32 research outputs found
Probing mechanisms by which cerebral vascular disease may influence cognitive impairment and dementia
Introduction
Vascular cognitive impairment (VCI) describes a full spectrum of cognitive deficits caused by
underlying cerebral vascular alterations, regardless of the specific mechanisms involved.
Several factors such as ageing, stroke, hypertension and cerebral hypoperfusion are
associated with an increased risk of developing VCI. Vascular dementia (VaD) is the second
most common cause of dementia after Alzheimer’s disease (AD). It is now recognised that
considerable overlaps exist between the features of VaD and AD. Key pathological and
neuroimaging features including cerebral amyloid angiopathy (CAA), white matter lesions
(WML), microinfarcts and microbleeds are evident in both VaD and AD. Furthermore, brain
infarction has been reported to influence the presence and severity of clinical expressions
such as cognitive performance of AD, suggesting a common pathophysiological mechanism
that contributes to the development of cognitive deficits. However, gaps remain in
understanding the exact mechanisms by which vascular risk factors contribute to cognitive
decline and neurodegenerative processes leading to dementia. Given the importance of
blood supply to the brain for maintaining its structural and functional integrity, it has been
proposed that vascular risk factors may affect the cerebral haemodynamic and alter the
vascular function resulting in damages to the brain. These changes may involve altered
neurovascular coupling that is a critical mechanism for regulating the dynamic changes of
local cerebral circulation. Further, impaired vascular function, amyloid-β (Aβ) accumulation in
the cerebral vasculature and disrupted neurovascular unit are found in VCI. The glymphatic
pathway, a clearance route for removing soluble waste from the brain to periphery, has been
proposed to play a role in the pathogenesis of VCI.
Mounting evidence has suggested that cerebral hypoperfusion, by large vessel occlusion
and stenosis, is emerging as a major contributor to cognitive impairment. This has led to the
development of mouse models of bilateral common carotid stenosis (BCAS), a model
narrowing both common carotid arteries by placing microcoils. The BCAS model has been
reported to produce many features of VCI, including white matter damage, microglial
activation, gliovascular disruption, increased oxidative stress (by increased NADPH oxidase
2 (NOX2) levels) as well as memory impairment. To investigate whether there is an
interaction between cerebral hypoperfusion and microvascular Aβ accumulation, a mixed
model that demonstrates both microvascular amyloid (Tg-SwDI mouse model) and BCAS
has been developed. In this thesis, it is hypothesized that the complex interaction of Aβ and
BCAS leads to cognitive impairment via an impaired glymphatic function in addition to
perfusion deficits that promote vascular related lesions and neurodegenerative changes.
Second to this, given clear links between NOX2, hypoperfusion and amyloid accumulation, it
was further hypothesised that NOX2 is a central mechanism leading to VCI.
Methods
Mice were given BCAS surgery to mimic cerebral hypoperfusion for a period of 3 months. In
vivo laser speckle imaging was performed to evaluate the changes in cortical blood flow.
This was followed by additional CBF measurements using arterial spin labelling (ASL)-based
magnetic resonance imaging (MRI), which gave a non-invasive access to CBF information in
the cerebral cortex, hippocampus and thalamus. Neurovascular coupling was assessed by
performing whisker stimulation. Barnes maze was used to assess the spatial learning and
memory function at 3 months following BCAS or sham surgery. For the examination of
glymphatic function, in vivo intracisternal injection and ex vivo imaging of CSF fluorescent
tracers were performed. Histological assessment and immunohistochemistry were used to
examine vascular related pathology, Aβ burden, astrogliosis and basement membrane
changes following BCAS.
Results
Part 1: To examine the effect of BCAS on cerebral perfusion deficits, glymphatic
function and cognition in Tg-SwDI mice compared to wild-type mice.
