Relationship between enlarged perivascular space in hippocampus and P300 event-related potential in patients with acute lacunar stroke

This study aimed to investigate the relationship between enlarged perivascular space in hippocampus (H-EPVS) and P300 event-related potential (ERP) in patients with acute lacunar stroke. 2020 were collected. All the patients performed the P300 auditory oddball task and a full set of cognitive function tests. We recorded P300 latency and amplitude by EMG evoked potential instrument. H-EPVS showed round, oval and linear structures on MRI T2-weighted images of hippocampus, with clear boundaries and consistency in the direction of the perforating arterioles. Dividing the total number of H-EPVS ≥ 7 into extensive H-EPVS group (n = 61) and non-extensive H-EPVS group (n = 53). ROC curve was used to analyze the relationship between P300 ERP and cognitive function of extensive H-EPVS in patients with acute lacunar stroke.

SD, -0.41 ± 0.50) was signi cantly lower than in non-extensive H-EPVS (mean and SD, 0.47 ± 0.54, t = 9.017, P < 0.01). Further analysis shows that in patients with acute lacunar stroke, P300 latency was positively associated with H-EPVS counts (r = 0.726, P = 0.000). H-EPVS counts was negatively associated with cognitive function score (the eld of verbal uency) (r=-0.705, P = 0.000). ROC curve analysis showed that the index of cognitive impairment diagnosed by P300 in patients with acute lacunar stoke was 0.796.

Conclusion
Extensive H-EPVS has a high incidence in patients with acute lacunar stroke. The P300 latency may be used as an early indicator to evaluate the verbal uency function of extensive H-EPVS patients with acute lacunar stroke.
Background Page 3/15 The perivascular spaces (PVS) is a potential space around the perforating branch of the brain, the products of brain metabolism can be cleared through this space [1]. At present, it is believed that the PVS visible in MRI is enlargement perivascular spaces (EPVS), and diameter is usually < 3 mm.
Previous studies have found that EPVS in the basal ganglia (BG-EPVS) and central semioval (CSO-EPVS) is the most common area. The occurrence of EPVS in hippocampus (H-EPVS) has been paid more and more attention. The hippocampal vascular system is the basis for maintaining the normal function of the hippocampus. However, the diameter of the artery that directly supplies the hippocampus and its branches is very small [2,3]. Physiological (aging) or pathological (stroke, Alzheimer' s disease) cases, the blood vessels in the hippocampus may be more easily affected by decreased blood perfusion and hypoxia, resulting in hippocampal neuronal dysfunction [3].
The hippocampal formation is closely related to cognitive function. P300 may be an important index to evaluate the hippocampus function. P300 event-related potential (ERP) is considered the most valuable neurophysiological index to re ect the process of individual cognitive processing [4]. P300 (or 'P3b') is related to the information processing and contextual memory updating when brain performing tasks [5].
Unfortunately, the relationship between H-EPVS and P300 in patients with acute lacunar stroke is still unclear.
In this study, we aim to understand the incidence of H-EPVS in patients with lacunar stroke and we want to determine whether P300 ERP is associated with H-EPVS cognitive function.

