Exogenous Glutamate Modulates Porcine Retinal Development in vitro

Abstract

Embryogenesis of the retina is a complex event orchestrated by a multitude of physical and biochemical signals. To study the impact of intrinsic developmental cues, the retinal tissue can be isolated in culture which also enables modulation of normal development for other purposes, i.e. transplantation of specific neuronal cell types. In the present experiment, cell type development of immature porcine retinal tissue kept in culture was explored using specific immunohistochemical markers. Retinal explants were either kept under standard culture conditions or supplemented with glutamate and their morphology was compared with in vivo controls of corresponding age. After 15 days in vitro (DIV), E45 retinal explants displayed several signs of atypical development when compared with E60 in vivo controls. First, an accelerated photoreceptor differentiation was evident, seen in sections labeled with antibodies directed against recoverin, rhodopsin and synaptophysin. Second, apoptotic cells in the inner retina were more prevalent in the cultured retinas (TUNEL). Rod photoreceptor differentiation as well as inner retinal apoptosis was even more pronounced in glutamate-supplemented specimens in which they occurred already at 8 DIV. Müller cell, vimentin and GFAP expression was not affected in any of the cultured retinas. These results suggest that normal retinal embryogenesis is more dependent on tissue extrinsic factors than what has been deduced from previous small animal experiments. Glutamate, which has been identified as an important regulator of cell cycle exit, may also be important for photoreceptor differentiation and induction of developmental apoptosis. Insights into retinal cell type differentiation under in vitro conditions is of interest from a biological standpoint, and the possibility of modulation of this process is valuable for research directed towards cell replacement in retinal degenerative disease.

Feet on the Ground: Physical Support of the Inner Retina Is a Strong Determinant for Cell Survival and Structural Preservation In Vitro

“…The overall morphology and immunohistochemical characteristics of the normal adult porcine retina has been well described previously but will be summarized here [4][5][12][13][14][15][16][17][18]. Hematoxylin and eosin staining of in vivo controls revealed clearly defined nuclear and plexiform layers as previously described (Fig.…”

“…In addition, it was believed that the support provided by the culture membrane would mimic physical RPE-outer segment interaction in vivo. This approach has been used successfully for the study and modulation of immature tissue which has been found to survive well with several signs of normal development [9][10][11][12][13]. In contrast, as mentioned above, isolated adult retinal sheets cultured under standard conditions, display gliosis and neuronal degeneration very early [14][15][16][17].…”

Current Advancements in the Development and Characterization of Full-Thickness Adult Neuroretina Organotypic Culture Systems

“…This approach has been used successfully for the study and modulation of immature retinal tissue, which has been found to survive well and even displays several signs of normal development [Caffé et al, 1989;Ogilvie et al, 1999Ogilvie et al, , 2000Engelsberg and Ghosh, 2007;Ghosh et al, 2012]. Cultures of adult retinal tissue, on the other hand, have been found to be much more limited in their ability to survive in vitro.…”