Exploration of the Mechanism Underlying the Association of Incident Microinfarct and Motor Deficit: A Preliminary Functional MRI Study

Participant recruitment, cognitive and motor function assessment

The Medical Ethics Committee approved the study of the Second Affiliated Hospital, Zhejiang University School of Medicine. We obtained written informed consent from all participants. We retrospectively reviewed middle-aged and elderly outpatients who subjectively complained of mild dizziness, headache, dysphagia, and fatigue in the Neurology Department between July 2018 and December 2020. Neurologists first performed a preliminary physical examination and inquired about medical history. Each participant was 1) muscle strength grading 5 (muscle activation against maximum resistance of examiner, full range of motion); 2) activities of daily living (ADL) function degree (eating, dressing, indoor mobility, bathing, using the toilet, and continence) fully independent and no assistance required [22]. After the outpatient screening, those participants who had written informed consent underwent head MRI scanning and behavioral assessment (motor function and cognitive function, the interval between examinations did not exceed one week.). Specifically, inclusion criteria are as follows: 1) age > 55 (middle-aged and elderly); 2) visible white matter hyperintensities on fluid attenuated inversion recovery (FLAIR, i.e., Fazekas score 1–3 in periventricular or deep white matter); 3) non-demented, no subjective complaint of cognitive decline, and Mini-Mental State Examination (MMSE) > 24; 4) normal vision and hearing. We excluded participants with: 1) MRI-DWI showed hyperintensity > 5 mm, including lacunar stroke; 2) incomplete demographics, motor assessment, and imaging data (severe head motion, artifacts during MRI scanning; 3) secondary causes of white matter lesions (e.g., demyelinating, metabolic, immunological, toxic, infectious, and other causes); 4) abnormal MRI findings (e.g., head trauma, hemorrhage, non-lacunar infarction, and other space-occupying lesions); 5) definitive peripheral neuropathy and spinal cord disease; 6) psychiatric illness (e.g., major depression); 7) use of medication known to influence cerebral function; 8) alcohol or drug abuse; 9) left-handedness. We finally included 133 participants (silent CSVD patients) who had no noticeable motor or cognitive symptoms.

We used the short physical performance battery protocol (SPPB) [23, 24]. The battery comprises three simple parts involves gait and balance. Each participant had one trial per task condition. Amongst, gait speed was measured based on 10 meters walk; balance is tested by scoring stand, semi-tandem stand, tandem stand, and repetitive chair-stand. Each set of performance measures were assigned a score of 0 to 4, and the total score of SPPB is 12. A higher SPPB score means better global motor function. The cognitive assessment included Trail Making Test Part A and B (TMT-A/TMT-B), symbol digit modalities test (SDMT) [25–27], forward and backward digital span (DSF/DSB).

MRI data acquisition

MRI data were acquired from the 3T (Discovery 750, GE Healthcare) scanner using an 8-channel brain phased array coil [28]. We acquired axial DWI with TR = 5000 ms; TE = 86 ms; voxel size =0.94×0.94×4 mm³; b = 1000 s/mm², and gradient recalled echo-planar imaging sequence for rsfMRI with time point = 180 volumes, TR = 2000; TE = 30 ms; slice thickness = 4mm; More MRI data acquisition detail in Supplementary Material 1.

Acute cerebral microinfarct detection

Based on MRIcron (https://www.nitrc.org/projects/mricron/), acute CMIs were rated by visual inspection according to previously proposed criteria by two experienced radiologists without knowledge of clinical information [8, 30]. CMIs was defined as: 1) small hyperintense lesions on DWI at any brain parenchymal location, with the most significant dimension≤5 mm; 2) lesions should be isointense or hypointense in apparent diffusion coefficient to exclude T2 shine-through of high T2 signal; 3) lesions did not show hypointensities on susceptibility-weighted imaging (Fig. 1). We divide participants into the 34 CMIs carriers and 99 controls according to the presence of CMIs or not.

