5.4.1 OUTCOMES
The neurodegenerative stressors one encounters in the world are not isolated, but most often an isolated stressor is the clearest to study. Once we established how the
interconnectivity of the brain contributes to the broad impact of neurodegeneration, we then
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needed to establish how this paradigm would impact an individual impacted by multiple stressors. If resources are drawn away from one region of the brain to support another, does that then render the originating tissue vulnerable? To investigate this, we utilized a staggered pattern of IOP elevation at the precise timepoint we previously determined to be the inflection point for resource redistribution.
We utilized KO and KO control mice to address directly how astrocyte connexins serve to maintain or diminish neuronal function in each paradigm. Here, we examined active anterograde transport function, one of the earliest neuronal functional deficits established in our model153. One week of unilateral IOP elevation did not significantly reduce anterograde transport function in control mice in either visual stream, and two weeks resulted in a 24.3%
functional loss (Figure 4.4). In KO mice, one week of unilateral IOP elevation reduced anterograde transport function by a remarkable 40.9% in the stressed optic projections, a significant reduction both from the contralateral naïve projections and corresponding control projections. Two weeks of IOP elevation in KO mice caused a 45.6% loss in anterograde transport function compared to the contralateral naïve nerve. Certainly, astrocyte connexins play a role in the maintenance of neuronal function at this early stage of neurodegeneration.
The 2-1 staggered paradigm produced intriguing results in both control and KO mice. In control mice, the 2-week and 1 week projections both exhibited roughly 50% intact transport.
Remarkably, despite the fact that one eye was exposed to elevated IOP for double the length of time, the two projections do not exhibit a significant difference in transport ability between one another. However, the projections exposed to 1 week of elevated IOP demonstrate significantly
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diminished anterograde transport function compared to the corresponding unilaterally injected 1 week control projections. This demonstrates the impact localized neurodegeneration can have on nearby regions; certainly, the degenerative program was accelerated because the contralateral eye had already been exposed to stress.
The same staggered paradigm in KO mice, however, did result in a statistically significant difference in anterograde transport function between the two projections. Remarkably, the KO projections exposed to 1 week of elevation in the staggered paradigm demonstrated a nearly identical percentage of intact anterograde transport to the unilateral KO 1 week exposed, demonstrating how isolated these neurons become after astrocyte communication through connexins is limited. Additionally, transport ability between the KO projections exposed to 2 weeks of elevation in the staggered paradigm and the unilateral KO 2 week projections was not statistically significant. However, the trend toward increased transport loss in the 2-1 paradigm may reveal other mechanisms involved in regulating degeneration between the two optic tracts. These additional mechanisms may include other astrocyte connexins or remodeling patterns that may be differentially impacted by a bilateral model; these factors should be examined in future experiments.
Finally, we examined the functional ability of the visual system as a whole, and how visual function during neurodegeneration is differentially impacted in control and KO mice exposed to the staggered paradigm of neurodegeneration. Six days following IOP elevation we detected diminished visual function in stressed visual streams compared to contralateral visual streams in both control and KO mice; however, visual function loss was exaggerated in KO mice.
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Intriguingly, this pattern of loss in control mice appears to precede the loss of active anterograde transport function, meaning that there is likely another earlier component to neurodegeneration. KO mice still exhibit remarkably exaggerated deficits compared to control mice at this timepoint, further underlining the critical importance of astrocyte connexins in maintaining visual function. Intriguingly, neither group exhibits altered visual function in the contralateral visual stream. Likely, the loss of astrocyte glycogen stores without a second stressor does not stress the system in any way significant enough to impede functional ability;
thus, in the event that a single stressor occurs without any further stressors, metabolite
redistribution serves to protect the stressed region at no functional cost at this early timepoint.
On the seventh day, the contralateral eye was injected with microbeads in all mice. Two days following this second microbead injection, control mice exhibited moderate visual function loss in both visual streams compared to respective baseline values. Thus, functional loss is accelerated in the fellow eye due to the initial injection. Two days following the initial injection, no functional loss was detected in any group. The previously stressed visual stream still
exhibited significantly greater visual acuity loss than its contralateral partner that has been stressed for seven fewer days; however, at this time point the gap in visual acuity loss is rapidly closing. In knockout mice, the previously stressed visual stream continued to rapidly decline in visual acuity compared to baseline and the levels exhibited on day 6. Intriguingly, the newly stressed visual stream did not exhibit any alteration from baseline values, just as two days after the initial injection no functional loss could be detected. For this brief period of time after the second IOP elevation, it appears that the relative isolation inflicted upon the contralateral KO nerve protects it from immediate visual acuity loss in KO mice.