In this study, non-hydrocephalic controls demonstrated that GFAP expression was highest within deep white matter regions, lowest within the deep cortex, and was slightly higher within periventricular regions in comparison to those which were superficial cortex. Similar relative differences were demonstrated by AQP4 expression within controls, the only difference being that slightly higher levels of AQP4 expression were detected within superficial cortex compared to periventricular locations. Histological analysis demonstrated that a delay in treatment time resulted in significant decreases in GFAP expression in SC and PV regions compared to controls after chronically treated hydrocephalus. Similar patterns were observed in AQP4 expression where late treated animals demonstrated significant decreases in AQP4 expression within SC and PV regions compared to controls, while DWM remained stable. Interestingly, unlike GFAP, AQP4 expression within DC exhibited a significant increase. Our findings demonstrate that within the brains of hydrocephalic neonatal felines, a significant redistribution of GFAP and AQP4 occurs in a region dependent manner. Since these changes are localized to specific paravascular sites, our results support an involvement of an impaired glymphatic system within the hydrocephalic brain.
Expression of key glymphatic biomarkers, GFAP and AQP4, were altered significantly in our model of chronic neonatal hydrocephalus. Although our group and others have employed experimental models of delayed shunting and non-treated hydrocephalus in (14-16), to the best of our knowledge, this study is the first to follow chronic hydrocephalus for extended periods in a neonatal model and evaluate glymphatic disturbance in various treatment time points. Also important in analyzing the results of this study is the degree of ventricular enlargement which through the use of clinically indicated tapping criteria, demonstrated progressive increases in ventricular size without causing animal mortality. The degree of ventricular enlargement was similar to that seen in premature infants with post-hemorrhagic hydrocephalus(17, 18) and those born with congenital hydrocephalus (19, 20) who at times develop severe ventriculomegaly.
Control animals without hydrocephalus demonstrated GFAP expression that was highest within DWM regions (i.e. internal capsule), lowest within DC (deep cortical mantle along hemispheric convexity) and was higher within SC regions than PV regions. Control animals exhibited similar expression patterns in AQP4, the only difference being that AQP4 expression was slightly higher within periventricular vs SC regions. These baseline patterns demonstrated significant differences between brain regions but maintained ratios of GFAP/AQP4 which were similar among brain ROIs. Our chronic reservoir-treated hydrocephalus groups displayed surprising differences in the GFAP and AQP4 expression depending on the initial timing of reservoir treatment. Early-treated animals maintained relatively stable levels of GFAP throughout all brain ROIs without significant differences compared to controls. However, in late-treated animals, when signs of progressive hydrocephalus had fully developed in addition to grossly enlarged ventricles, GFAP levels were significantly altered at sacrifice 4 months later. Our a priori expectations were that GFAP and AQP4 levels may follow similar patterns in both treatment groups given the chronicity of the treatment timeline, however we noted a surprising decrease in overall GFAP expression in the late-treated animals in all ROIs with the greatest drop in SC and PV regions. By contrast, the levels of AQP4 in the DC of the late-treated animals doubled while SC and PV AQP4 dropped similar to that seen in the GFAP levels in the same cohort of late-treated animals. The overall drop in GFAP and rise in AQP4 in the DC demonstrated the first indication that the glymphatic unit of astrocyte endfeet, AQP4 water channels and perivascular spaces, were disrupted in chronically treated neonatal hydrocephalus.
Our previous studies have demonstrated a link between delayed reservoir treatment and larger ventricular volumes, altered DWM diffusion tensor imaging (DTI), neurological outcomes and decrease motor accuracy scores (12, 21). It is noteworthy that once reservoirs were placed, the criteria for tapping was based on the same clinically-modeled assessment algorithm in both early and late treatment groups resulting in a typical tapping frequency of once or twice a day(12). Therefore, differences noted in the brains of early versus late animals can be traced to the decision to intervene before or after clinical signs of progressive hydrocephalus arise.
