A novel organotypic culture model of the postnatal mouse retina allows the study of glutamate-mediated excitotoxicity.
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 PKCalpha, 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.
Xin
Hua
H
University of North Texas Health Science Center, Department of Pharmacology & Neuroscience, 3500 Camp Bowie Blvd., Fort Worth, TX 76107-2699, USA.
Yannazzo
Jo-Ann S
JA
Duncan
R Scott
RS
Gregg
Elaine V
EV
Singh
Meharvan
M
Koulen
Peter
P
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D003470:Culture Media; D004305:Dose-Response Relationship, Drug; D018690:Excitatory Amino Acid Agonists / Q000633:toxicity; D018698:Glutamic Acid / Q000502:physiology*; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D008959:Models, Neurological; D016202:N-Methylaspartate / Q000633:toxicity; D009410:Nerve Degeneration / Q000473:pathology; D009924:Organ Culture Techniques / Q000379:methods*; D012160:Retina / Q000166:cytology / Q000502:physiology*; D012165:Retinal Ganglion Cells / Q000187:drug effects / Q000502:physiology
D003470:Culture Media; D018690:Excitatory Amino Acid Agonists; D018698:Glutamic Acid; D016202:N-Methylaspartate