The first studies in the thesis sought to examine the effect of BCAS and microvascular
amyloid on the extent of cerebral perfusion deficits and cognitive impairment. The first step
was to validate whether the BCAS model has an effect on cerebral perfusion. Cortical
cerebral blood flow (CBF) was examined by laser speckle imaging. This revealed sustained
reductions of CBF at 24 hours, 1 and 3 months following the establishment of BCAS
(p<0.001) but no effect of the microvascular Aβ was found to affect cortical perfusion
(p>0.05). To further explore the CBF changes in other brain regions following BCAS, Arterial
spin labelling (ASL), a technique widely used in clinical imaging, was performed. A
significant effect of BCAS was confirmed in the dorsolateral cortex and hippocampus
(p<0.001, respectively) but no genotype effect of the microvascular Aβ or any interaction
was found (p>0.05, respectively). In order to investigate whether long-term carotid stenosis
has a further effect on cognitive function in the experimental animals, assessment of Barnes
maze demonstrated that BCAS mice spent longer escape latency than the sham mice in
both wild-type and Tg-SwDI animals (p<0.05, respectively) indicating visuo-spatial learning
was significantly impaired at 3 months following BCAS. To determine the effect of BCAS and
Aβ on long-term memory, a probe test was taken to examine whether mice remember the
previous training target after a period of time. This test revealed that all groups spent a
significantly higher percentage of time than chance (25%). Exclusively in wild-type BCAS
mice, the percentage of time spent in the target quadrant was significantly lower than by
chance (p<0.05). In addition, there was no significant effect of BCAS or Aβ on the
percentage of time spent in the correct quadrant (p>0.05, respectively). These results
suggested long-term memory was not impaired in BCAS and the presence of amyloid.
Further to enhance the detection of spatial learning and memory impairment, reversal trials
were taken to evaluate the ability of experimental animals to learn a new location. Compared
to wild-type mice that still learned the new tests showing significantly improved performance
over time (p<0.05), both the Tg-SwDI sham and BCAS mice no longer learned the task
(p>0.05). The long-term memory tested in reversal tests showed impairment in both wildtype and Tg-SwDI BCAS as well as in the presence of amyloid after increasing the
difficulties in reversal probe tests. The results indicated the only mice from wild-type sham
(37.40 ± 12.63) (p<0.05) spent a significantly higher percentage of time by 12.40 (95%CI,
1.84 to 22.96) than by chance, t(7)=2.8, p=0.027 and a significantly higher percentage of
time than Tg-SwDI BCAS mice (p<0.05) with all the other groups spending a lower
percentage of time than chance (wild-type BCAS: 27.57 ± 11.12%, Tg-SwDI sham: 20.36 ±
15.50%, Tg-SwDI BCAS: 26.79 ± 16.79%).
To further explore the potential mechanisms by which BCAS causes cognitive impairment,
the glymphatic entry was further assessed. This revealed that the global influx of CSF
tracers was different across the anatomical levels (p<0.001) but unaltered post-BCAS in
wild-type and Tg-SwDI mice (p>0.05, respectively). To explore whether BCAS influences
CSF glymphatic influx, ex vivo images of the CSF tracer influx in the dorsolateral cortex (DL
CTX) and hippocampus (CA1-DG molecular layer) on the D-3 tracer were measured. The
results showed in both regions, altered CSF influx was found in the BCAS and Tg-SwDI
mice due to the main effect of BCAS (p=0.037 and p=0.011, DL CTX and CA-DG regions
respectively) but not Aβ (p>0.05, respectively). Taken together, these first studies support
the original hypothesis that BCAS causes cognitive impairment via reduced cerebral
perfusion and impaired glymphatic function. However, there was no exacerbation of these
effects in Tg-SwDI mice.
Part 2: To examine the effect of BCAS on neurovascular function, degenerative changes
and amyloid accumulation in Tg-SwDI mice compared to wild-type mice.
To begin with, responses of cortical blood vessels to whisker stimulation were recorded and
quantified as the mean CBF percentage increase from the baseline. There was a significant
effect of BCAS (p<0.001), whereby impaired neurovascular coupling was observed in the
BCAS mice from both wild-type and Tg-SwDI mice. However, there was no significant effect
of Aβ in these mice (p>0.05). Vascular related lesions including microinfarcts and
microbleeds were compared by measuring the frequency in experimental animals. No
vascular lesions were detectable in wild-type and Tg-SwDI sham mice. 4/10 mice were
found to have vascular lesions in the wild-type BCAS mice following 3 months of surgery.
6/10 mice were identified with vascular lesions in the Tg-SwDI mice. No significant difference
in proportions (p>0.05) was found between Tg-SwDI BCAS and wild-type BCAS mice. To
discern the mechanisms by which BCAS and microvascular amyloid may impact on the
glymphatic function, the extent of astrogliosis was further studied. GFAP immunostaining
was undertaken to investigate the extent of reactive gliosis post-BCAS. Increased
astrogliosis following BCAS was found (p<0.05), but no effect of Aβ or interaction was found
in the dorsolateral cortex. The hippocampal CA1-DG molecular layer was further analysed,
and this showed a significant effect of Aβ (p=0.002) but no effect of BCAS (p>0.05) and
interactions (p>0.05) on astrogliosis. Further, Aβ load was evaluated in the cortex and colabelled with collagen 4 (COL4) (a marker of the basement membrane of blood vessels) to
enable the assessment of microvascular amyloid in the Tg-SwDI mouse model. A significant
increase in the total amount of amyloid as well as the percentage of vascular amyloid was
detected post-stenosis (p<0.05, respectively). No changes of COL4 levels were found in the
mice post-BCAS (p>0.05). In summary, these results demonstrated that BCAS impaired
neurovascular coupling and promoted amyloid accumulation in the cerebral
microvasculature.
