Discrepancies of more than one grade unit were resolved by mutual consensus after reexamination of sections involved. A separate analysis of 5 of the genes that were most regulated in elevated IOP eyes demonstrated that mRNA levels in control eyes from the same animal were not significantly affected by damage in the contralateral eye data not shown. Results for retinas with elevated IOP are grouped according to optic nerve injury grade.
ANOVA with Dunnett's multiple comparison post-test and regression analysis were used to test for significant changes in message and protein levels. Glaucoma model data presented are from tissues analyzed at five weeks following injections to elevate IOP, with the exception of the changes in message level following short-term IOP elevation which are presented at the end of the results section. The average nerve injury grade for the injected eyes was 3. As shown in Fig. IOP histories and correlation of optic nerve injury grade to pressure exposure. Graph showing the high correlation between cumulative IOP and optic nerve injury grade for all eyes evaluated for mRNA expression at five weeks post-injection.
Graph of the daily mean IOP for the fellow eyes and each of the three injury grade groups, with best fit lines superimposed. The lines demonstrate little, if any, overlap between the groups after day 10, when pressure elevation is generally established in this model. For these analyses, data from retinas were placed into four groups depending on the degree of nerve injury: Table 2 gives the mean optic nerve injury grade and the range of cumulative exposure to elevated IOP for each of these groups.
A graph of the daily mean IOP for each group is shown in Fig. Each of these groups segregates into a distinct level of mean pressure over the experimental period, with remarkably little overlap, suggesting that our results provide an accurate reflection of the effects of elevated IOP. Brn3a, b, and c are transcription factors specifically localized to RGC nuclei Xiang et al. This provides context for interpreting the other findings of this study. Responses of retinal neuronal and glial marker mRNA in glaucoma model eyes. By contrast, Nefh mRNA is significantly depressed in retinas with both focal 1.
No significant alterations are observed at any extent of injury in markers for photoreceptors Rho , horizontal cells Calb1 or amacrine and horizontal cells Stx1A. We also evaluated possible damage in other retinal cell types due to elevation of IOP by measuring mRNA levels of the following neuronal cell type markers: As illustrated in Fig.
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The upregulation of Gfap mRNA is consistent with our previously reported microarray and immunohistochemical data Ahmed et al. We found no significant difference in mRNA level between fellow and glaucoma model groups in any of these NTs. RGC apoptosis following optic nerve injury has been suggested to result from the loss of target-derived neurotrophic support to RGC and a number of strategies to increase BDNF levels in the retina have resulted in increased RGC survival Castillo et al.
The levels of mature, 14kD BDNF protein in the retina were not significantly different from fellow eye values in glaucoma eyes with either mild to moderate grade 1. Retinal BDNF protein levels in glaucoma model retinas. Representative gel for quantification of mature BDNF, showing standard curve units reflect relative protein load and results from 4 retinas with increasing injury grades.
Representative gel for 32 kD proBDNF demonstrating reduced protein levels with increasing nerve injury. ProBDNF is quantitated on a separate set of gels from an electrophoresis of lesser amounts of total protein, due to the relative abundance of proBDNF in the retina. There was no significant correlation between injury and the mRNA levels of the two truncated forms of the TrkB receptor, T1 and T2 both: Response of retinal mRNA levels of full-length Trk receptors in a glaucoma model. By immunohistochemistry, full-length TRKB protein appeared primarily located within the RGC and nerve fiber layer, and did not appear to change with pressure-induced optic nerve damage Fig.
Response of TRKB receptor protein in experimental glaucoma.
Full-length TRKB receptor protein does not change significantly with increasing optic nerve damage, while the activated form, pTRKB, increases linearly with increasing optic nerve damage. Upper green band is pTRKA, which is more abundant in the retina. Standard curve units reflect relative protein load. For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.
Protein levels were unchanged in glaucoma model eyes. Representative example of western blot documenting lack of consistent change in protein levels with increasing pressure-induced nerve damage. Immunolabeling of retinas from fellow eyes demonstrated an inner retinal distribution similar to that of TRKB protein Fig. Injected eyes with nerve injury grades from 1 to 5 did not reveal any consistent difference in this pattern of labeling. Lingo1 is an alternative p75 NTR adaptor protein, and when in complex with p75 NTR and the NOGO receptor, is believed to inhibit the regenerative potential of axons in response to myelin protein Mi et al.
The further downstream signaling pathways regulating p75 NTR -mediated neuronal death are not clearly understood, but the JNKpBax pathway is thought to play a crucial role, a process that is potentiated by Bim Putcha et al. JNK activation also plays important roles in cell motility and multiple cellular stress response pathways.