EPVS and other markers
We used GE 3.0T MR (Discovery MR750, USA) imaging system scan the T1WI, T2WI and T2 Flair sequence of all subjects. EPVS showed round, oval and linear structures with clear boundaries consistency in the direction of perforating arterioles (due to differences in location and section) on MRI. T1WI and FLAIR sequences showed low signal intensity, T2WI showed high signal intensity, and was the same as cerebrospinal uid signal. EPVS usually occurs in BG, CSO, hippocampus and brainstem. Visual quanti cation was used to count the largest number of EPVS in unilateral BG and CSO plane [6] : grade 0, was non-EPVS; grade 1 was EPVS≤ 10; grade 2 was EPVS 11~20; grade 3 was EPVS 21~40; grade 4 was EPVS>40. Grade 0~1 was de ned as mild EPVS, Grade 2~4 was de ned as moderate -severe EPVS [6] (Fig1 B~C). We calculate total count of left and right hippocampus, H-EPVS≥ 7 was de ned as extensive H-EPVS, H-EPVS< 7 was de ned as non-extensive H-EPVS [7,8] (Fig1 A). We only calculate the maximum diameter < 3mm EPVS, because the EPVS of > 3mm may have different pathogenesis.
The white matter hyperintensities (WMH) was the speckled or patchy high signal changes of the paraventricular or deep white matter on the FLAIR sequence. Fazekas score ≤ 2 was de ned as mild WMH, Fazekas score ≥ 3 as moderate-severe WMH [9] (Fig1 D). Lacunar infarction usually shows marginal high signal intensity on T2WI sequence with a diameter of ≥ 3 mm.
Cognitive assessment All subjects were evaluated by the same physician trained by the formal uni ed neuropsychological scale in the same environment. The test scale includes overall cognitive function, memory, information processing speed, executive function and verbal uency function. Convert the each test scores into standardized Z-score (individual test score -mean test score) / standard deviation [10]. Finally, the test Zscore of each cognitive domain is averaged, and the compound Z-score of the cognitive domain is obtained. The higher the Z-score, the better the performance.
The test scale includes: (1) Overall cognitive function: Montreal cognitive assessment (MoCA) has been widely used in vascular cognitive impairment and Mild cognitive impairment screening. Compared with mini-mental state examination (MMSE), it has higher sensitivity and speci city [11]. P300 ERP data acquisition The P300 wave was generated from the keypoint EMG evoked potential instrument produced by Dandy Company. The reference electrode was placed in the posterior mastoid of both ears, the recording electrode was placed at the Cz point, and the ground electrode was placed in the center of the forehead. It was suitable for scrub to reduce the resistance of each electrode to less than 5K Ω, and the sensitivity was 5μV. Auditory Oddball mode was selected, which consists of two different frequencies of sound. One was target stimulus, also known as treble stimulus, the occurrence rate was 20%. The other was nontarget stimulus, also known as bass stimulus, the occurrence rate was 80%. The two stimuli occur irregularly alternately, the interval of sound stimulation was 1.5s, superimposed 200 times. Keystroke responses to Target stimuli is monitored when the patients stays quiet and with closed eyes. The reaction time and hit rate of the subjects were recorded, the test was repeated twice, and the average value was taken.
In this study, basic waveform on the Cz points were recorded, the P300 latency (the straightline distance from the start of stimulation to the peak of the maximum P300 amplitude) and amplitude (the vertical distance from the baseline to the peak of P300) were analyzed in both groups.  (Table 1). Second, Spearman correlation analysis was used to analyze the correlation between information processing speed Z-score, speech uency Z-score, P300 latency and H-EPVS counts (Fig 2). After adjusting the covariates of age, sex and education years, further analysis the relationship between H-EPVS count and P300 latency. The results were expressed in terms of β and 95% con dence interval (Table 2). Finally, receiver operating characteristic (ROC) curves was generated, and analyze the speci city and sensitivity of P300 latency in predicting cognitive impairment with extensive H-EPVS in patients with acute lacunar stroke.
The information processing speed Z-score and Verbal uency Z-score in extensive H-EPVS group were lower than non-extensive H-EPVS group (P < 0.01). The two subscales AVLT immediate recall and delayed recall Z-score re ecting memory function in the extensive H-EPVS group were also lower than those in the non-extensive H-EPVS group, but there was no signi cant difference in the Z score of memory function and executive function between the two groups (P > 0.05) ( Table 1).

ROC analysis predictive of H-EPVS cognitive function
ROC curve analyses (Fig 3) were used to identify that the area under the curve (AUC) for the prediction of verbal uency function with extensive H-EPVS patients using P300 latency was 0.796 (95% CI 0.711 0 .881, P < 0.01).

