Neuronal integration in an abutting-retinas culture system.
OBJECTIVE
Limited integration is consistently observed between subretinal transplants and host retinas. In the current study, an in vitro model system for studying connections forming between two abutting retinas was developed.


METHODS
Neuroretinas were dissected from normal wild-type (WT) mice and green fluorescent protein (GFP) transgenic mice (obtained at postnatal days [P]0, P5, or P60), as well as from adult rd mice. Pieces from two different retinas (WT-WT, GFP-WT, GFP-rd) were placed side-by-side (contacting each other at the margins) or overlapping each other in organ cultures for 7 or 12 days. The abutting retinal pieces derived from animals of the same age (P5-P5; P60-P60) or of different ages (P0-P60; P5-P60). Retinal cells and fibers were visualized in wholemount preparations and in cross sections by immunocytochemistry using antibodies against neurofilament (NF+), neuronal nitric oxide synthase (NOS+), and protein kinase C (PKC+) and by GFP fluorescence (GFP+).


RESULTS
In side-by-side pairs (WT-WT, GFP-WT), numerous horizontal cell fibers (NF+) and amacrine cell fibers (NOS+) crossed the interface between the two pieces, forming continuous plexiform layers. In overlapping pairs, NF+, NOS+, and PKC+ fibers displayed parallel plexiform layers, and no crossover of fibers was observed in any of the pair combinations examined (WT-WT, GFP-WT, GFP-rd). Some integration was seen only in small areas where the structure of both retinal pieces was disrupted at the interface.


CONCLUSIONS
The results demonstrate the ability of neurites to extend between abutting retinas and to make appropriate target choices when they are placed side-by-side. However, this ability is limited when they overlap each other, similar to that observed in subretinal transplantation.
Zhang
Yiqin
Y
Wallenberg Retina Center, Department of Ophthalmology, Lund University, Lund, Sweden.
Caffé
A Romeo
AR
Azadi
Seifollah
S
van Veen
Theo
T
Ehinger
Berndt
B
Perez
Maria-Thereza R
MT
D000818:Animals; D019084:Fluorescent Antibody Technique, Indirect; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009434:Neural Pathways / Q000502:physiology*; D016501:Neurites / Q000502:physiology*; D016900:Neurofilament Proteins / Q000378:metabolism; D009457:Neuroglia / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I; D009924:Organ Culture Techniques; D011493:Protein Kinase C / Q000378:metabolism; D012160:Retina / Q000166:cytology* / Q000378:metabolism; D012162:Retinal Degeneration / Q000378:metabolism / Q000473:pathology
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D016900:Neurofilament Proteins; D049452:Green Fluorescent Proteins; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496321:Nos1 protein, mouse; D011493:Protein Kinase C