Retinal thickness in the wild-type mice remained constant in the left control eye (mean ± SD, 219 ± 36 μm; range, 206–225 μm; 17% thinning) at 21 days after injury but decreased significantly in the treated eye (mean ± SD, 182 ± 30 μm; range, 175–200 μm; P < 0.05). There was only a mild change in retinal thickness after injury in the Bax-knockout mice (mean ± SD, 329 ± 30 μm vs 338 ± 31 μm of the left control eye; range, 3%), and the final value was significantly higher for the treated wild-type mice (P < 0.05). The retinas of the heterozygous mice were thicker than those of the wild-type mice, measuring 333 ± 34 μm in the left eye (range, 284–369 μm) and 299 ± 31 μm in the right eye (range, 247–384 μm; 11% postinjury thinning).
Maximal apoptosis of the cells in the retinal ganglion layer was detected on days 1 or 3 after injury in the heterozygous mice (12% and 20%, respectively) and the wild-type mice (15% and 35%, respectively). In the transgenic knockout mice, no apoptotic cells were detected at these time points (Fig. 2, Table 2).
Apoptotic Gene Expression
Bax expression in the wild-type mice was elevated on day 1 and returned to baseline on day 3; in the heterozygous mice, it remained low on days 1 and 3; in the knockout mice, it was undetectable (Table 3). Levels of Bcl-2 and Bcl-X expression in the wild-type mice were elevated on day 1 and then returned to baseline (Bcl-X) or below (Bcl-2). In the heterozygous mice, Bcl-2 levels were below baseline, like the Bax levels. The Bax/Bcl-2 ratio was higher in the wild-type group than that in the heterozygous group on day 1, with a trend for an increase in both the groups on day 3. The Bax/Bcl-X ratio increased on day 1, but decreased to 0.8 on day 3. Caspase-3 expression was higher on day 1 in the wild-type group than in the heterozygous group and decreased toward baseline on day 3. The knockout mice showed an opposite trend, with a decrease in caspase-3 to below baseline on day 1 and its return toward baseline thereafter.
Optic nerve crush damage leads to retinal cell death. Apoptosis is known to play a key role in cell death after retinal ischemia. Apoptosis has been described in various models of ischemic ocular injuries, including crush (10,11), glaucoma (6), retinal ischemia (9,16), ischemic optic neuropathy (13,17), and photoreceptor light damage (18). Studies in transgenic animals suggest that the mitochondrion-mediated apoptosis pathway is involved in ischemia-related cell death. This pathway is triggered by activation of proapoptotic members of the Bcl-2 family of genes (2,10). Accordingly, Bcl-2, Bax, and Bcl-X, as well as activated caspases, may be necessary players in the control of programmed cell death in the RGC layer (19). Studies have reported an upregulation of Bax in RGCs after injury (5); increased survival of injured RGCs when Bax or caspase-3 was inhibited (19); and prevention of RGC death by ablation of the Bax protein (19). In line with these findings, the present study showed that survival of retinal cells of the inner retina, especially in the RGC layer, is increased in Bax-knockout mice subjected to crush injury compared to wild-type and heterozygous Bax mice and that Bax ablation protects the RGCs from apoptosis.
It has been proposed that the ratio of Bcl-2 to Bax or other members of the Bcl-2 family may govern the sensitivity of cells to apoptotic stimuli. Therefore, we investigated the relationship of the Bax (apoptosis promotor)/Bcl-2 (apoptosis inhibitor) ratio and the Bax/Bcl-X (apoptosis inhibitor) ratio and compared the findings with TUNEL staining for apoptosis. On day 1, both the Bax/Bcl-2 and Bax/Bcl-X ratios were upregulated. On day 3, the Bax/Bcl-2 ratio remained high, but the Bax/Bcl-X returned to baseline, in agreement with our histological finding of cell preservation. This suggests that the level of Bcl-X may better reflect the apoptosis state of the RGC layer than the level of Bcl-2 (10). It is of note that we did not find significant changes in these ratios. In a previous ischemia study (20), the Bax/Bcl-2 and Bax/Bcl-X ratios were not modified early after injury, but both were downregulated in the recovery phase, 24 hours later.
Some investigators have suggested that in ganglion cells and other cell types of the retina, Bcl-X serve as the major antiapoptotic gene (10), being at least 16-fold more abundant than Bcl-2. Although Bcl-X may better reflect the antiapoptotic state, our results showed only a mild difference in the level of expression between Bcl-2 and Bcl-X at 3 days after optic nerve crush injury.
