T. van Veen

Spectral characteristics of visible radiation penetrating into the brain and stimulating extraretinal photoreceptors

SummarySupravital recordings of spectral transmission in the brains of two species of teleosts (Anguilla anguilla, Ictalurus nebulosus), an amphibian (Rana temporaria), a reptile (Lacerta muralis), two species of birds (Passer domesticus, Columba livia), and a mammal (Phodopus sungorus) indicate that photons of longer wavelengths (700–750 nm) penetrate approximately 1,000 times more effectively into the hypothalamus than photons of shorter wavelengths (400–450 nm). The decrease in transmission from 750 to 400 nm is slightly interrupted by a plateau around 500 to 540 nm because of the transmission characteristics of hemoglobin. There is a small, ill-defined transmission minimum around 430 nm corresponding to the transmission minimum of melanin and hemoglobin (soret band). The high light sensitivity of deep diencephalic photoreceptors involved in the control of photoneuroendocrine events characteristic of some non-mammalian vertebrates suggests the occurrence of photopigment-containing receptors and nerve cells summating the input of several photoreceptors. However, in addition to photopigments, there may also exist other photosensitive compounds that mediate non-visual photoneuroendocrine responses.

Significant photoreceptor rescue by treatment with a combination of antioxidants in an animal model for retinal degeneration

The purpose of this study was to investigate the presence of oxidative DNA damage in the photoreceptors of the rd1 mouse, an animal model for retinitis pigmentosa, and to determine if antioxidants could delay the progress of photoreceptor cell death. Retinas of rd1 mice and congenic wild type controls were examined for DNA oxidation and fragmentation. To study the rescue effect of antioxidants on retinal degeneration, rd1 retinas were studied in vitro and in vivo using lutein, zeaxanthin, alpha lipoic acid and reduced l-glutathione. For the in vitro studies, antioxidants were added to the culture medium. For the in vivo studies, postnatal day (PN3) pups of rd1 mice were fed antioxidants either individually or in combination and control rd1 animals received vehicle alone. Histological evaluation was performed using hematoxylin/eosin and avidin staining, as well as terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Many of the rd1 rod photoreceptors at PN11 displayed oxidative DNA damage and TUNEL positive reaction which co-localized in a subset of rod photoreceptors. Avidin-labeled rod photoreceptors were more abundant than the TUNEL positive photoreceptors of the rd1 mouse, indicating that oxidative DNA damage precedes fragmentation. The number of TUNEL positive and avidin positive cells was considerably decreased upon treatment with the combination of the antioxidants. Rescue of rd1 photoreceptors was significant at PN18 and PN17, respectively, in the in vitro and in vivo studies. In conclusion individual antioxidants had no significant rescue effect but the combination slowed down the rd1 rod photoreceptor degeneration, indicating an additive or synergistic effect.

Mouse retina explants after long-term culture in serum free medium

The neonatal mouse retina remains viable as an explant in serum-supplemented growth media for more than 4 weeks. Interpretation of drug effects on this tissue is compromised by the enigmatic composition of the serum. We sought to remove this ambiguity by culturing neonatal as well as late postnatal mouse retina in serum-free nutrient medium. In this study three important observations were made, (1) there is histotypic development of neonatal as well as preservation of late postnatal mouse retinal structure during long-term culture in serum-free medium, although the late postnatal tissue tends to show some loss of cells in the outer nuclear layer. (2) Protein expression in explant photoreceptor cells was similar to that in the litter-matched ones, except for green cone opsin and interphotoreceptor retinoid-binding protein, although mRNA of the latter is present at similar amounts as in age-matched in vivo controls. (3) Cells of the inner retina stained by antibodies to calcium-binding proteins display some novel sprouting of processes. The results show that the mouse retina can be cultured as an explant for more than 4 weeks in a serum-free medium. This represents an important step forward because, (1) the possibility of interference of drug effects by unknown serum factors has been eliminated; and (2) the spent culture medium can be analyzed to investigate biomolecules released by the retina in vitro.

Excessive HDAC activation is critical for neurodegeneration in the rd1 mouse

Inherited retinal degenerations, collectively termed retinitis pigmentosa (RP), constitute one of the leading causes of blindness in the developed world. RP is at present untreatable and the underlying neurodegenerative mechanisms are unknown, even though the genetic causes are often established. Acetylation and deacetylation of histones, carried out by histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively, affects cellular division, differentiation, death and survival. We found acetylation of histones and probably other proteins to be dramatically reduced in degenerating photoreceptors in the rd1 human homologous mouse model for RP. Using a custom developed in situ HDAC activity assay, we show that overactivation of HDAC classes I/II temporally precedes photoreceptor degeneration. Moreover, pharmacological inhibition of HDACs I/II activity in rd1 organotypic retinal explants decreased activity of poly-ADP-ribose-polymerase and strongly reduced photoreceptor cell death. These findings highlight the importance of protein acetylation for photoreceptor cell death and survival and propose certain HDAC classes as novel targets for the pharmacological intervention in RP.