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Fig. 1

Illustrates four examples of diffusion-weighted imaging-based CMIs diagnosis from our research sample. Case 1:79-year-old male with left basal ganglia cerebral microinfarcts (CMIs), short physical performance battery (SPPB, 10); Case 2:83-year-old male with right basal ganglia CMIs (SPPB 12, left picture), after a 1-year follow-up, the lesion disappears (right picture); Case 3:70-year-old female with lobar CMIs (SPPB 9); Case 4:62-year-old male with lobar CMIs (SPPB 11).

White matter hyperintensity assessment

The white matter hyperintensity (WMH) lesion map was automatically created for each subject based on the 3D T1-weighted and FLAIR images using the Lesion Segmentation Toolbox (Lesion prediction algorithm) [31]. Lesion masks were manually edited by one experienced radiologist (MMZ). We then co-registered the 3D T1W, FLAIR, and corrected masks to the standard atlas (UNC adult brain atlas template, http://www.nitrc.org). We finally calculated each WMH volume of the individual.

MRI preprocessing

Statistical analysis

All analyses in our study were performed using SPSS (version 26.0). We described quantitative variables as the mean and standard deviation, while categorical variables are absolute and relative frequencies. We used two-sample t-tests to examine differences in demographical information, motor and cognitive performance, and WMH burden. The Chi-square test was used to examine sex and a series of vascular risk factors (hypertension, diabetes, hyperlipidemia, smoking, excessing alcohol drinking). Then, linear regression models with entering method (p < 0.05 for significance) were used to determine the association between motor function score (dependent variable, 0–12) and CMIs (yes/no), as well as a series of independent variables. Specifically, WMH burden (ml) was entered into the regression model to test whether CMIs have an independent effect on motor function because WMH is the most commonly used imaging marker of CSVD and is related to motor function [37–39]. Other subjects included in the regression model included cognitive function (MMSE, 0–30), demographics (age, years; sex, female/male; education, years), and common vascular risk factors (hypertension, diabetes, hyperlipidemia, smoking, excessing alcohol drinking; yes/no). Further, each motor subfield score (tests of balance, gait speed, and chair stand) was tested as a dependent variable (0–4) in the regression mentioned above model. The standardized coefficients (β) with 95% confidence intervals (CI) were reported when statistical analyses were significant (p < 0.05).

The statistical analysis of the imaging is based on the DPABI [32]. We performed a two-sample T-test to explore differences between the CMIs carriers and controls in a voxel-wise manner regarding DCM and ECM. Further, we adjusted for the effects of age, gender, education, and WMH burden in subsequent analyses. Multiple comparisons correction was performed using Gaussian random field (GRF), p < 0.05 at height (i.e., minimum Z value > 2.3), p < 0.05 at the cluster level, two tails [40]. Further, Pearson correlations between the motor function (SPPB, balance, gait speed, chair stands) and functional connectivity metrics (EC and DC) were performed (p < 0.05, Bonferroni corrected) in regions with a between-group difference.

CMIs carriers had worse motor function than non-carriers

In this study, we identified 34 CMIs carriers (66.2±7.9 years) and 99 non-carriers (64.1±8.5 years) as controls, based on MRI-based DWI in a middle-aged and elderly population (64.6±8.3 years) with CSVD. Among 34 CMIs carriers, there were 19 lobar CMIs carriers (67.8±4.8 years) and 15 BG CMIs carriers (64.1±10.5 years), and all CMIs were supratentorial (Supplementary Material 3). Between the CMIs carriers and non-carriers, no significant differences existed in terms of demographic characteristics (age, sex, education), the incidence of hypertension (26.5% versus 39.4%), diabetes (5.9% versus. 6.1%), hyperlipidemia (5.9% versus 6.1%), smoking (20.6% versus 35.4%), and alcoholism (17.6% versus 28.3%).

Regarding cognitive function, CMIs carriers matched well with controls in MMSE and multiple cognitive domains. CMIs carriers had worse motor function (SPPB score), heel and toe (p < 0.001), and chair stand (p < 0.05) than controls (Table 1). Further, CMIs carriers had more WMH burden than controls (p < 0.05).

