RESUMO
PURPOSE: Although it is known that Müller cells express the glial fibrillary acidic protein (GFAP) in response to acute retinal damage, the regulatory mechanism is not completely understood. α(2)-Macroglobulin (α(2)M) and its receptor, low-density lipoprotein receptor-related protein 1 (LRP1), have also been found in injured retinas. Herein, the authors examined the involvement of the α(2)M/LRP1 system in GFAP expression in Müller cells using in vitro and in vivo experimental models. METHODS: Using Western blot analysis and immunocytochemistry, the authors evaluated the effect of α(2)M* on GFAP expression in the Müller cell line MIO-M1, which constitutively expresses LRP1. Intracellular signaling pathways activated by α(2)M* were examined by Western blot analysis. The effect of α(2)M* on GFAP expression in the mouse retina was examined by intravitreal microinjection of α(2)M* in mouse eyes. RESULTS: These data demonstrate that α(2)M* induced GFAP expression in the MIO-M1 cell line, which was selectively blocked by RAP, an antagonist of LRP1 binding ligands. In addition, α(2)M* induced JAK/STAT pathway activation, determined by STAT3 phosphorylation (p-STAT3), which was also blocked by RAP. Finally, the authors showed that GFAP was expressed in the retinas of mice, preferentially in Müller cells at 3 and 6 days after a single intravitreal α(2)M* injection, whereas p-STAT3 staining increased at day 1 in both the ganglion cell layer and the inner nuclear layer. CONCLUSIONS: These results demonstrate that α(2)M* induces GFAP expression in retinal Müller cells through LRP1, which could be mediated by JAK/STAT pathway activation.
Assuntos
Proteína Glial Fibrilar Ácida/metabolismo , Neuroglia/efeitos dos fármacos , Receptores de LDL/metabolismo , Proteínas Supressoras de Tumor/metabolismo , alfa-Macroglobulinas/farmacologia , Animais , Western Blotting , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Humanos , Imuno-Histoquímica , Janus Quinases/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Neuroglia/metabolismo , Ratos , Fator de Transcrição STAT3/metabolismo , Vimentina/metabolismoRESUMO
OBJECTIVE: To describe the functional and structural characteristics of the cornea in healthy Guinea pigs. ANIMALS STUDIED: Healthy male and female pigmented and albino Guinea pigs (Caviaporcellus) aged 3-5 months old were used. PROCEDURES: The animals' corneas underwent different in vivo studies including: slit-lamp biomicroscopy, fluorescein staining (FS), break-up time test (BUT), confocal microscopy and pachymetry. The corneas were also studied histopathologically with light microscopy, immunohistochemistry and transmission electron microscopy. RESULTS: No significant differences were found between pigmented and albino animals, male and female, OD and OS in any study performed. The differences on corneal thickness values were not significant among central (227.85 +/- 14.09 microm) and upper and temporal peripheral regions (226.60 +/- 12.50 and 225.70 +/- 14.40 microm, respectively). All histological studies performed permitted identification and precise description of the different corneal structures in Guinea pigs: the stratified epithelium (45.52 +/- 5.26 microm), Bowman's layer (2.23 +/- 0.38 microm), stroma (163.69 +/- 4.90 microm), Descemet's membrane (3.96 +/- 0.46 microm) and the endothelium (5.09 +/- 0.71 microm). Combining results from all eyes mean and SD from corneal BUT values was 4.98 +/- 1.67 s. Corneas often showed discrete superficial erosions being the FS positive in both eyes from all the animals. CONCLUSION: This study provides a detailed in vivo and postfixed histological description of the Guinea pig's cornea and information about the physiological tests.
Assuntos
Córnea/anatomia & histologia , Córnea/fisiologia , Cobaias/anatomia & histologia , Cobaias/fisiologia , Animais , Feminino , Masculino , MicroscopiaRESUMO
The low-density lipoprotein receptor-related protein-1 (LRP-1) is a high-molecular weight receptor of the LDL receptor gene family. Its ability to bind and internalize both proteinases and proteinase-inhibitor complexes from the extracellular space suggests that it has a major role in modulating uncontrolled retinal cell proliferation. In order to test this assumption, we investigated the expression of LRP-1 and receptor-associated ligands in a rat model of oxygen-induced retinal neovascularization. Wistar albino rats were placed into incubators at birth and exposed to an atmosphere alternating between 50% and 10% of oxygen every 24 h. After 14 days, the animals were allowed to recover in room air and sacrificed at postnatal day 20 (P20). The protein expression of LRP-1 and alpha2-macroglobulin (alpha2M) in the retina from unexposed and hyperoxia-exposed rats was investigated by Western blot. The localization of LRP-1 after neovascularization was assessed by immunohistochemical staining. The activity of metalloproteinases (MMPs) was determined by zymography. Histological analysis was done to quantitate the neovascular response in these animals. Western blot analysis showed that LRP-1 was expressed, along with alpha2M, in the retina of rats with oxygen-induced neovascularization at P20. By immunohistochemical analysis, positive staining for LRP-1 appeared in cells extending from the inner limiting membrane (ILM) to the outer limiting membrane (OLM). The cells of the retina that expressed LRP-1 were identified by immunofluorescence as Müller cells. Zymographic analysis demonstrated increased activity of MMP-2 and MMP-9 under neovascular conditions. This is the first demonstration of the involvement of LRP-1 in retinal neovascularization. In retinas of rats with oxygen-induced neovascularization, the expression of LRP-1 and alpha2M was increased along with an enhanced activity of MMPs, suggesting that LRP-1 expression may play a role in modulating retinal neovascularization by regulating proteolytic activity.