positive (+1) in the Exg slide. After methylation, both groups have shown negative reaction, with pH=2.5 alcian blue staining. The lamina propria staining became negative after methylation in both groups, followed by saponification and enzymatic digestion on slide. CONCLUSIONS: there is clear predominance of sulphated glycosaminoglycans in the Cg as compared to the Exg and a small amount of identified carboxylated glycosaminoglycans in the Exg. The changes evidenced in the extracellular matrix have suggested that the hyaluronidase injected in the uterine cervix has promoted biochemical changes compatible with cervix maturation.