A novel organotypic culture model of the postnatal mouse retina allows the study of glutamate-mediated excitotoxicity

Hua Xin, Jo Ann S. Yannazzo, R. Scott Duncan, Elaine V. Gregg, Meharvan Singh, Peter Koulen

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

A novel organotypic culture method of mouse retina explants is being introduced and characterized to evaluate its usefulness in studying glutamate excitotoxicity. Retinal whole-mounts were dissected from eyes of C57BL/6 mice aged P10-14 and transferred to poly-d-lysine/laminin coated round coverslips. After 7 days in vitro, retina explants were treated with varying concentrations of l-glutamate and cell death was accessed with TUNEL histochemistry. Neurofilament-68 kDa immunoreactivity was used to identify retinal ganglion cells (RGC) with immunohistochemistry. Additional cell markers were used to further characterize the cytoarchitecture of the organotypic retina cultures. Retina explants attached very well to the coated coverslips allowing for experimental manipulation and pharmacological access to the tissue. Hematoxylin-Eosin (HE) staining of vertical cryostat sections of retina explants demonstrated well preserved intact cytoarchitecture under organotypic culture conditions and PKCα, Calbindin, GABA, Rhodopsin, GFAP and neurofilament immunoreactivities identifying rod bipolar, horizontal, amacrine, photoreceptor, glial, and retinal ganglion cells, respectively, were not different from freshly isolated mouse retina. Dose dependent glutamate toxicity and accompanying RGC apoptotic cell death were determined by TUNEL histochemistry. In contrast to previously published methods using slice or floating whole-mount cultures, the ex vivo culture system presented here combines accessibility to experimental manipulation, and adherence of whole-mount cultures to a substrate with a significant preservation of retinal cell types, numbers and morphology. The described retina explant culture on glass coverslips allows for effective pharmacological manipulation including the study of neuronal cell death and RGC physiology.

Original languageEnglish
Pages (from-to)35-42
Number of pages8
JournalJournal of Neuroscience Methods
Volume159
Issue number1
DOIs
StatePublished - 15 Jan 2007

Fingerprint

Retina
Glutamic Acid
Retinal Ganglion Cells
Cell Death
Intermediate Filaments
In Situ Nick-End Labeling
Pharmacology
Calbindins
Cell Physiological Phenomena
Rhodopsin
Laminin
Hematoxylin
Eosine Yellowish-(YS)
Inbred C57BL Mouse
Neuroglia
gamma-Aminobutyric Acid
Lysine
Glass
Cell Count
Immunohistochemistry

Keywords

  • Glutamate-mediated excitotoxicity
  • Organotypic culture
  • Retina explants
  • Retinal ganglion cells
  • TUNEL

Cite this

Xin, Hua ; Yannazzo, Jo Ann S. ; Duncan, R. Scott ; Gregg, Elaine V. ; Singh, Meharvan ; Koulen, Peter. / A novel organotypic culture model of the postnatal mouse retina allows the study of glutamate-mediated excitotoxicity. In: Journal of Neuroscience Methods. 2007 ; Vol. 159, No. 1. pp. 35-42.
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A novel organotypic culture model of the postnatal mouse retina allows the study of glutamate-mediated excitotoxicity. / Xin, Hua; Yannazzo, Jo Ann S.; Duncan, R. Scott; Gregg, Elaine V.; Singh, Meharvan; Koulen, Peter.

In: Journal of Neuroscience Methods, Vol. 159, No. 1, 15.01.2007, p. 35-42.

Research output: Contribution to journalArticle

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