N-methyl-D-aspartate (NMDA)--induced apoptosis in rat retina.

PURPOSE: The involvement of apoptosis in N-methyl-D-aspartate (NMDA)-induced excitotoxicity in adult rat retinas was examined. METHODS: Excitotoxic loss of inner retinal elements was induced by intravitreal injections of various concentrations of neutralized NMDA in adult albino Lewis rats. Tissue responses were quantified by measuring the inner retinal thickness (IRT) in plastic sections of the retinas and cell counts in the retinal ganglion cell layer in flatmount preparations of the whole retinas. Internucleosomal DNA fragmentation, a hallmark of apoptosis, was assayed with agarose DNA gel electrophoresis. The in situ TdT-mediated biotin-dUTP nick end labeling (TUNEL) method was used to locate nicked DNA in paraffin sections of the retinas. Ultrastructural changes of the degenerating cells were examined by electron microscopy. The efficacy of Ac-Tyr-Val-Ala-Asp-CMK (YVAD-CMK), a peptidyl caspase inhibitor, and 3-aminobenzamide (ABA), an inhibitor of poly(ADP-ribose) polymerase (PARP), in ameliorating the loss of inner retinal elements was evaluated using morphometry to examine the apoptotic pathways. RESULTS: Intravitreal injection of NMDA induced a dose-dependent loss of inner retinal elements as evidenced by the measurements of IRT and RGCCs. There were time- and dose-related appearances of internucleosomal fragmentation of retinal DNA and a time-related appearance of TUNEL-positive nuclei in the inner retinas after intravitreal NMDA injection. Ultrastructural features consistent with classic apoptotic changes were noted in degenerating cells in the retinal ganglion cell layer and the inner nuclear layer. Control retinas given vehicle, N-methyl-L-aspartate (the L-isomer of NMDA), or NMDA plus MK-801, a specific antagonist, did not show these changes. Simultaneous administration of NMDA and YVAD-CMK or ABA abolished or attenuated the loss of RGCCs in the posterior retinas. CONCLUSIONS: NMDA-induced excitotoxicity involved apoptosis and caspases and PARP may play important roles in the pathways.

Apoptosis and caspases after ischemia-reperfusion injury in rat retina.

PURPOSE: Extensive cell loss in the retinal ganglion cell layer (RGCL) and the inner nuclear layer (INL) was noted in a rat model of retinal ischemia-reperfusion injury by transient elevated intraocular pressure (IOP). The possible involvement of apoptosis and caspases was examined in this model of neuronal loss. METHODS: Transient elevated IOP was induced in albino Lewis rats through the insertion of a needle into the anterior chamber connected to a saline column. Elevated IOP at 110 mm Hg was maintained for 60 minutes. Groups of animals were euthanatized at various times after reperfusion, and their retinas were evaluated by morphology, agarose gel electrophoresis of DNA, in situ terminal deoxynucleotidyl transferase-mediated biotin-deoxyuridine triphosphate nick-end labeling (TUNEL), immunohistochemistry of caspases II (ICH1) and III (CPP32), and morphometry. YVAD.CMK, a tetrapeptide inhibitor of caspases, was used to examine the involvement of caspases. RESULTS: A marked ladder pattern in retinal DNA gel analysis, typical of internucleosomal DNA fragmentation and characteristic of apoptosis, was present 12 and 18 hours after reperfusion. Labeling of nuclei in the RGCL and the inner nuclear layer (INL) by TUNEL was noted between 8 and 18 hours after reperfusion. Histologic and ultrastructural features typical of apoptosis were also observed in the inner retina after ischemia. YVAD.CMK administered during the ischemic period inhibited apoptotic fragmentation of retinal DNA and ameliorated the tissue damage. When administered intravitreally 0, 2, or 4 hours after reperfusion, YVAD.CMK was also effective in preserving the inner retina but had no significant effect when administered 6 or 8 hours after reperfusion. The inner retina showed transient elevated immunoreactivity of caspases II and III 4 and 8 hours after reperfusion. CONCLUSIONS: Retinal ischemia-reperfusion after transient elevated IOP induced apoptosis of cells in the retinal ganglion cell layer and the INL. Caspases may have a pivotal role in the early events of the apoptotic pathway(s). Rescue by using anti-apoptotic agents after ischemia-reperfusion is feasible.