CMIs were independently contributed to motor deficits in middle-aged and elderly

The linear regression model showed that general motor function (SPPB score) was significantly associated with WMH burden (β=–0.28, p = 0.002) and presence of CMIs (β= –0.21, p = 0.01, Table 2). For sub-item, we found that balance test was related with WMH burden (β= –0.21, p = 0.02) and presence of CMIs (β= –0.27, p = 0.001); age (β= –0.25, p = 0.001, Supplementary Material 4); while chair stand test was associated with WMH burden (β= –0.23, p = 0.02, Supplementary Material 5).

Brain functional deficit and compensation co-exist in CMIs carriers

CMIs carriers had increased DC at the local level in the right middle temporal gyrus (MTG) and left inferior frontal gyrus (IFG); CMIs carriers had decreased EC in the left precuneus at the global level while increased EC in the right MTG (p < 0.05, GRF corrected). Results remained primarily unchanged, adjusting for age, sex, education, and WMH burden (p < 0.05, GRF corrected, Fig. 2, Table 1). We also explored the effects of CMIs location on the brain network, and the preliminary results are located in Supplementary Material 3.

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Fig. 2

Illustrates regions showing differences of degree centrality (DC) and eigenvector centrality (EC) between cerebral microinfarcts (CMIs) carriers and non-carriers (GRF corrected, p < 0.05 at height, and p < 0.05 at the cluster level, two tails). Hot and cold areas represent DC/EC decrease and increase when CMIs carriers compared to controls, respectively. A) CMIs carriers versus controls, mapping of EC (GRF corrected); B) CMIs carriers versus controls, mapping of DC (GRF corrected); C) CMIs carriers versus controls, ECM (GRF corrected, adjusting for age, sex, education, WMH burden); D) CMIs carriers versus controls, ECM (GRF corrected, adjusting for age, sex, education, WMH burden).

Degree centrality and eigenvector centrality metrics were related to motor function

Pearson correlation results showed that EC of precuneus positively related to SPPB (r = 0.25, p = 0.004) and balance performance (r = 0.27, p = 0.002), while DC (r = –0.25, p = 0.003) and EC (r = –0.25, p = 0.003) of MTG was negatively related with SPPB in all participants (Fig. 3).

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Fig. 3

Correlation relationship between imaging metrics and motor function. Across groups, the Pearson correlation results showed that eigenvector centrality (EC) of precuneus positively related to short physical performance battery (SPPB, r = 0.25, p = 0.004, A) and balance performance (r = 0.27, p = 0.002, B). In contrast, DC (r = –0.25, p = 0.003, C) and EC (r = –0.25, p = 0.003, D) of MTG was related with SPPB.

CMIs independently related to motor function impairment

Apart from skeletal muscle bulk, accumulating studies have demonstrated that CSVD surrogates, such as WMH, contributed to the late-life impaired gait, balance, and postural stability [41, 42]. We also demonstrated that WMH independently contributed to motor dysfunction. Notably, for the first time, our work suggested that CMIs were related to the motor function in vivo, results independent of WMH burden and vascular risk factors. Our results were primarily in line with neuropathological work documenting that CMIs were associated with the motor function before death [5]. However, their results reported that CMIs are related to gait speed rather than balance [5]. The potential interpretations of the discrepancy include the difference in sample age and the ceiling effect of the balance test in the very elderly population (mean age 88.5 years).

Presence of CMIs linking both increase and decrease of centrality connectivity to motor impairment

We found that the CMIs carriers had decreased global centrality in the precuneus. After correcting for age, sex, education, and WMH, our results remained unchanged. Based on the hub vulnerability hypothesis, brain functional hub regions are usually the first to be affected in the presence of pathological factors [43, 44]. As a core hub, the precuneus has a pivotal role in the frontoparietal network [45]. Anatomically, the precuneus and prefrontal cortex are interconnected. This circuit covers different executive control functions, including task-elicited awareness, visuospatial function, and motor imagery, contributing to the cognitive control of posture, such as holding something in mind [39, 46]. We inferred that CMIs might preferentially damage the hub regions of the frontoparietal network via perilesional and remote effects [11, 12]. As a result, the global connectedness of precuneus is disrupted, leading to cognitive control dysfunction and balance impairment. Previous work consistently showed that the executive function mediated the relationship between WMH and the impaired motor [47, 48]. Another possible explanation is that decreased functional connection in precuneus merely represents the most salient damage in the presence of CMIs; balance impairment might be attributed to the widespread invisible CMIs, which affect the corticostriatal or thalamostriatal fiber directly [49–51].