The ependymal epithelium is both functionally and mechanically important to the developing neonatal CNS (22-24). Pioneering work by the Rodriquez and Jimenez laboratories have demonstrated ependymal denudation after experimental hydrocephalus, while Kahle and others have demonstrated a strong association between the functional unit of the normal ventricular ependyma with motile cilia and the maintenance of non-hydrocephalic states. These findings all demonstrate a critical role of ependymal cells facilitating normal CSF distribution (25-27). The single-cell ependymal layer also forms the physiologic boundary separating ventricular CSF from the developmentally important stem cells residing in the adjacent subventricular zone (28, 29). Ependymal injury from progressive ventricular dilatation compromises this fluid barrier exposing subependymal structures, including the PV glymphatic system to CSF.
We found that substantial decreases in PV AQP4 expression were present within both early and late treatment groups compared with control animals. Given that the early reservoir treated animals never developed clinical signs of significant ICP elevation but did have demonstrable ventricular enlargement on MRI, physical stress in the form of stretch injury to the ependymal lining and compressive injury to the immediate PV brain may have initiated an injury cascade permitting prolonged changes in glymphatic proteins at sacrifice. Our group and others have demonstrated that even short periods of time under pathological pressure can be detrimental to not only ependymal cilia, but also damage developing brain cells (12, 21, 30-38). When comparing the two treatment groups, late-treated animals demonstrated even more substantial decrease in AQP4 within the PV region, as well as significantly diminished GFAP signal in the same region, compared with early and control groups. In this model of neonatal hydrocephalus, investigation of early decisions can be longitudinally followed for durability and impact chronically. One explanation of these findings is that heightened vulnerability of PV cells, paired with disruption in CNS fluid homeostasis chronically facilitates an inflammatory cycle preventing full recovery in chronic progressive hydrocephalus.
Much like AQP4 within the CNS, GFAP is widely regarded as being expressed primarily by astrocytes, although its presence has been noted in immature ependymal cells (28, 39-41). While the primary function of GFAP is to maintain the structural integrity of astrocytes, this protein is also functionally important to the CNS immune response. Astrogliosis in response to cellular injury may be characterized by increased astrocyte reactivity resulting in GFAP upregulation, changes in cell morphology and phenotypic polarization of astrocytes (42-47). Importantly, GFAP accumulation is integral in facilitating the physical isolation of damaged CNS tissue from viable tissue in adjacent regions (48-51). We found that while PV GFAP expression within the early treatment group was unchanged, within the late treatment group GFAP expression declined to one-third of non-hydrocephalic controls. We surmise that earlier intervention helped minimize cellular disturbances resulting in astrocyte preservation and increased neuronal viability immediately adjacent to the dilated ventricles. Polarization of perilesional astrocytes into a phenotypically reactive state is well-documented phenomenon after CNS injury (42, 45).
Therefore, it is reasonable that the loss of AQP4 on the endfeet of injured astrocytes may result from early injury before neurological deterioration or noticeable white matter degradation. We propose that the substantial decreases in both PV GFAP and AQP4 within the late treatment group could be due to astrocyte loss and glial scar formation. Late treatment animals demonstrated substantially thickened GFAP+ glial scars without identifiable astrocyte cell bodies or processes. Such scarring likely does not harbor functional components of the glymphatic unit and therefore can explain lack of AQP4+ water channels.