A developmental study of interphotoreceptor retinoid-binding protein (IRBP) in single and double homozygous rd and rds mutant mouse retinae

Interphotoreceptor retinoid-binding protein (IRBP) was studied using immunochemical and immunocytochemical techniques in retinae of mice with allelic combinations at the rd and rds loci at different stages of development and degeneration. Until postnatal day 7 (P7), IRBP is located intracellularly in developing retinae of the different genotypes. Thereafter, IRBP is present mainly in the interphotoreceptor matrix. As previously noted, cell death is slowest in the heterozygous +/+,rds/+ mutant with loss increasing in order in +/+,rds/rds, rd/rd, rds/rds and rd/rd,+/+ animals. The IRBP content of the total retina also approximates this pattern, with lowest amounts by far in rd/rd, rds/rds and rd/rd,+/+ mutants (after P14). Interestingly though, IRBP loss significantly precedes visual cell loss in the rd/rd,rds/rds retina. In all the mutants, the remaining rod cells in the outer nuclear layer exhibit synthesis of intracellularly located IRBP at late stages of degeneration. In the single homozygous rd/rd,+/+ and the double homozygous rd/rd,rds/rds mutants, IRBP is present intracellularly during the entire degenerative process with somewhat less intracellular IRBP in the rd/rd,rds/rds mutant. Retinae of homozygous +/+,rds/rds and heterozygous +/+,rds/+ animals exhibit a normal distribution pattern of IRBP immunoreactivity until loss of photoreceptor cells becomes pronounced at later stages of the disease. Many of the remaining cells at this time are probably cone elements although they are structurally changed. Double labeling with IRBP and S-antigen demonstrates, in many but not all, the presence of both proteins in the same cell body. Immunocytochemistry clearly demonstrated the presence of IRBP in remaining photoreceptor cells at late stages of the disease. Thus, the biochemically measured loss of IRBP appears to be a complex process neither directly dependent on the loss of photoreceptor outer segments and reduced interphotoreceptor matrix space (e.g. there is a sustained IRBP level in rodless rds mutants) nor simply due to cell death (e.g. in the rd/rd,rds/rds mutant, IRBP loss significantly precedes cell loss). That this IRBP is mainly intracellular, however, may indicate an abnormality in secretion which, combined with other factors, induces a degenerated and less differentiated phenotype.

Lens epithelium-derived growth factor (LEDGF) delays photoreceptor degeneration in explants of rd/rd mouse retina

Lens epithelium derived growth factor (LEDGF) has been shown to rescue embryonic chick photoreceptor cells from serum starvation and heat stress, light damaged photoreceptor cells in Lewis rats, and photoreceptor cells in RCS rats. The aim of our study is to study the rescue effect of LEDGF on photoreceptor cells in the rd/rd mouse using our long-term serum free organ culture. At the end of this culture period of 21–26 days LEDGF treated rd mouse retina showed an increased photoreceptor survival compared to the untreated controls. LEDGF has no effect on expression and localization of opsin and arrestin in the rod photoreceptor cells when RPE is present. The protective potency of LEDGF on the retinal photoreceptor cells is similar to that of BDNF. LEDGF is known to activate heat shock proteins (Hsps) and the elevated Hsps are also reported to suppress apoptosis.

DECREASED GLUTATHIONE TRANSFERASE LEVELS IN rd1/rd1 MOUSE RETINA: REPLENISHMENT PROTECTS PHOTORECEPTORS IN RETINAL EXPLANTS

Currently much attention is focused on glutathione S transferase (GST)-induced suppression of apoptosis. The objective of our studies was therefore to see if GST isoenzymes rescue photoreceptors in retinal explants from rd1/rd1 mice, in which photoreceptors degenerate rapidly. Eyes from C3H rd1/rd1 and +/+ mice were collected at various time points between postnatal day (PN) 2 and PN28. Localization and content of alpha-GST and mu-GST was investigated by immunofluorescence and semi-quantitative Western blot analysis, respectively. In addition, PN2 and PN7 retinal explants were cultured till PN28, during which they were treated with 10 ng/ml alpha-GST or mu-GST. The spatiotemporal expression of both GST isoforms was closely similar: early presence in ganglion cell layer after which staining became restricted to Muller cells (particularly in the endfeet) and horizontal cell fibers in both rd1/rd1 and +/+. Doublets of alpha-GST and mu-GST were detected by Western blot analysis. Densitometry of these bands indicated steady reduction of alpha-GST content in rd1/rd1 retina starting from the second postnatal week. When alpha-GST and mu-GST were added exogenously to rd1/rd1 explants, photoreceptor rescue was produced that was more prominent in PN2 than in PN7 explants and more effective by alpha-GST than mu-GST. We propose that alpha-GST neuroprotection is mediated by reduction of tissue oxidative stress.

Photoreceptor degeneration and loss of immunoreactive GABA in the Abyssinian cat retina.

GABA (gamma-amino butyric acid) and its synthesizing enzyme, GAD (glutamate decarboxylase; EC 4.1.1.15) were localized in the retina of Abyssinian cats homozygous for a recessively inherited retinal degenerative disorder which in several respects is similar to the human disease, retinitis pigmentosa. Clinically normal mongrel cats and heterozygous Abyssinian cats were studied for comparison. The GABA and GAD immunoreactive neurons of the heterozygous or young homozygous (clinically unaffected animals) had the same distribution and morphology as normal mongrel European type cats. The neuronal GABA immunoreactivity in both the inner and outer parts of the retina gradually disappeared in the course of the disease, with little or no loss of GAD immunoreactive neurons. Early in the disease, the changes were most severe in patches in the mid periphery of the eye and then spread both centrally and peripherally. Loss of photoreceptors was a prerequisite for the loss of GABA immunoreactivity. The observations show that retinal changes are not limited to the photoreceptors. The GABA loss is not likely to be due to a loss of neurons, because of the persistence of GAD immunoreactive neurons.