Introduction: A significant proportion of stroke patients exhibits impaired reperfusion (1), which manifests in different clinical outcomes including cognitive impairment. Although effective treatments to reverse cognitive decline following stroke are still not available, interventions to control risk factors implicated in the disease onset may help to reduce the burden of dementia (2). One such modifiable risk factor is hyperhomocysteinemia (HHcy) or elevation in plasma total homocysteine (tHcy), which has been extensively studied for its cerebrovascular effects (3). HHcy is independently associated with increased incidence of stroke as well as dementia (4-7).  

Objective: We wanted to determine if Hcy exacerbates post-stroke memory impairment through a NMDAr dependent mechanism.

Methods: From 4 weeks of age, mice were fed either a control diet or a high methionine/low folate (HM/LF) diet that increases the blood plasma level of Hcy leading to HHcy. Inhibition of NMDAr was achieved using memantine. The stroke was modelled using middle cerebral artery occlusion, followed by the assessment of learning and memory impairment using a battery of behavioral tests. Blood brain barrier (BBB) leakage was assessed using Evans Blue extravasation. We performed repeated measures for time dependent assays and the analysis of variance (ANOVA) with Tukey’s post-hoc analysis for multiple comparisons within several groups. For unpaired comparisons, with a standard deviation of 30% and a minimum expected difference between groups of 40%, 10 mice were included in each group to achieve 80% likelihood of detecting significance at the 0.05% level. Values presented as Mean

Results: In fear conditioning, I  observed that overall freezing time  was significantly less (p<0.05) in mice after stroke (84.75±5.99) compared to naive mice (111.75±11). Next, I observed significantly less freezing in HHcy (Control (84.75±5.99; HHcy 61.75±5.69, p<0.05), suggesting that it can exacerbates loss of memory post-stroke. Next, we assessed if HHcy can promote learning and memory impairment in novel object recognition. Mice acquired a memory of the familiar object by exploring two identical objects. 24 hours after acquisition, we assessed NOR by replacing one of the familiar objects with a novel object and assessing the amount of time that mice spent interacting with the novel and familiar objects. Surprisingly, when NOR was assessed 24 h later, we found that mice with HHcy failed to discriminate between the novel and familiar objects lower discrimination index s compared to controls (Control 71.35±3.63; HHcy 54.25±6.27; p<0.05). We further made an interesting observation that HHcy leads to BBB leakage that was reversed by the administration of memantine (Control; 2.65±0.36 µg/g; HHcy 4.2±0.38; HHcy with memantine 2.57±0.53; p value for HHcy vs Control<0.05; p value for HHcy vs HHcy with memantine<0.05).

Conclusion: Altogether, the findings from the  project suggests that HHcy exacerbates cognitive and BBB leakage following stroke. The  findings from the project will allow us to initiate clinical trials to determine if stroke patients with higher level of Hcy are more vulnerable to the development of cognitive dysfunction and need a memantine or Vitamin B12 supplementation as prophylactic treatment.