Padgett, Matthew
(2024)
It’s in your blood: understanding memory loss when blood vessels malfunction.
PhD thesis, University of Nottingham.
Abstract
The bilateral carotid artery stenosis model, (BCAS), is a popular method in which to model vascular cognitive impairment in the mouse. This model has gained in popularity due to reports of selective white matter deficits accompanying cognitive decline. However, physiological and behavioural deficits have not always been replicated across different research groups, suggesting high levels of variability in the model. The overall aim of this thesis was to assess cognitive decline in the BCAS model in the lab and across the literature, as well as assessing biological changes to the visual system and cerebral white matter.
The first step to address the primary aim of understanding cognitive deficits after BCAS was to conduct a meta-analysis. This identified the most used cognitive tests in the model. Four tests were identified as being used across the model in more than 10 separate papers: the radial arm maze, Morris water maze, y maze and novel object recognition task. All tests showed a large effect size, with BCAS mice performing worse than shams, suggesting all tests are sensitive to cognitive deficits. The largest effect was seen in the radial arm maze, indicating that spatial working memory is the most impaired domain in BCAS mice. Interestingly, the second largest effect size was seen in the novel object recognition task, a measure of long-term recognition memory. However, there are issues of publication bias and non-reporting of null results across the sample that affect the confidence of the researcher to recommend these tests as efficacious in detecting cognitive deficits after BCAS.
Practical lab work was also conducted using the BCAS model. The first study conducted focussed primarily on understanding damage to the visual system after BCAS in acute and chronic timepoints. For this analysis 18 mice (n=8 sham, and n=10 BCAS) were humanely euthanised and their optic nerves placed in an aglycemic environment to assess optic nerve function. This demonstrated no differences between mice in time to compound action potential failure, or peak velocity of action potentials. However, there was marked variability in BCAS mice, with only 4 out of 10 optic nerves able to generate the standard 3 compound action potential peaks. This warrants further investigation to assess the underlying causes of variability between BCAS mice. A secondary aim in this study was to assess miRNA changes after BCAS surgery. This was examined using the same cohort of mice. Brains were first dissected, and the white matter, cortex and hippocampus removed and sequenced separately. Samples were sent for sequencing from the white matter at 7 days (n=2, sham, and n=3 BCAS) and at 23 days (n=3 sham and n=3 BCAS). Additional samples were also sent from the hippocampus at 7 days (n=0 sham and n=2 BCAS), and at 23 days (n=3 sham and n=1 BCAS). Sequencing was conducted by Genewiz (Azenta Life Sciences). The results revealed several interesting miRNAs for future analysis, with the most promising being mmu-miR-30d-5p, which was upregulated in both the white matter and hippocampus 7 days after surgery. This was the only miRNA which showed changes in expression in separate brain regions. A final aim of this experiment was to pilot behavioural testing in the radial arm maze, Morris water maze and novel object recognition task. However, this was conducted at an early time point (beginning 10 days after surgery) and found no deficits on any task. The failures in cognitive testing in this study were used to inform cognitive testing protocols in later experiments.
The final study was a large behavioural study assessing all the cognitive tests identified in the meta-analysis in 36 mice (n=18 sham and n=18 BCAS) 4 weeks after surgery. Before conducting this study, optimisation of the RAM was carried out in 7 healthy mice over a 25-day period to ensure optimal food deprivation protocols to motivate performance before scaling up. In this study there were no differences between sham and BCAS mice on any measure on any of the four cognitive tests conducted (the radial arm, Morris water and y mazes, and the novel object recognition task). This was in direct contrast to the results of the meta-analysis which had suggested deficits were present on all these cognitive tests across published research. Finally, the mice were assessed for the presence of 3 nitro-tyrosine (a prominent oxidative stress marker) using immunohistochemistry. Unfortunately, differences in BCAS mice could not be quantified, but appeared to show increased levels of 3NT in the cortex, midbrain and hippocampus. However, counter to our hypothesis, there was only minimal evidence of 3NT staining in the white matter (optic tract, corpus callosum and striatum) in BCAS mice.
In conclusion, the BCAS model was very mild in this thesis. In two studies there was no evidence of functional deficits on any of the most used cognitive tests across the literature. It is hard to make conclusions surrounding the validity of the model in replicating the cognitive deficits seen in VCI, as the results here are in a direct contrast to published literature. However, the meta-analysis and experimental results presented suggests that the large effect sizes often reported are not a complete picture. Future research should instead focus on conducting more sensitive tests of executive function, as opposed to the more classic memory tests assessed here. These tests may hopefully be more reliable in detecting cognitive deficits in BCAS mice. Further, there were no differences in optic nerve function after BCAS, although increased variability in BCAS mice suggests additional research is needed. There was also no evidence of oxidative stress in the optic tract of BCAS mice, as well as other prominent white matter tracts, demonstrating our failure to replicate the white matter damage reported in previous research. Finally, there are promising miRNA targets that can be assessed in the future as potential biomarkers in the model and provide avenues for future intervention studies. To conclude, this thesis casts some doubt on the ability of BCAS model to replicate VCI, with no functional deficits present in either of our studies. There was also a lack of obvious biological changes, particularly in the white matter.
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