Interestingly, we found that CMIs carriers had both regional and global function connection increases in MTG compared to controls. We thus interpreted that increased centrality in MTG may reflect compensatory effects on motion function in CMIs carriers. In other words, the MTG of the CMIs carriers generated additional activation to finish the comprehensive motion assessment. Many previous animals or functional MRI studies showed that MTG is related to motion observation, such as memory trace consolidation processes and enhancing memory consolidation of actions [52]. One fMRI study showed that MTG takes part in action-feedback monitoring, which is an essential process that aids in motor learning and distinguishes self-generated from externally generated stimuli [53]. Our assumption supported by our correlation analyses showed that DC and EC of MTG were negatively related to global motor function. This negative correlation further indicates that the increased regional and global centrality in MTG is a negative contribution. Notably, in most previous studies, MTG function is more related to language processing; but tends to occur in the language-related dominant hemisphere (i.e., the left MTG).

Another interesting finding is that CMIs carriers had increased DC in IFG relative to non-carriers. We inferred that this result might reflect a compensatory enhancement of functional connections. IFG is a region that plays a pivotal role in motor control and program [54, 55]. Previous studies have frequently reported that compensatory IFG functional connectivity increases when the task becomes more complex [56, 57]. It became understandable that CMIs carriers compensatively activated the IFG region to complete a series of integrated movements when the functional hub region (i.e., precuneus) was impaired. The correlation analysis, however, did not find the association between IFG and SPPB and its sub-item. The possible reason is that IFG is more involved in fine movement, so a more comprehensive evaluation of motor function is necessary for the future [58].

No differences in cognitive function between CMIs carriers and non-carriers

Although cognitive function was not the main objective of our study, we unexpectedly found no significant differences between CMIs carriers and non-carriers in general cognitive function and each sub-item. Two possible explanations may account for the negative result. First, motor symptoms are common after a stroke (> 5 mm); nearly 30% of stroke patients develop cognitive impairment, such as attention and executive function, within 1 year of stroke onset [59]. CMIs are a special type of infarction (< 5 mm) essential; thus, they may affect motor function prior to cognition abilities. We hypothesized that cognitive decline might occur in CMIs carriers after a period of follow-up. Another interpretation is that the current neuropsychological battery is not wide enough, such as lacking memory-related scale.

The study has some limitations. First, future longitudinal studies are still required to understand the causal relationship between CMIs and motor function; the effects of CMIs on cognitive function might also be found in longitudinal cohorts. Second, although we adopted the proposed diagnosis criterion of acute CMIs, it should be noted that the size of DWI-based lesions still depends on field strength and the parameters of the DWI sequence [60]. Our results thus need to be further verified when the scanning consensus is determined. Third, movement evaluations in our study are still insufficient, only involving the lower extremities. On the other side, our study did not quantify arthritis, falls, and frailty conditions, which may affect SPPB score, although the neurologists tried to control these physical factors on motor function at the initial screening stage. Manual dexterity and strength, and quantitative physical factors should also be included in future research. Finally, vascular risk factors are not enough in our study; previous work has shown that abnormalities of cardiac function (e.g., atrial fibrillation, ischemic heart diseases, and congestive heart failure) may be an important factor in the occurrence of CMIs [61]. Future studies need to consider more vascular risk factors.

In conclusion, our study demonstrated that CMIs are an independent risk factor for motor function impairment, especially balance. Further, CMIs are related to brain network hub region functional connectivity decrease and compensatory connectivity increase in the motor-related region. CMIs might affect motor function via damaging motor-related cognitive control or fiber tracts directly.