The study was approved by the Ethics Committee of Pu’er People’s Hospital, and all subjects signed an informed consent form. Thirty patients with CSVD who met the inclusion criteria in the Department of Neurology, Pu’er People’s Hospital from October 2018 to November 2018 were selected, all of which have had the complete magnetic resonance imaging (MRI) and susceptibility weighted imaging (SWI) of the crania. According to the result of cranial MRI examination, they were divided into lacunar infarction (LI) group and multiple lacunar infarction (MLI) combined with white matter lesions (WML) group (Fig. 1). Thirty samples from healthy volunteers without abnormalities after head MRI examination of Pu’er People’s Hospital were selected as the control group. There were eight patients with LI, including six males and two females, aged 49–68 years old (58.72±7.34); 22 patients with MLI combined with WML, 15 males and seven females, aged 48–71 years old (60.04±7.08); healthy volunteers in the control group of 20 males and 10 females, aged 48–69 years old (59±6.71). There were no significant differences between the three groups in gender and age (p<0.05), which were comparable.

At present, there is no unified conclusion on the diagnostic criteria for CSVD. This study refers to the “Chinese CSVD diagnosis and treatment consensus” [3] and combined with the common performance of MRI to establish the following criteria : 1) LI : MRI showed a circular, elliptical or fissure-like cavity with a clear boundary, 3–15 mm in diameter, a significantly low signal on T1WI and a high signal on T2WI (Fig. 1A); 2) WML : manifested as punctate or flaky abnormal signals in the basal ganglia, semi-oval center, and radiation crown, T1WI showed low signal, T2WI and MRI liquid attenuation inversion recovery sequence was high signal, lesion boundary was blurred and the diameter was more than 5 mm (Fig. 2A); 3) cerebral microbleeds : MRI T2WI gradient echo or SWI showed uniform circular low-signal lesion with a diameter of 2–5 mm, surrounded by no edema (Fig. 2B); and 4) expansion of the perivascular space : T2WI had a high signal, diameter <3 mm, circular or linear, located in white matter and deep gray matter, and a low signal was visible on T1 (Fig. 2C).

Inclusion criteria : 1) those with complete MRI and SWI data; 2) those who meet the above criteria for diagnosis of CSVD (diagnostic criteria 1 and/or diagnostic criteria 2); 3) MRI indicates that the infarct is LI or MLI combined with WML; and 4) those who have no communication problems and agree to participate in the study.

Exclusion criteria : 1) MRI showed that the diameter of the infarct was >20 mm; 2) computed tomography angiography of the head and neck showed stenosis of the intracranial and extracranial aorta, intracranial hemorrhagic disease or previous cerebral hemorrhage, cardiogenic cerebral embolism; 3) those with tumors, severe heart, lung, liver and kidney diseases and blood diseases; 4) those with genetic, metabolic, poisoning and rheumatic immune system diseases; and 5) those with central nervous system infections, multiple sclerosis and other central systems inflammatory demyelinating diseases, brain trauma, brain tumors, etc.

In the early morning, the peripheral arterial blood of the elbow artery was taken on fasting, and the blood sample was stored in a centrifuge tube containing heparin. After the sample was collected for 30 minutes, it was centrifuged at 2000 r/min at 4°C for 15 minutes. The serum was separated and stored in a refrigerator at -80°C for reserve. Real-time quantitative polymerase chain reaction was used to detect the expression level of miR-155 in serum. U6 RNA primers (internal reference primers) and miR-155 stem-loop primers were purchased from Guangzhou RiboBio Co., Ltd (Guangzhou, China). The relative quantity of gene expression was expressed by 2^-ΔΔCt.

SPSS ver. 20.0 statistical software (IBM, Guangzhou, Guangdong, China) was used for data analysis. The normal distribution of measurement data was expressed by mean±standard deviation. Analysis of variance was used. Two independent samples t test were used for comparison between the two groups, and Spearman correlation analysis was adopted. p<0.05 was considered statistically significant.

Compared with the control group, the levels of miR-155 in the serum of the LI group increased (p<0.05), and MLI combined with WML group increased (p<0.001). Because the number of lesions in LI is not directly related to cerebral small vessels, it does not need to be considered (Table 1 and Fig. 3) [26].

The level of miR-155 in serum was positively correlated with the increase of imaging lesions (p<0.05) (Table 1).