Message levels for activating transcription factor 3 Atf3 were also determined. This is a marker for neuronal injury Vlug et al.
Atf3 has been shown to increase following optic nerve transection Takeda et al. As a result, both mRNAs were significantly higher in the severest injury group compared to fellow retina values: In contrast, Jund mRNA levels were unaffected. No significant changes were found in the message levels of Bax and Bim. Signaling via JNK activation.
Message level for p53, although significantly correlated to injury, is not significantly different between injury groups. Representative immunostaining for p ser63 JUN pattern, comparing a fellow and grade 5 retina, illustrating a clearly heavier label of RGCs in the glaucoma model retina. Immunolabeling for p ser63 JUN demonstrated a marked increase in intensity in the RGC layer in glaucoma model eyes with little or no labeling of other regions of the retina Fig. This activated form of JUN has been associated with apoptosis following withdrawal of trophic support Li et al.
A similar pattern was seen when retinal sections were immunostained for p ser73 JUN. Immunolabeling for BAX demonstrated a generally uniform staining pattern that did not differ between fellow and glaucoma model retinas not shown. Several pathways have been implicated in Trk receptor mediated survival signaling in neurons.
Akt activation inhibits pro-apoptotic p53 and enhances expression of Bcl2 and Bcl-xl, two typical anti-apoptotic Bcl2 family proteins.
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Erk5, the newest member of the MAP kinase family Lee et al. Downstream of Trk receptor activation are survival pathways mediated by Akt and Erk kinases. Mitochondrial pro-survival mRNA levels for Bcl2 are unchanged, while levels of Bcl-xl are slightly, but significantly reduced in retinas with injury greater than 4. NT-signaled neuronal survival can also be mediated by p75 NTR.
To attempt to capture early, potentially important, gene expression responses to IOP elevation, we examined the same message levels in retinas collected at one week following the initial IOP measurement of 35 mmHg or more. Description of the cumulative IOP history and optic nerve injury grades associated with these retinas is shown in Table 3. For Lingo1, a significant negative correlation between injury grade and message level was detected r 2 0. Originating in the superior colliculus, NT complex with their Trk receptors and are ultimately delivered by retrograde transport to the RGC body.
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Because the receptors are produced in the RGC, and must first be transported in orthograde fashion to the superior colliculus, it seems reasonable to expect that elevated IOP, which can obstruct both retrograde and orthograde axonal transport Pease et al. Because of this, an analysis of the simultaneous responses of its major components in an intact glaucoma model is essential in order to determine if predictions based on this theory are indeed supported. If all pertinent RGC NT were derived from the superior colliculus, we would expect that their retinal levels would be diminished in eyes with chronic IOP elevation.
However, we found no significant alteration in the mature form of BDNF protein in the whole retina. Although we previously found immunohistochemical evidence for reduced inner retinal labeling in specimens shortly after IOP elevation, we also noted a return of RGC label in eyes with prolonged pressure elevation Johnson et al. These results strongly suggest that depletion of mature BDNF is not seen in eyes with chronic moderate pressure elevation. Although proBDNF was significantly decreased, this likely represents ongoing conversion to the mature, active form. Both studies, along with other work Vecino et al.
Chen and colleagues have reported that intravitreal application of BDNF can decrease retinal message and protein levels for full-length TrkB Chen and Weber, This suggests that production of TrkB receptor is sensitive to feedback based on retinal levels of NT. Instead, we found that TrkB message levels were reduced.
Rudzinski, using the vein cautery model, found no change in retinal TrkB Rudzinski et al. Again, neither model produced the result predicted by the NT hypothesis. Our finding of a concurrent reduction in message for TrkC as well as TrkB is interesting. However, its significance is uncertain, since message for its specific NT, NT-3, did not change.
These results suggest that further understanding of the responses and function of this NT and its receptor with regard to RGC survival in glaucoma should be pursued. Our observation of a significant increase in message for the p75 NTR receptor, with no apparent change in protein level, provides an additional indication of the complexity of the role of NT signaling in glaucoma. The p75 NTR receptor is capable of triggering multiple pro-apoptotic and pro-survival pathways and interactions, as well as regulating axon regenerative potential Barker, ; Mandemakers and Barres, Rudzinski has also noted a slight increase in message for p75 NTR and suggested that this increase may contribute to RGC death in glaucoma Rudzinski et al.