In our study, RT-QPCR analysis performed 1 day after induction of ischemia yielded increased levels of Bax, Bcl-2, and Bcl-X in the wild-type mice, which decreased by day 3 (to a lesser extent for Bcl-X). Similarly, in a previous report on ischemia-induced apoptosis (20), a time-dependent decrease was noted in Bax, Bak, and Bcl-X messenger RNA (mRNA) expression, but there was no alteration in Bcl-2 mRNA. Although we did not measure protein levels, the earlier study (20) reported that the levels of the proapoptotic Bax and Bak proteins remained unchanged, whereas the Bcl-2 and Bcl-X proteins were significantly upregulated.
Caspase-3 expression was associated with an increase in cell loss in the wild-type group. At 1 day after injury, caspase-3 levels increased in the wild-type group, and to a lesser extent in the heterozygous group, but there was no change in the knockout group. These results are similar to those reported earlier in ischemic retinas 24 hours after reperfusion (20). It is noteworthy that caspase-3 mRNA expression may not reflect the level of the activated protein.
An earlier study examined the retinas of Bax-knockout rd mice for cell death during development, with specific attention to photoreceptor degeneration (21). Although Bax deficiency had no effect on photoreceptor and retinal degeneration, the total number of RGCs rose to 226% of normal. Our findings of an increase in retinal thickness and cell number in the retina are in line with this report. However, in our study, the increase in retinal thickness was associated with an increased number of nuclei in all retinal layers, including the outer nuclear layer. The sham-operated Bax-null mice had a significantly greater nuclear volume in the retina than the wild-type mice, similar to the findings reported by Vecino et al (22) in developing retina and the findings of Tehranian et al (1) of increased brain volume in Bax−/− mice. We noted a mean retinal thickness of 241 μm (range, 216–257 μm) in the Bax−/− mice compared to a mean retinal thickness of 219 μm in the wild-type mice (range, 206–225 μm). After injury, values in the inner nuclear layer were reduced, but not in the Bax-knockout mice, where there was no evidence of cell loss.
Isenmann et al (4) reported rapid elevation of Bax protein expression in rat retinas following crush injury. Levels peaked at 3 days and remained upregulated for at least 1 week thereafter. The same group (5) also reported upregulation of the Bax protein in rat retinas as early as 2 hours following axotomy, again peaking at 3 days after lesion induction. However, these findings were based on immunofluorescent studies, before it was understood that during apoptosis, the Bax protein translocates and concentrates in the mitochondria. Thus, staining may give the appearance of an increase in expression because of the aggregation phenomenon. Since the report of Isenmann et al (4), there has been at least 1 independent study describing an increase in Bax mRNA expression in the axotomy paradigm (23) and 2 studies that failed to detect any significant increase (24,25). In the present study, Bax mRNA levels were increased in the wild-type group as early as 1 day after injury.
Semaan et al (24) performed gene dosage experiments in mice, yielding a single wild-type Bax allele. Their findings indicated that genetic background influences the cell death phenotype, including an RGC cell line. This study supports the assumption that quantitative expression of the Bax gene is important in neuronal susceptibility to damaging stimuli. Further support was provided by the elevated Bax mRNA expression in the susceptible species. In the present study, Bax mRNA expression levels were elevated in the wild-type mice at day 1 after crush injury and not in the Bax+/− mice. Both the groups, on day 3, showed almost same (baseline) levels of Bax expression.
The decreased apoptosis rate in the inner retina of the transgenic Bax-knockout mice corresponds to observations in a glaucoma model (6) and in ischemic brain (2), liver (26), and heart (12) tissues in other transgenic mouse models. The greater tolerance of the RGCs in the Bax-knockout mice after crush injury also agrees with earlier studies of ischemic ocular injury (20). In wild-type mice in these studies, the number of TUNEL-positive cells reached maximum at 24 to 72 hours after injury (8,11,13,16,17). In contrast, following light damage, Bax−/− mice showed no reduction in the number of TUNEL-positive photoreceptor nuclei at 24 hours or 7 days (18). The higher percentage of surviving cells in the RGC layer in our knockout group at 21 days following crush parallels the results reported in the hippocampal region in a transgenic mouse model of neuronal posttraumatic damage (1). Together, these findings support the notion that deletion of the Bax gene ameliorates RGC death after crush injury.
The finding that apoptosis is a significant mechanism of cell death in various eye diseases (ischemia, trauma, or glaucoma, as many other conditions) may have important implications for the development of new treatments that specifically block or interfere with RGC apoptosis. Although this means preventing the result of the disease, not managing its cause, in many patients, ganglion cell death has already been stimulated by the time of diagnosis and continues to progress after conventional treatment.
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