Time-course of glial changes in the hyperhomocysteinemia model of vascular cognitive impairment and dementia (VCID)

Abstract

Vascular cognitive impairment and dementia (VCID) is the second leading cause of dementia behind Alzheimer's disease (AD) and is a frequent co-morbidity with AD. Despite its prevalence, little is known about the molecular mechanisms underlying the cognitive dysfunction resulting from cerebrovascular disease. Astrocytic end-feet almost completely surround intraparenchymal blood vessels in the brain and express a variety of channels and markers indicative of their specialized functions in the maintenance of ionic and osmotic homeostasis and gliovascular signaling. These functions are mediated by end-foot enrichment of the aquaporin 4 water channel (AQP4), the inward rectifying potassium channel Kir4.1 and the calcium-dependent potassium channel MaxiK. Using our hyperhomocysteinemia (HHcy) model of VCID we examined the time-course of astrocytic end-foot changes along with cognitive and neuroinflammatory outcomes. We found that there were significant astrocytic end-foot disruptions in the HHcy model. AQP4 becomes dislocalized from the end-feet, there is a loss of Kir4.1 and MaxiK protein expression, as well as a loss of the Dp71 protein known to anchor the Kir4.1, MaxiK and AQP4 channels to the end-foot membrane. Neuroinflammation occurs prior to the astrocytic changes, while cognitive impairment continues to decline with the exacerbation of the astrocytic changes. We have previously reported similar astrocytic changes in models of cerebral amyloid angiopathy (CAA) and therefore, we believe astrocytic end-foot disruption could represent a common cellular mechanism of VCID and may be a target for therapeutic development.

Keywords: astrocyte; cerebrovascular; dementia; microhemorrhage; neuroinflammation.

Figure 1. The two-day radial arm water maze task shows progressive cognitive deficits in HHcy mice relative to mice on control diet

Panels A–C show mean number of errors per trial for mice receiving either control diet (red lines) or HHcy-inducing diet (blue lines). Each block number is the average of errors for three individual trials. Individual groups of mice were tested after being on diet for 6 weeks (A), 10 weeks (B), and 14 weeks (C). * indicates P<0.05; ** indicates P<0.01 for the given block comparison between mice on control diet for the same time.

Figure 2. Microhemorrhages are increased in number after 10 and 14 weeks on HHcy inducing diet

The images show representative Prussian blue positive microhemorrhages from either mice on control diet for 14 weeks, or mice on HHcy inducing diet for 6, 10 and 14 weeks. Microhemorrhages are shown as blue staining on the neutral red counterstained background. The graph shows the mean number of microhemorrhages per section for each group of animals. The mean is calculated from counts of 16 sections per animal. * indicates P<0.05; ** indicates P<0.01 when compared to mice on control diet for the same time.

Figure 3. HHcy does not induce a significant astrogliosis in the brain

The images show representative GFAP immunohistochemistry in the dentate gyrus of the hippocampus from either mice on control diet for 14 weeks, or mice on HHcy inducing diet for 6, 10 and 14 weeks. The graph shows percent area occupied by GFAP-positive immunostaining in the frontal cortex and hippocampus of mice receiving control diet (red bars) or HHcy-inducing diet (blue bars).

Figure 4. Astrocytic end-foot expression of AQP4 is significantly reduced following 10 and 14 weeks of HHcy-inducing diet

The images show representative AQP4 immunohistochemistry in the frontal cortex from either mice on control diet for 14 weeks, or mice on HHcy inducing diet for 6, 10 and 14 weeks. The graph shows mean number of AQP4-positive vessels in the frontal cortex (FCX) and hippocampus (CA3 and CA1 regions) of mice receiving control diet (red bars) or HHcy-inducing diet (blue bars). * indicates P<0.05; ** indicates P<0.01 when compared to mice on control diet for the same time.

Figure 5. Astrocytic end-foot expression of Dp71 is significantly reduced following 10 and 14 weeks of HHcy-inducing diet

The images show representative Dp71 immunohistochemistry in the frontal cortex from either mice on control diet for 14 weeks, or mice on HHcy inducing diet for 6, 10 and 14 weeks. The graph shows mean number of Dp71-positive vessels in the frontal cortex (FCX) and hippocampus (CA3 and CA1 regions) of mice receiving control diet (red bars) or HHcy-inducing diet (blue bars). ** indicates P<0.01 when compared to mice on control diet for the same time.

Figure 6. Expression of Kir4.1 and MaxiK is decreased after 10 and 14 weeks of HHcy induction

The hippocampus was used for gene expression analysis and the frontal cortex for the protein analysis. The graph in A shows fold-change from the mice on control diet of Kir4.1 and MaxiK gene expression analysis by qPCR. The image in B show representative images of Western blots for MaxiK, Kir4.1 and b-actin. These are the same samples for all 3 images. The graph in C shows the quantification of band density for Kir4.1 and MaxiK normalized to the density of the corresponding b-actin band. Each bar is the average of all animals for each time-point. For graphs, * indicates P<0.05; ** indicates P<0.01 when compared to mice on control diet for the same time.

Figure 7. HHcy activates microglia at all time-points examined

The images show representative CD11b immunohistochemistry in the dentate gyrus of the hippocampus from either mice on control diet for 14 weeks, or mice on HHcy inducing diet for 6, 10 and 14 weeks. The graph shows percent area occupied by CD11b-positive immunostaining in the frontal cortex and hippocampus of mice receiving control diet (red bars) or HHcy-inducing diet (blue bars). * indicates P<0.05; ** indicates P<0.01 when compared to mice on control diet for the same time.

Figure 8. Pro-inflammatory mediators are increased by HHcy

qPCR analysis for TNFα, IL-1β, IL-12A and IL-6 was performed and the graphs show the fold change at 6, 10 and 14 weeks relative to mice on control diet for the same amount of time. * indicates P<0.05; ** indicates P<0.01 when comparing HHcy mice to mice on control diet for the same time.