A reliable method for organ culture of neonatal mouse retina with long-term survival.

Organ culture systems of the central nervous system have proven to be useful tools for the study of development, differentiation, and degeneration. Some studies have been limited by the inability to maintain the cultures over an extended period. Here we describe an organ culture technique for the mouse retina. This method uses commercially available supplies and reproducible procedures to maintain healthy retinas with normal architecture for 4 weeks in vitro. The system is amenable to quantitative analysis. It can be used with both normal and retinal degeneration (rd) retinas to study of the role of various factors in photoreceptor degeneration in retinal cell fate determination and development.

Age-related distribution of cones and ON-bipolar cells in the rd mouse retina.

PURPOSE: Retinal dystrophic (rd) mice lose most of their rod photoreceptors within the first three weeks after birth. We determined the age-related distribution of peanut agglutinin lectin (PNA)-labeled cones during the first 12 months of age. We also investigated whether the density of ON-bipolar cells expressing L7 protein was affected by their loss of photoreceptor inputs. METHODS: rd mice were selected from a transgenic strain which expresses an L7-beta-galactosidase fusion gene localized to ON-bipolar cells. Cones were stained with PNA and ON-bipolar cells with bluo-gal (halogenated indolyl-beta-D-galactoside). Retinas were flat-mounted and observed at 1, 2, 3, 6 and 12 months of age. RESULTS: PNA-labeled cones are distributed unevenly across the retina at 1 month postnatal. Their concentration decreases first in the central and far peripheral retina, leaving a ring of labeled cells in the midperipheral region. At 3 months, a larger patch of cones remains in the supero-temporal midperipheral region and a smaller patch in the infero-nasal retina. By 6 months, few cones remain in the infero-nasal retina; by 1 year approximately 100 cones remain in the entire retina, localized to the superior midperipheral region. ON-bipolar cells appear evenly distributed at 1 month. By 2-3 months, relatively more bluo-gal staining is seen in the midperipheral regions underlying dense cone populations. At 6-12 months, bluo-gal label is distributed in a spotty pattern with little or no staining seen in areas of apparent neovascularization. CONCLUSIONS: (1) PNA-labeling of cones in the rd retina deteriorates in a distinct spatial pattern with the longest cone survival in the midperipheral superior retina. (2) ON-bipolar cells are more densely labeled in regions of high cone density during the early months of cone degeneration and, in later stages, show relative decreases in regions of apparent neovascularization.

Growth factors in combination, but not individually, rescue rd mouse photoreceptors in organ culture.

The rd mouse retina is an animal model for human retinal dystrophy in which the rod photoreceptors undergo apoptosis during the first 4 weeks in vivo or in organ culture. We have examined the effect of different families of trophic factors on the survival of rd mouse photoreceptors in organ culture. Retinas were harvested from rd mice at postnatal day 2 and grown in organ culture for 27 days in vitro (DIV) in DMEM with 10% fetal calf serum. Ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), fibroblast growth factor-2 (FGF2), glial cell line-derived neurotrophic factor (GDNF), neurturin, and persephon were added individually or in combination to the medium at a dose of 50 ng/ml or less. CNTF + BDNF in combination resulted in photoreceptor survival comparable to wild-type retinas after 27 DIV. CNTF + FGF2 or CNTF + GDNF produced a partial prevention of photoreceptor death. Photoreceptor degeneration was not blocked by any of the trophic factors added individually. A significant increase in photoreceptor survival was seen with forskolin added to CNTF, but not to BDNF, FGF2, or GDNF. These results demonstrate that trophic factors promote photoreceptor survival through a synergistic interaction. Increased understanding of receptor interactions and signaling pathways may lead to a potential therapeutic role for combinatorial trophic factors in treatment of photoreceptor dystrophies.

Progression of cochlear and retinal degeneration in the tubby (rd5) mouse.

Mice homozygous for a defect of the tub (rd5) gene exhibit cochlear and retinal degeneration combined with obesity, and resemble certain human autosomal recessive sensory deficit syndromes. To establish the progressive nature of sensory cell loss associated with the tub gene, and to differentiate tub-related losses from those associated with the C57 background on which tub arose, we evaluated cochleas and retinas from tub/tub, tub/+, and +/+ mice, aged 2 weeks to 1 year by light and electron microscopy. Cochleas from mice of all three genotypes show progressive inner (IHC) and outer hair cell (OHC) loss. Relative to tub/+ and +/+ animals, however, tub homozygotes show accelerated OHC loss, affecting the extreme cochlear base (hook region) by 1 month, and the apex by 6 months. IHC loss in tub/tub animals is accelerated in the basal half of the cochlea, affecting the hook region by 6 months. Spiral ganglion cell losses were observed only in tub/tub mice, and only in the cochlear base. Retinas of tub/tub mice are abnormal at maturity, exhibiting shortened photoreceptor outer segments by 2 weeks, and progressive photoreceptor loss thereafter. Because the tub mutation causes degeneration of sensory cells in the ear and eye but has no other neurological effects, tubby mice hold unique promise for the study of human syndromic sensory loss.

Photoreceptor transplants increase host cone survival in the retinal degeneration (rd) mouse.

Retinal transplants offer a potentially interesting approach to treating human retinal degenerations, but so far little quantitative data are available on possible beneficial effects. We isolated photoreceptor layers from normal-sighted mice and grafted them into the subretinal space of retinal degeneration (rd) mice lacking rod photoreceptors. At 2 weeks after surgery, the numbers of residual host cone photoreceptors outside the graft zone were quantified following specific labelling. Examination of operated retinas revealed highly significantly greater numbers of surviving cones (mean of 38% more at 2 weeks) within the central field compared to sham-operated paired control retinas (p < 0.01). These are the first quantified data indicating a trophic effect of transplanted photoreceptors upon host cone cells. As cone cells are responsible for high acuity and colour vision, such data could have important implications not only for eventual therapeutic approaches to human retinal degenerations but also to understanding underlying interactions between retinal photoreceptors.