This results, in part, in activation of PI3K-Akt signaling pathway, which mediates signaling of neuronal survival Miller and Kaplan, b as well as enhanced expression of anti-apoptotic Bcl2 and Bcl-xl. Previous work in the vein cautery model has demonstrated upregulation of message for both Bcl2 and Bcl-xl Kim et al. While we did not find a change in message for Bcl2 and Akt, there appeared to be a slight decrease in retinal Bcl-xl message and protein, with only a suggestion of increased immunolabeling for AKT in the RGC layer in eyes with elevated IOP, even after five weeks.
Again, these discrepancies may reflect differences in the methods used to elevate IOP Nissirios et al. We did, however, detect a significant increase in Erk5 message, representing what we believe to be the first report on this signaling molecule in the retina. While this is consistent with increased survival signaling by retrograde transport Watson et al.
Although beyond the scope of the present study, detailed evaluations of potentially altered levels, intracellular distribution or activation states of these and other intriguing proteins should stimulate future investigations. Our finding of an increase in the activated form of TRKB protein further suggests that, following chronic IOP elevation, the NT receptor system is tilted in favor of survival, rather than apoptotic pathways.
However, these authors also acknowledge that Jun may contribute to cell survival as well as cell death Levkovitch-Verbin et al. Along these lines, we found a nearly 5-fold increase in message for Atf3, representing the most dramatic response of all of the messages that we evaluated. While Levkovitch and colleagues reported no change in the related Atf2 in their glaucoma model, our study appears to represent the first report of retinal Atf3 expression changes following chronic IOP elevation. Atf3 is an immediate early response gene induced by a wide variety of stresses in neuronal and non-neuronal cells.
It can contribute to apoptosis as well as cell survival, depending on the cell type Mei et al. In the eye, Takeda and colleagues have demonstrated increased expression of Atf3 in RGC, colocalized with phosphorylated Jun, following intraorbital optic nerve crush Takeda et al. Because this expression appears mostly during the initial regenerative period, these authors suggest that Atf3 heterodimerizes with Jun and functions in a supportive, or anti-apoptotic role. The marked upregulation that we observed in our model suggests that Atf3 deserves closer scrutiny to identify its cellular localization and potential relationship to Jun and its role in RGC death or survival.
Although our data do not support the concept that reduced delivery of NT plays a major role in RGC damage due to elevated IOP, other trophic factors could still contribute to this process. Deficits of these, or other trophic factors, may contribute to glaucomatous RGC loss. It is also important to note that these results reflect findings from the whole retina, and that responses unique to RGCs may be diluted by contributions from non-RGC layers of the retina.
More refined analyses, based on analysis of the RGC layer alone, are likely to reveal changes on the NT signaling system more specific to RGC damage in glaucoma and are currently in progress in our laboratory. This study of the NT system in an intact model of chronic IOP elevation indicates a higher than anticipated level of complexity and suggests that responses of many of its components enhance survival, rather than apoptosis.
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This may explain why interventions based on our current understanding of the role of NT in RGC survival and the assumption that an NT deficit underlies glaucomatous RGC loss so far have not provided lasting benefits. Inappropriate NT therapy may result in unexpected and, potentially undesirable consequences. National Center for Biotechnology Information , U. Author manuscript; available in PMC Dec 1. Author information Copyright and License information Disclaimer. The publisher's final edited version of this article is available at Exp Eye Res.
See other articles in PMC that cite the published article. Abstract Reduced retrograde transport of neurotrophins NT and their receptors has been hypothesized to contribute directly to retinal ganglion cell RGC loss in glaucoma. Introduction Progressive loss of retinal ganglion cells RGCs , a characteristic of primary open-angle glaucoma, is attributed to apoptosis of the RGCs Quigley et al.
Open in a separate window. Materials and methods 2. IOP history For each injected eye, a cumulative IOP dose was determined as the area under the curve of the plot of days post-injection vs. Table 1 Primers used for qPCR. Results Glaucoma model data presented are from tissues analyzed at five weeks following injections to elevate IOP, with the exception of the changes in message level following short-term IOP elevation which are presented at the end of the results section.
Table 2 Histories for five-week glaucoma model retinas analyzed by qPCR. JNK activation and pro-apoptotic signaling The further downstream signaling pathways regulating p75 NTR -mediated neuronal death are not clearly understood, but the JNKpBax pathway is thought to play a crucial role, a process that is potentiated by Bim Putcha et al.
NT receptor mediated pro-survival signaling Several pathways have been implicated in Trk receptor mediated survival signaling in neurons. Message level changes following short-term exposure to elevated IOP short-term glaucoma To attempt to capture early, potentially important, gene expression responses to IOP elevation, we examined the same message levels in retinas collected at one week following the initial IOP measurement of 35 mmHg or more. Provide feedback about this page. There's a problem loading this menu right now.
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