Superficial Cortex Region
In the SC region, the effects of chronic HCP and ventriculomegaly resulted in diminished GFAP and AQP4 expression. However, unlike the significant decline in AQP4 observed in the PV region of both early and late treatment groups, AQP4 decrease within the SC region was only statistically significant in the late group, nearly one-fourth that seen in non-hydrocephalic controls. Notably, while AQP4 and GFAP levels within the PV and SC regions of the late treatment group seemed to decline proportionately relative to one another, a break in this pattern was observed in the expression of AQP4 with the PV region of early group. We reason that lower early group SC AQP4 loss compared with PV region could be partly due to relative anatomical positioning away from the expanding ventricles. As CSF accumulates within the ventricles from chronic hydrocephalus, the resulting injury mechanism may be simplified into two phases and may affect the PV regions differently compared with other CSF-containing regions of the brain (i.e. superficial subarachnoid, interstitial spaces etc.). Initially, as the ventricles enlarge, and the ependymal lining may give way to pressure, small gaps in the ventricular wall facilitates CSF exiting into the PV, and eventually DC regions of the brain. This pathological phenomenon demonstrated on most brain imaging is transependymal flow (52-54). Chronic hydrocephalus and sustained ventricular dilatation also facilitate compression injury of the cortical mantle against the rigid skull. These mechanisms are outlined by Del Bigio et. al. through work done on hydrocephalic rats, where MRI demonstrated focal, not diffuse changes in brain water content after kaolin-induced hydrocephalus in a neonatal model (55). We propose that with early HCP treatment (i.e. early reservoir treatment) before clinical signs of injury and significant cellular damage occurs, glymphatic units remained structurally intact longer maintaining closer to normal AQP4 levels within regions of the brain distant from the enlarged cerebral ventricles. Importantly, while the SC region seemed to offer a protective advantage in the early group, this anatomical advantage was nullified in the late group by delaying HCP treatment until pathological signs of disease became apparent through worsening neurological deficit scores. Moreover, diffuse injury to cells within the PV and SC regions in the late group were likely from both chronic edema in the first phase of post-hydrocephalic injury, and chronic progressive compression against the hard surface skull in the second phase of injury. To some extent, this compressive injury may be dampened within the neonatal skull through its ability to expand due to open fontanelles and unfused sutures. However, the efficacy of these compensatory mechanisms diminishes as CSF accumulates, the ventricles expand and as ICP increases.
Deep White Matter Regions
The internal capsule was used as representative DWM in our study. In previous studies, we and others have utilized many other white matter structures such as the crus cerebri, corpus callosum, optic nerves/chiasm/tracts and corona radiate (12, 21, 56). The decision to use deeper internal capsule fibers when comparing white matter to other structures such as the PV region, was to analyze a region that was relatively protected against direct ventricular pressure and anatomic distortion with severe thinning and compression against the rigid skull. Animals receiving reservoirs at the early treatment period demonstrated ventricular dilatation without clinical signs of elevated ICP, and our previous work concluded that internal capsule fibers were not as damaged by progressive hydrocephalus in the early treatment cohort through intact fractional anisotropy values and DTI (12, 21, 57). Histologically, neither GFAP nor AQP4 levels were significantly altered within the DWM of the early treatment group compared with controls. In the late treatment group, GFAP dropped compared with control and early treatment groups, yet AQP4 levels continued to increase. Although these trends did not achieve statistical significance, this may be secondary to low animal numbers and we believe deserves more attention in our future experiments. We may be witnessing that indeed DWM is partially protected during progressive hydrocephalus, however with chronicity and enough severity such as the late treatment groups, injuries in DWM regions are also possible.
Noteworthy is that our findings of DWM GFAP signal in progressive hydrocephalus are contrary to those found in neonatal HCP rats with congenital hydrocephalus, where reactive microglia were found predominantly in the crus cerebri and thalamic relay nuclei (56). Involvement of both microglia and astrocytes in neuroinflammation is well documented, and thus differences between these findings may be attributed to variation in cell specific activating factors such as timing, or differential expression in specific cellular phenotypes resulting from the injuries that accompany chronic HCP. However, given the discordant results, more detailed evaluation of DWM in pediatric hydrocephalus is needed.
The expression of GFAP and AQP4 within PV and SC regions seemed to follow a trend in which chronic injury led to decreased GFAP expression accompanied by proportional decreases in AQP4 expression in the early treatment group. Furthermore, increasing HCP severity from delayed treatment translated to further protein loss in the late treatment group. However, AQP4 within the DWM regions of both early and late treatment groups exhibited expression patterns which were opposite of those seen within PV and SC regions. Chronic HCP and ventriculomegaly resulted in increased DWM AQP4 expression in both early and late treatment groups. Interestingly, delayed treatment resulted in further increases in DWM AQP4 expression.