Part 3: To determine whether targeting NOX2 has an effect on cerebral perfusion,
degenerative changes and cognitive impairment in Tg-SwDI mice compared to wildtype mice.
The third aim of the thesis was to determine the effect of NOX inhibitor (apocynin) on the
previously reported cerebral hypoperfusion, impaired neurovascular coupling, development
of neurodegenerative pathologies and cognitive deficits caused by BCAS in the Tg-SwDI
mice. Following BCAS surgery, mice were immediately fed with either apocynin or vehicle in
their drinking water for 3 months. Cortical CBF changes after the treatment of apocynin were
assessed using laser speckle imaging, in apocynin treated mice, a recovery of CBF from the
BCAS apocynin group after 3 months of treatment was found. The further investigation of
neurovascular coupling revealed that apocynin restored vascular function following carotid
stenosis. A significant interaction between BCAS surgery and apocynin treatment (p<0.05)
was found after 3 months of treatment. The mice that received 3 months of apocynin
treatment showed a robust response during the stimulation. The frequency of vascular
lesions was counted to compare whether inhibiting NOX activity could provide any beneficial
effect on the development of vascular pathology. However, there was no significant
difference in proportions between the mice treated with vehicle and apocynin (p>0.05). The
cortical amyloid load was assessed by double labelling of COL4 and 6E10. The results
revealed no effect of treatment on the Aβ burden and vessel densities compared to vehicle
treated group (p>0.05). Finally, the cognitive function was assessed using Barnes maze. It
demonstrated that apocynin did not improve spatial learning and memory in the behavioural
tests (p>0.05, respectively).
Conclusions
The findings in this thesis demonstrate novel evidence of how carotid stenosis damages the
cerebral microcirculation and structure, contributing to the pathogenesis of cognitive
impairment. Specifically, long-term BCAS caused chronic cerebral hypoperfusion and
impaired glymphatic function, which is likely to contribute to the accumulation of Aβ in the
microvasculature. Additionally, carotid stenosis caused sustained cerebral hypoperfusion
and led to impaired neurovascular coupling, neurodegenerative changes and cognitive
deficits. However, despite evidence supporting a basis for targeting NADPH oxidase, there
was only a modest beneficial effect of the NOX inhibitor on neurovascular function.
Collectively, this thesis provides evidence that following the carotid stenosis, while reducing
cerebral perfusion, the glymphatic drainage pathway may be affected, leading to cognitive
impairment. This new data adds credence to a growing body of human studies that alternate
mechanisms may exist in addition to cerebral hypoperfusion leading to VCI. The treatment
with non-selective NOX inhibitor successfully restored blood perfusion and vascular function
with no ultimate improvement in cognitive function, suggesting a limited role by targeting
NOX to restore the full pathological processes in VCI. Thus, further studies using more
specific method targeting post-carotid stenosis events will help to understand the proposed
mechanisms and provide a therapeutic strategy
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Impaired Glymphatic Function and Pulsation Alterations in a Mouse Model of Vascular Cognitive Impairment
ACKNOWLEDGMENTS Schematic diagrams in Figures 2, 8 are created withBiorender.com. FUNDING We gratefully acknowledge the grant support from the Alzheimer’s Society (152 (PG-157); 290 (AS-PG-15b-018); 228 (AS-DTC-2014-017), 314 (AS –PhD-16-006), and Alzheimer’s Research United Kingdom (ART-PG2010-3; ARUK-PG2013- 22; ARUK-PG2016B-6), and The University of Edinburgh Centre for Cognitive Ageing and Cognitive Epidemiology, part of the cross council Lifelong Health and Wellbeing Initiative (G0700704/84698). ML and JB are funded by an Alzheimer’s Society Scotland Doctoral Training Programme and RS Macdonald Trust. ML was also funded by a China Scholarship Council (CSC)/University of Edinburgh scholarship.Peer reviewedPublisher PD
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