MRI-visible enlarged perivascular spaces: imaging marker to predict cognitive impairment in older chronic insomnia patients.

OBJECTIVE: Perivascular spaces (PVS), components of the glymphatic system in the brain, have been known to be important conduits for clearing metabolic waste, and this process mainly increases during sleep. Sleep disruption might result in PVS dysfunction and cognitive impairment. In this study, we aim to explore whether MRI-visible enlarged perivascular spaces (EPVS) could be imaging markers to predict cognitive impairment in chronic insomnia patients. METHOD: We obtained data from 156 patients with chronic insomnia and 79 age-matched healthy individuals. Using T2-weighted MRI images, visible EPVS in various brain regions were measured and analyzed. The associations between EPVS numbers and cerebrospinal fluid (CSF) ß-amyloid 42 (Aß42), total tau (t-tau), and phosphorylated tau (p-tau) level in chronic insomnia patients were evaluated. RESULT: Our results showed that MRI-visible EPVS in the frontal cortex, centrum semiovale, basal ganglia, and hippocampus of chronic insomnia patients with impaired cognition (ICG) significantly increased than that in normal cognition (NCG) patients. The increased MRI-visible EPVS in the frontal cortex, centrum semiovale, and basal ganglia were also associated with the increased CSF Aß42, t-tau, and p-tau level in ICG patients. MRI-visible EPVS in the basal ganglia and centrum semiovale had high sensitivity and specificity in distinguishing ICG chronic insomnia patients from those with NCG. CONCLUSION: Our study indicated that MRI-visible EPVS in the basal ganglia and centrum semiovale might be valuable imaging markers to predict cognitive impairment in chronic insomnia patients. It will be meaningful to discern those cognitive decline patients in preclinical stage and take some measures to prevent disease progression. KEY POINTS: • Increased MRI-visible EPVS were associated with the increased CSF Aß42, t-tau, and p-tau level in older chronic insomnia patients with impaired cognition.

Exosomes derived from bone marrow mesenchymal stem cells protect the injured spinal cord by inhibiting pericyte pyroptosis.

Mesenchymal stem cell (MSC) transplantation is a promising treatment strategy for spinal cord injury, but immunological rejection and possible tumor formation limit its application. The therapeutic effects of MSCs mainly depend on their release of soluble paracrine factors. Exosomes are essential for the secretion of these paracrine effectors. Bone marrow mesenchymal stem cell-derived exosomes (BMSC-EXOs) can be substituted for BMSCs in cell transplantation. However, the underlying mechanisms remain unclear. In this study, a rat model of T10 spinal cord injury was established using the impact method. Then, 30 minutes and 1 day after spinal cord injury, the rats were administered 200 µL exosomes via the tail vein (200 µg/mL; approximately 1 × 106 BMSCs). Treatment with BMSC-EXOs greatly reduced neuronal cell death, improved myelin arrangement and reduced myelin loss, increased pericyte/endothelial cell coverage on the vascular wall, decreased blood-spinal cord barrier leakage, reduced caspase 1 expression, inhibited interleukin-1ß release, and accelerated locomotor functional recovery in rats with spinal cord injury. In the cell culture experiment, pericytes were treated with interferon-γ and tumor necrosis factor-α. Then, Lipofectamine 3000 was used to deliver lipopolysaccharide into the cells, and the cells were co-incubated with adenosine triphosphate to simulate injury in vitro. Pre-treatment with BMSC-EXOs for 8 hours greatly reduced pericyte pyroptosis and increased pericyte survival rate. These findings suggest that BMSC-EXOs may protect pericytes by inhibiting pyroptosis and by improving blood-spinal cord barrier integrity, thereby promoting the survival of neurons and the extension of nerve fibers, and ultimately improving motor function in rats with spinal cord injury. All protocols were conducted with the approval of the Animal Ethics Committee of Zhengzhou University on March 16, 2019.

Inhibition of microRNA-29b suppresses oxidative stress and reduces apoptosis in ischemic stroke.