Cyr61 and Fisp12 are both ECM-associated signaling molecules: activities, metabolism, and localization during development.

cyr61 and fisp12 are homologous immediate-early genes that are transcriptionally activated upon growth factor stimulation in fibroblasts. Their gene products belong to an emerging family of secreted proteins with a high degree of sequence homology, including conservation of all 38 cysteine residues in their secreted portions. We have recently shown that Cyr61 is an extracellular matrix (ECM) signaling molecule that promotes cell proliferation, migration, and adhesion. We describe herein the first purification of the Fisp12 protein and we compare the activities of purified Cyr61 and Fisp12, their metabolism, targeting, and their localization during development. Although Fisp12 is the mouse homolog of the human connective tissue growth factor (CTGF), it has no detectable mitogenic activity by itself. Rather, Fisp12 enhances fibroblast growth factor-induced DNA synthesis. The activities of Fisp12 and Cyr61 are nearly indistinguishable in three cell types tested: fibroblasts, endothelial, and epithelial cells. Both proteins are found in the ECM, although Cyr61 associates with the ECM more strongly and binds heparin with higher affinity. Fisp12, but not Cyr61, is also found in the culture medium, suggesting that Fisp12 might be able to act at a distance from its site of secretion, whereas Cyr61 might act more locally. Both secreted proteins are internalized and degraded through the lysosomal pathway, suggesting interaction with cell surface receptors. Both Cyr61 and Fisp12 are found in the placenta and the circulatory system as detected by immunohistochemistry, whereas Cyr61, but not Fisp12, is found in the skeletal and nervous systems. Fisp12, but not Cyr61, is found in secretory organs. Taken together, we propose that Cyr61 and Fisp12 are both signaling cell adhesion molecules that have similar or overlapping activities, and their differential sites of localization and targeting may dictate specificity in their biological roles.

Unscheduled DNA replication precedes apoptosis of photoreceptors expressing SV40 T antigen.

Expression of simian virus 40 T antigen (Tag) in the rod photoreceptors of transgenic mice leads to cell death that is completed by the end of the third week of postnatal development. To understand the mechanistic link between Tag expression and the death of the expressing photoreceptors, cell cycle activity was followed in a transgenic mouse family that expresses Tag directed by the mouse opsin promoter. Tag-expressing photoreceptors also expressed rhodopsin suggesting that these cells were differentiated. The presence of Tag in the photoreceptors induced the expression of both proliferating cell nuclear antigen (PCNA) and thymidine kinase (TK). The abnormally high levels of PCNA and TK continued until the complete disappearance of the cells expressing Tag. Photoreceptor cell death was also associated with continued DNA synthesis that ceased shortly after postnatal day 16. The specific loss of the rod photoreceptors that re-entered the cell cycle accounted entirely for the loss of photoreceptors from the outer nuclear layer. The antiproliferative nature of the mature retina is directly involved in the apoptotic death of photoreceptors expressing Tag.

A comparison of continuous versus intermittent light exposure on apoptosis.

