Abnormal amplitude of low‐frequency fluctuations and functional connectivity of resting‐state functional magnetic resonance imaging in patients with leukoaraiosis

Abstract Introduction This study aimed to investigate the cerebral function deficits in patients with leukoaraiosis (LA) and the correlation with white matter hyperintensity (WMH) using functional MRI (fMRI) technology. Materials and Methods Twenty‐eight patients with LA and 30 volunteers were enrolled in this study. All patients underwent structural MRI and resting‐state functional MRI (rs‐fMRI) scanning. The amplitude of low‐frequency fluctuations (ALFF) of rs‐fMRI signals for the two groups was compared using two‐sample t tests. A one‐sample t test was performed on the individual z‐value maps to identify the functional connectivity of each group. The z values were compared between the two groups using a two‐sample t test. Partial correlations between ALFF values and functional connectivity of the brain regions that showed group differences and Fazekas scores of the WMH were analyzed. Results Compared with the control group, the LA group showed a significant decrease in the ALFF in the left parahippocampal gyrus (PHG) and an increased ALFF in the left inferior semi‐lunar lobule and right superior orbital frontal gyrus (SOFG). The patients with LA showed an increased functional connectivity between the right insular region and the right SOFG and between the right calcarine cortex and the left PHG. After the effects of age, gender, and years of education were corrected as covariates, the functional connectivity strength of the right insular and the right SOFG showed close correlations with the Fazekas scores. Conclusion Our results enhance the understanding of the pathomechanism of LA. Leukoaraiosis is associated with widespread cerebral function deficits, which show a close correlation with WMH and can be measured by rs‐fMRI.


| INTRODUCTION
Cognitive impairment is characterized by impaired cortical functions such as memory, orientation, computation, understanding, and visual spatial skills. The increasing prevalence of cognitive impairment with age seriously affects the health and lives of the elderly and is attracting more attention from research investigators (Hastings et al., 2014).
Leukoaraiosis (LA) is the main pathological mechanism of vascular cognitive impairment (Longstreth et al., 2005;van Straaten et al., 2006). It commonly appears in subcortical atherosclerotic encephalopathy, chronic cerebral insufficiency, and other diseases and often affects the white matter in the periventricular and subcortical areas of the brain.
The proportion of LA in patients who are aged 60 years and older is as high as 30%, as detected by brain magnetic resonance imaging (Launer et al., 2006;Nichtweiss, Weidauer, Treusch, & Hattingen, 2012).
According to the standards developed by Fazekas, Chawluk, Alavi, Hurtig, and Zimmerman (1987), the white matter hyperintensity (WMH) severity is assessed on fluid-attenuated inversion recovery (FLAIR) images using a grading scale (grade 0, absent; grade 1, punctate; grade 2, early confluent; grade 3, confluent). Presently, the mechanism underlying WMH that affects brain function is not clear. With the development of neuroimaging, functional magnetic resonance imaging (fMRI) can study brain activity by detecting blood oxygen levels in the brain using high spatial resolution. fMRI includes task correlation and resting-state fMRI (rs-fMRI). Spontaneous neural activity can be detected in the brain, which is helpful for understanding the pathological mechanisms of nervous system diseases and mental diseases from the baseline level Wu, Lai, Zhang, Yao, & Wen, 2015). Because rs-fMRI is simple and easy to perform, is highly consistent, does not require task design, and facilitates patient cooperation, it has been commonly used in recent fMRI studies and is meaningful for clinical diagnosis and treatment evaluation. The amplitude of low-frequency fluctuations (ALFFs) of rs-fMRI can assess the amplitude of resting-state spontaneous brain activity by calculating the square root of the power spectrum (typically in the frequency range of 0.01-0.08 Hz; Zang et al., 2007;Zou et al., 2008). Rs-fMRI has been proven effective for reflecting the spontaneous neural activity in both animals and humans (Goncalves et al., 2006;Leopold, Murayama, & Logothetis, 2003). Our hypothesis is as follows: (1) changes in intrinsic brain activity patterns in patients with LA could be located in multiple brain regions; and (2) the features of ALFF and functional connectivity in specific regions correlate with the disease severity of LA.