Deep Cortex Region
The DC region is composed of parenchyma deep to the gyral folds along the lateral convexity of the brain, containing neurons, superficial white matter and a paucity of astrocytes. Vasculature in this region is typically small arterioles, venules, and capillaries. Astrocytes within the DC demonstrate faint GFAP expression, tend to be protoplasmic subtype and lack extended processes (28, 58, 59). Baseline low GFAP expression made detection of differences between experimental groups challenging. Deep cortex AQP4 expression, however, demonstrated significant increase within the late treatment group, more than doubling compared with controls. While the mechanisms of AQP4 regulation are not fully understood, osmotic stress has recently been linked to overall AQP4 expression (60-62).
Injury mechanisms in progressive hydrocephalus are multifactorial (i.e. stretch, compression, hypoxia/ischemia, edema, etc.) and often present simultaneously. Comparing injury between early and late reservoir groups highlights an important pathophysiology in chronic hydrocephalus; namely, significant decreases in AQP4 and GFAP within both the SC and PV regions of the late reservoir group, upregulation of DC AQP4, and mismatch between GFAP positive astrocytes and AQP4 water channels. Such derangements were not noted in the early treatment group, and thus we propose a threshold theory of early onset injury, below/before which progressive ventricular dilatation will not continue to cause harm. We have previously reported similar thresholds of injury using our model’s Neurological Deficit Score (NDS) and ventricular volumes when evaluating potential white matter injury using DTI (57). In the first two-weeks after HCP induction, the rate of ventricular dilatation within the late treatment group was nearly twice that seen in the early treatment group secondary to earlier initiation of ventricular reservoir tapping. Since tapping criteria was held constant and clinically driven, ventricular volumes were monitored as a function of time and treatment group. Over time, the ventricular volumes in the early group slowly intersected that of the late group, and at sacrifice they were equivalent. Any histopathological differences seen in the brains of early versus late treatment groups is a function treatment timing during the first couple weeks of progressive hydrocephalus. With respect to essential glymphatic system constituents, treatment prior to obvious neurological deficits and signs of elevated ICP during critical neurodevelopmental periods facilitates histological outcomes (AQP4 and GFAP expression) that more closely resemble patterns in non-hydrocephalic controls.
One proposed mechanism of injury is that of early ependymal injury and subsequent consequences. Higher rates of ventricular dilatation early in development, paired with longer delays in treatment (late treatment group) resulted in more severe damage to the PV region, namely the ependymal epithelium. Delaying treatment also exposed these particular animals to longer periods of pathophysiological stress during developmentally critical times. We suspect that as a result of early damage to the ependymal lining compounded by subsequent chronic hydrocephalus-induced injury, flux of ventricular CSF into the interstitial parenchyma diluted extracellular contents creating a state of extracellular hypotonicity. Previous studies have demonstrated the importance of AQP4 in astrocyte migration (61-63). Furthermore, they have demonstrated osmotic gradients to accelerate migration speed in the direction of the hypoosmotic region of the brain (64). If this fact holds true, then chronic hydrocephalus in the late treatment group would have caused a larger initial insult to the ependymal wall from more rapid increases of ventricular size and longer exposure to injurious conditions including pathological spread of CSF into the brain parenchyma from the ventricles toward the cortical surface. Moreover, continued chronic hydrocephalus causing stretch and compressive injury elevated levels of stress throughout the brain. To counteract these multiple injurious mechanisms, we see AQP4 is redirected into the regions of the brain with the largest total surface area for CSF removal, namely the DC capillaries. The decline of PV and SC AQP4 demonstrated only in the late group lends credibility to this potential pathophysiological mechanism.