PURPOSE: We recently found that continuous light exposure at a moderate intensity triggered apoptosis of photoreceptor cells. Since intermittent light exposure is known to cause more severe retinal damage than is continuous light exposure, we sought to determine if intermittent light exposure also triggered apoptosis of photoreceptor cells. METHODS: Lewis albino rats were reared, for 2 weeks, in cyclic light and dark adapted for 24 hr before light exposure. Rats were exposed to intermittent light or continuous light for 6 or 9 hr, respectively. Light-exposed rats were killed by lethal injection at three timepoints: immediately after light exposure, after 6 hr of dark recovery following light exposure and after 24 hr of dark recovery following light exposure. Retinal damage after light exposure was evaluated by morphology, morphometry, the terminal transferase-mediated biotin dUTP nick end labeling (TUNEL) technique for identification of nicked/cleaved nuclear DNA and agarose gel electrophoresis of retinal DNA. RESULTS: Evaluation of morphology confirmed that intermittent light exposure caused more photoreceptor cell damage than did continuous light exposure of the same duration and intensity. The TUNEL technique showed that photoreceptor nuclei contained nicked or cleaved DNA after either intermittent or continuous light exposure, although more TUNEL-positive nuclei were observed after intermittent exposure. Agarose gel electrophoresis of retinal DNA showed internucleosomal DNA fragmentation, which is associated with apoptosis in samples from intermittent light exposure. CONCLUSIONS: These data demonstrated that intermittent light exposure triggered apoptosis in more photoreceptor cells than did continuous light exposure of the same intensity and duration.

Photic injury triggers apoptosis of photoreceptor cells.

Apoptosis, a highly regulated and energy-dependent process of cell death, plays an important part in normal tissue development. Its role in pathological conditions may vary. Earlier morphologic studies suggest that apoptosis may be an important mechanism in light-induced photoreceptor degeneration. In this study, we determined if photic exposure triggers apoptosis in photoreceptor cells in an established model of photoreceptor degeneration by light exposure. Twenty eight Lewis albino rats were divided into seven groups of 4 animals each: one group served as the unexposed control; and the other groups were exposed continuously to green fluorescent light (320 foot-candles) for 3, 6, 9, 12, 18 or 24 hours, respectively and killed for histopathological examination, biochemical isolation of retinal DNA; and in situ analysis of nicked nuclear DNA by terminal transferase biotin-dUTP nick end labeling (TUNEL). Histopathological study revealed morphological changes comparable to previous reports on photic injury. Electrophoretic analysis of DNA showed internucleosomal DNA cleavage as early as 12 hours of light exposure. The fluorescent intensity of DNA fragments, which were monomers and multimers of 180-200 base pairs, increased with the duration of light exposure. TUNEL technique, which localized DNA cleavage to the photoreceptor cell nuclei as early as 6 hours of light exposure, showed that the number of TUNEL positive nuclei increased with light exposure, and revealed more DNA degradation in the superior quadrant. Our findings confirmed earlier morphologic observations that photic exposure triggers apoptosis of photoreceptor cells.

Apoptosis leads to photoreceptor degeneration in inherited retinal dystrophy of RCS rats.

PURPOSE: To determine the pathogenetic mechanism of photoreceptor cell degeneration in the inherited retinal dystrophy in Royal College of Surgeons (RCS) rats. METHODS: The dystrophic retinas of the pink-eyed RCS (RCS-rdy-p) rats were examined for DNA fragmentation by agarose gel electrophoresis of retinal DNA and by TdT-mediated biotin-dUDP nick-end labeling (TUNEL) in paraffin sections. Rats ranging in age from 3 to 60 days were examined. RESULTS: Agarose gel electrophoresis of retinal DNA isolated from animals 25, 30, 35, and 40 days old showed a ladder pattern of degradation with bands corresponding to multiples of 180 to 200 base pair subunits. TUNEL study showed increasing labeling of photoreceptor cells with progression of the retinal dystrophy of the RCS rats. CONCLUSIONS: Apoptosis is the dominant mechanism of photoreceptor degeneration in the RCS rat, which has a genetic defect in the phagocytic activity of retinal pigment epithelium. The onset of the degeneration appeared to vary between rod cells in the different regions of the eye.

cDNA sequence of a growth factor-inducible immediate early gene and characterization of its encoded protein.

We report the cDNA sequence of 3CH134, an immediate early gene whose transcription is rapidly and transiently stimulated by serum growth factors. 3CH134 encodes a 367 amino acid protein that does not share significant sequence similarity with any known protein. 3CH134 is inducible through multiple signal transduction pathways, and in the adult mouse is expressed predominantly in the lung. Using affinity-purified antibodies, we have identified the 3CH134 protein in serum-stimulated Balb/c 3T3 cells and determined that it has a short half-life.