| Subjects
This study was approved by the Medical Ethics Committee at our institution. Informed consent was obtained from each subject.
Twenty-eight patients diagnosed with brain ischemic diseases such as subcortical arteriosclerotic encephalopathy or chronic cerebral circulation insufficiency were recruited from the Neurology Department of our hospital (

Control subjects (n = 30) p Value
Gender ( p Values for gender distribution between the two groups was obtained using a two-tailed chi-squared test. b p Values were obtained using a two-sample two-tailed t test.

| Image processing and analysis
The structural data were obtained with an optimized VBM8 proto-

| Statistical analysis
Amplitude of low-frequency fluctuation comparisons between patients with LA and healthy controls were performed using two-sample t tests. A one-sample t test was performed on the individual z-value maps to examine functional connectivity of each group. The twosample t test was used to compare the z values in each voxel of the two groups. We considered the whole brain gray matter volume, mean FD, age, gender, and education years as covariates. The relationship between functional connectivity and Fazekas scale scores was analyzed by partial correlation. The significance was set at p < .05 corrected with a multiple comparison correction.

| RESULTS
The patient and healthy control groups were not significantly different in age, gender, education levels, or mean FD (  Figure 1, Table 2). Brain regions that showed group ALFF differences were selected as seed regions. The patients with LA showed an increased connectivity between the right insular region and the right SOFG, as well as between the right calcarine cortex and the left PHG ( Figure 2). After the effects of age, gender, and years of education were corrected as covariates, the functional connectivity strength of the right insular and the right SOFG showed closed correlations with the Fazekas scores ( Figure 3).

| DISCUSSION
To date, given its better availability, noninvasiveness, and lack of radiation exposure, fMRI has been used as an effective technique for the study of central nervous system disorders ( human studies have provided evidence that fMRI effectively identifies cortical spontaneous neuronal function (Moosmann et al., 2003;Pelled & Goelman, 2004).
In this study, we used rs-fMRI to systematically investigate the changes in the ALFF of intrinsic brain activity in patients with LA.
Reduced ALFF was observed in the left PHG, and increased ALFF was observed in the left ISLL and right SOFG. These findings suggest that the left PHG, left ISLL, and right SOFG may be the areas most affected by ischemic WMH. The altered spontaneous ALFF values in these areas are possible characteristics of the neurological impairments of LA. In behavioral studies, LA patients have been found to have significant cognitive impairments of attention, memory, executive function, and information processing speed (Defrancesco et al., 2013;Yuan et al., 2012). The PHG is considered to be critical for memory function, including memory encoding and retrieval. It is also closely associated with human learning, emotions, and other activities. Previous studies have found that the hippocampus and PHG are susceptible to ischemia and lower blood volume (Chai, Ofen, Jacobs, & Gabrieli, 2010;Fein et al., 2000;Yang et al., 2010). The reduced ALFF activities in the PHG may indicate memory impairment in human subjects who have LA. The ISLL is part of the cerebellum, which has not been commonly reported in previous fMRI investigations. In traditional views, the cerebellum functions focus on control of motor behavior and coordination. Recently, the lateral cerebellum has been discovered to play an important role in memory function (Jantzen, Oullier, Marshall, Steinberg, & Kelso, 2007). The relationship between memory load and BOLD signal changes in the inferior cerebellum has also been proven (Kirschen, Chen, & Desmond, 2010;Ng et al., 2016;Tamagni et al., 2010). The SOFG has core components of the emotional processing network and is involved in the perceptual processing of emotions in facial expressions (Leppanen & Nelson, 2009). Furthermore, it has been linked to the experience of anger and aggression (Lindquist, Wager, Kober, Bliss-Moreau, & Barrett, 2012;Vytal & Hamann, 2010).
Given that the SOFG is associated with negative emotion processing,