MiR-155 belongs to one of the miRs and is a typical multifunctional gene localized to human chromosome 21. Located in the 3rd exon of the B-cell integration cluster (BIC) gene, its expression level is regulated by the transcriptional level of BIC gene and processing of other miRs.

This study [15] showed that the levels of miR-155 in the serum of LI group and MLI combined with WML were higher than those in the control group (p<0.05), suggesting that the level of miR-155 in serum of CSVD increased [29]. Further analysis of the correlation between the changes of serum miR-155 levels in patients with CSVD and the increase of imaging lesions found that the level of miR-155 in serum was positively correlated with the number of lesions (p<0.05), showing that the more imaging lesions in patients with CSVD, the higher the level of miR-155 in the serum [14].

BBB dysfunction is considered to be a biological marker of cerebral stroke, and increased cerebral vascular permeability and BBB leakage will lead to ischemic brain damage [1]. Zhang et al. [34] pointed out that miR-155 can be used as a negative regulator of BBB function. This study found that the inhibition of endogenous miR-155 attenuated some of the high permeability induced by cytokines [2]. In addition, miR-155 not only targets cell-complex molecules, such as annexin-2 and annexin-1, but also targets adhesion molecules, such as DOCK-1 and Synthin-1 to regulate brain endothelial function. Ago [1] found that Bcl-2 was an important anti-apoptotic protein in mice, which can reduce BBB destruction and cerebral infarction in mice after focal cerebral ischemia [4].

In recent years, a lot of research has shown that the maintenance and exercise of normal brain function mainly depend on the function and structure of sound and intact neurovascular units in the brain parenchyma [1]. The integrity of neurovascular unit function depends on the neuroglial cells and neurons in the process of neurotransmitter conversion and transmission [19], and then coordinates with each other to perform their functions; the integrity of neurovascular unit structure depends on the integrity of its multiple constituent cells-nerve and non-neural cell integrity [30]. Neurons and glial cells are involved in the formation of nerve cells, and glial cells contain microglia, astrocytes, and oligodendrocytes; nonneuronal cells include vascular smooth muscle cells and vascular endothelial cells, basement membrane and extracellular matrix [10]. Although the brain parenchymal damage caused by CSVD is small, it also destroys the function and structural integrity of the neurovascular unit.

Nitric oxide (NO) secreted by vascular endothelium is for vascular endothelial homeostasis. Decreased NO released by nitricoxide synthase leads to endothelial dysfunction. Hypoxia and ischemia reduce endothelial nitric oxide synthetase (eNOS) expression in endothelial cells through post-transcriptional mechanism, resulting in NOS3 transcriptional instability. In this study, we found that miRNA contributes to this mechanism [8]. By studying changes in the expression profile of miRs in ischemic stroke, it was found that the expression level of miR-155 in rat brain tissue was increased [36], indicating that miR-155 was closely related to ischemic stroke. In recent years, studies have shown that in the acute phase of stroke in mice, the inhibition of miR-155 protects cerebral vascular endothelial cells [12], attenuates immune inflammatory responses in ischemic mouse brain tissue [16], and is good for neurological rehabilitation and infarct size reduction in ischemic stroke mice [27]. Pena-Philippides research team used novel Locked Nucleic Acid technology [36] to inhibit miR-155 in ischemic stroke mice and found that the expressions of cytokines CCL12, CX-CL3 and inflammation-related molecules were significantly reduced in experimental mice. It promotes cerebral angiogenesis, reduces brain tissue damage, and improves neurological function recovery. In the mouse model of stroke, there are also studies that use acetylcholine ester to inhibit miR-155 and achieve good recovery [32]. The new treatments in these experiments will provide new ideas for the treatment of clinical patients, and further clinical trials are needed to confirm this result [6].

The level of miR-155 in serum plays a role in the occurrence, development and prognosis of CSVD [9]. The increase of serum miR-155 level in patients with CSVD may be one of its selfprotection mechanisms, and the strength of this self-protection mechanism may be positively correlated with the number of lesions. Further in-depth research on the level of miR-155 in the serum of CSVD will bring new ways and methods for the prevention and treatment of CSVD [20]. It is hoped that more researchers will contribute to the clearer relationship between miR-155 and inflammatory markers from different aspects, and further study of relationship between miR-155 and cerebral small vessels.