Discussion
This study found that the incidence of extensive H-EPVS was higher in patients with lacunar stroke. P300 latency was positively associated with H-EPVS counts. Extensive H-EPVS has a high incidence in patients with acute lacunar stroke. The P300 latency may be used as an early indicator to evaluate the verbal uency function of extensive H-EPVS patients with acute lacunar stroke.
It was reported that the incidence of H-EPVS in healthy people was 36.4-77%, and the incidence of H-EPVS in stroke people was as high as 84% [13,14]. Blood-brain barrier (BBB) is a unique structure in brain microcirculation, endothelial cells play an important role in maintaining tissue perfusion pressure and hemodynamics [15]. The endothelial cells damage of hippocampal artery leads to the increase of bloodbrain barrier permeability, the metabolite clearance disturbance in hippocampal artery and the interstitial uid circulation disturbance will form H-EPVS [16]. This study found that age was risk factors for extensive H-EPVS, which was consistent with previous studies [7,14]. Related studies found that WMH, lacunar infarction, BG-EPVS and CSO-EPVS was associated with H-EPVS [14,17]. However, no association with extensive H-EPVS was found in this study.
At present, the research conclusions on H-EPVS and cognitive performance are not consistent. A study based on the patients with hypertension shows H-EPVS was related to worse verbal reasoning ability [7].
In contrast, no correlation was found between H-EPVS count and cognitive performance with dementia risk in healthy elderly and community populations [14,17]. We found that the information processing speed of extensive H-EPVS was lower than non-extensive H-EPVS, but no correlation was found between H-EPVS count and information processing function. In addition, H-EPVS count was positively correlated with delayed P300 latency. P300 is a component that re ects the cognitive process, and the P300 latency re ects the brain recognizes information speed [5]. The origin of P300 in the brain is still not completely clear. It may represents the sum of complex activities of different generators in the limbic system and related regions of the cerebral cortex, and each generator may represent different cognitive performance [18]. Hippocampus as an important part of the marginal-cortical network, it is involved in the activity of the generator [18,19]. Previous research found P300 disappeared in patients with bilateral hippocampal lesions, P300 may re ect the hippocampal function damage. Scalp recording potentials involve related areas of the hippocampus and cerebral cortex [20]. H-EPVS was closely related to vascular neural unit dysfunction. Vascular neural units can dynamically re ect the synaptic activity and metabolism of neurons [21]. This may be due to extensive H-EPVS damage to the neuronal circuits of the hippocampus and medial temporal lobe as well as other marginal structures connected to it, which in turn leads to the delayed P300 latency.
Our results mean signi cant negative correlation between H-EPVS count and Verbal uency, which further supports the previous research conclusions [7]. Research in Macaques found that areas similar to human temporo-parietal junction (TPJ) have two-way connections with the parahippocampal gyrus and are related to learning and memory [22]. Recent evidence suggested that the hippocampus acts as a sequence generator, extracts information from memory and participates in rapid context learning [23][24][25]. Verbal learning and episodic memory tests use the Ray auditory language learning test (RAVLT, immediate and delayed). We found that the AVLT immediate and delayed function decreased in the extensive H-EPVS group, but had no effect on memory function. The hippocampus was related to the process of contextual update in P300 [26]. P300 processes upstream of the hippocampus, such as the frontal cortex and TPJ, may achieve partial contextual updates. The increase of H-EPVS count may re ect focal hippocampus atrophy [27,28], hippocampal atrophy was associated with verbal memory impairment, especially verbal recall delayed [29]. Extensive H-EPVS may cause the hippocampal function decrease, destroy its connection with frontal cortex, and then verbal uency function became worse. In addition,we also found that P300 latency was negatively associated with verbal uency function.
Therefore, the P300 latency may be an important index for early evaluation of verbal uency function decline in patients with lacunar stroke extensive H-EPVS.
The advantage of this study is that use objective P300ERP examination and complete neuropsychological tests. Comprehensively evaluate the potential effects of H-EPVS on cognitive function in patients with acute lacunar stroke, which has important clinical signi cance.
This study also has some limitations. First of all, the sample size was relatively small, which may affect the results. Secondly, because this study only counts the H-EPVS < 3 mm, but does not take into account the H-EPVS > 3 mm. We did not measure hippocampal atrophy associated with H-EPVS. Finally, this study was a cross-sectional study. In follow-up studies, we will follow up to observe the long-term effects of extensive H-EPVS on P300 latency and verbal uency function.

Conclusion
We demonstrated that the incidence of extensive H-EPVS was higher in patients with acute lacunar stroke. The P300 latency may be used as an early indicator to evaluate the verbal uency function of extensive H-EPVS patients with acute lacunar stroke.

Availability of data and materials
The data that support the ndings of this study are available from the corresponding author upon reasonable request.
Ethics approval and consent to participate The study protocol was approved by the Institutional Ethics Committees of the Changzhou No.2 People's Hospital A liated to Nanjing Medical University (NO. 2018-KY032-01). Verbal and written informed consent was obtained from all individual participants included in the study.

Consent for publication
Not applicable. Data are presented as mean ± standard deviation, n (%) or median (IQR).