MNI coordinate (mm)
Maximum t x y z Comparisons were performed at p < .01 and corrected for multiple comparisons using the AlphaSim program. x, y, and z are coordinates of peak locations in the MNI space. Maximum t shows ALFF differences between the leukoaraiosis group and healthy subjects. A positive maximum t-score represents an increase, and a negative maximum t-score represents a decrease. ALFF, amplitude of low-frequency fluctuations; MNI, Montreal Neurological Institute Coordinate System or Template; PHG, parahippocampal; ISLL, inferior semi-lunar lobule; SOFG, frontal superior orbital gyrus.
T A B L E 2 Regions showing ALFF value differences between patients with leukoaraiosis and control subjects F I G U R E 1 Brain regions with abnormal amplitude of low-frequency fluctuations (ALFF) in leukoaraiosis patients are shown. These regions are described in detail in Table 2. Statistical thresholds were set at p < .01 for individual voxels and a cluster size >1,080 mm 3 , which corresponds to a corrected p < .01 as determined by Monte Carlo simulations. Color bars represent the t value of the group analysis. A cool color represents decreased ALFF values, and a warm color represents increased ALFF values. PHG, parahippocampal; ISLL, inferior semi-lunar lobule; SOFG, frontal superior orbital gyrus the increased ALFF in these areas suggests that people with LA may experience more negative emotions. This speculation is in accordance with the finding that impaired white matter is linked to anxiety and depression (Coplan et al., 2010).
In addition to regional ALFF, rs-fMRI can provide information about functional connectivity. The analysis of cross correlations between the ALFF of spatially remote regions can examine the brain connectivity and networks. This is the first study to investigate the brain functional connectivity alterations in patients with LA. The patients with LA showed an increased connectivity between the right SOFG and the right insula, which also showed closed correlations with the Fazekas scores. The human insular has an important role in attention, language, speech, working memory, and memory (Kelly et al., 2012). The increased functional connectivity between insula and SOFG was earlier reported in the Alzheimer's Disease (Balthazar et al., 2014;Hafkemeijer et al., 2017).
Insula and SOFG play an important role in anterior frontoinsular-cinguloorbitofrontal network often called the salience network which is related with the ability to direct attentional resources and goal-relevant cognition (Menon, 2011). The Fazekas score was designed for the crosssectional rating of WMH. It is stable and reliable and has strong ties to WMH severity (Kapeller et al., 2003). The calcarine was suggested to be a key hub of the posterior compensatory network in cognitively proficient elderly with hippocampal atrophy in a spatial working memory task fMRI (Valenzuela et al., 2015). PHG is considered as the hub of the default mode network and plays a mediation role to convey information to the frontal cortex, where information is integrated. The disrupted functional connectivity between CAL and PHG has also been founded in MCI in functional brain network study (Lou et al., 2016).
Previously, increased connectivity was observed in other diseases.
Patients with restless legs syndrome showed significantly increased connectivity in the sensory thalamic, ventral and dorsal attention, basal ganglia-thalamic, and cingulate networks (Gorges et al., 2016). Patients with mild cognitive impairment showed increased connectivity in the default mode network and visual network (Cai et al., 2016). The precise mechanisms leading to the increased connectivity in LA are not clearly.
In summary, in this study, we examined the LA -related cerebral function deficits of the ALFF during the resting state. We found that several cerebral regions were significantly correlated with WMH, including the left PHG, the ISLL, and the SOFG. The patients with LA showed an increased connectivity between the right SOFG and the right insular, which was closely correlated with Fazekas scores and suggests a compensatory mechanism. These findings may contribute to our understanding of the mechanism of LA. The main limitation F I G U R E 2 Brain functional connectivity alterations of leukoaraiosis patients are shown. Increased functional connectivity was found among the right insular and the right SOFG, the right calcarine cortex and the left PHG (p < .05, FDR corrected). Brain graph created using BrainNet Viewer (http://www.nitrc.org/projects/bnv/). The brain network edges were extracted from the correlation matrix of rs-fMRI connectivity across the regions of interest. Ball locations represent the peak Montreal Neurological Institute Coordinate System coordinate of differences within clusters, and the size indicates the cluster size. PHG, parahippocampal; ISLL, inferior semi-lunar lobule; SOFG, frontal superior orbital gyrus; INS, insula; CAL, calcarine F I G U R E 3 Correlations between the functional connectivity strength of the left insular and the right SOFG with the Fazekas scores. The effects of age, gender, and years of education were corrected as covariates. Each dot represents the data from one study participant. SOFG, frontal superior orbital gyrus of this study is the relatively small sample size of patients with LA.