MicroRNAs (miRNAs) regulate protein expression by antagonizing the translation of mRNAs and are effective regulators of normal nervous system development, function, and disease. MicroRNA-29b (miR-29b) plays a broad and critical role in brain homeostasis. In this study, we tested the function of miR-29b in animal and cell models by inhibiting miR-29b expression. Mouse models of middle cerebral artery occlusion were established using the modified Zea-Longa suture method. Prior to modeling, 50 nmol/kg miR-29b antagomir was injected via the tail vein. MiR-29b expression was found to be abnormally increased in ischemic brain tissue. The inhibition of miR-29b expression decreased the neurological function score and reduced the cerebral infarction volume and cell apoptosis. In addition, the inhibition of miR-29b significantly decreased the malondialdehyde level, increased superoxide dismutase activity, and Bcl-2 expression, and inhibited Bax and Caspase3 expression. PC12 cells were treated with glutamate for 12 hours to establish in vitro cell models of ischemic stroke and then treated with the miR-29 antagomir for 48 hours. The results revealed that miR-29b inhibition in PC12 cells increased Bcl-2 expression and inhibited cell apoptosis and oxidative damage. These findings suggest that the inhibition of miR-29b inhibits oxidative stress and cell apoptosis in ischemic stroke, producing therapeutic effects in ischemic stroke. This study was approved by the Laboratory Animal Care and Use Committee of the First Affiliated Hospital of Zhengzhou University (approval No. 201709276S) on September 27, 2017.

Impaired meningeal lymphatic drainage in patients with idiopathic Parkinson's disease.

Animal studies implicate meningeal lymphatic dysfunction in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease (PD). However, there is no direct evidence in humans to support this role1-5. In this study, we used dynamic contrast-enhanced magnetic resonance imaging to assess meningeal lymphatic flow in cognitively normal controls and patients with idiopathic PD (iPD) or atypical Parkinsonian (AP) disorders. We found that patients with iPD exhibited significantly reduced flow through the meningeal lymphatic vessels (mLVs) along the superior sagittal sinus and sigmoid sinus, as well as a notable delay in deep cervical lymph node perfusion, compared to patients with AP. There was no significant difference in the size (cross-sectional area) of mLVs in patients with iPD or AP versus controls. In mice injected with α-synuclein (α-syn) preformed fibrils, we showed that the emergence of α-syn pathology was followed by delayed meningeal lymphatic drainage, loss of tight junctions among meningeal lymphatic endothelial cells and increased inflammation of the meninges. Finally, blocking flow through the mLVs in mice treated with α-syn preformed fibrils increased α-syn pathology and exacerbated motor and memory deficits. These results suggest that meningeal lymphatic drainage dysfunction aggravates α-syn pathology and contributes to the progression of PD.

Autonomic ganglionic injection of α-synuclein fibrils as a model of pure autonomic failure α-synucleinopathy.

α-Synucleinopathies are characterized by autonomic dysfunction and motor impairments. In the pure autonomic failure (PAF), α-synuclein (α-Syn) pathology is confined within the autonomic nervous system with no motor features, but mouse models recapitulating PAF without motor dysfunction are lacking. Here, we show that in TgM83+/- mice, inoculation of α-Syn preformed fibrils (PFFs) into the stellate and celiac ganglia induces spreading of α-Syn pathology only through the autonomic pathway to both the central nervous system (CNS) and the autonomic innervation of peripheral organs bidirectionally. In parallel, the mice develop autonomic dysfunction, featured by orthostatic hypotension, constipation, hypohidrosis and hyposmia, without motor dysfunction. Thus, we have generated a mouse model of pure autonomic dysfunction caused by α-Syn pathology. This model may help define the mechanistic link between transmission of pathological α-Syn and the cardinal features of autonomic dysfunction in α-synucleinopathy.

Long-read sequencing identified intronic repeat expansions in SAMD12 from Chinese pedigrees affected with familial cortical myoclonic tremor with epilepsy.

BACKGROUND: The locus for familial cortical myoclonic tremor with epilepsy (FCMTE) has long been mapped to 8q24 in linkage studies, but the causative mutations remain unclear. Recently, expansions of intronic TTTCA and TTTTA repeat motifs within SAMD12 were found to be involved in the pathogenesis of FCMTE in Japanese pedigrees. We aim to identify the causative mutations of FCMTE in Chinese pedigrees. METHODS: We performed genetic linkage analysis by microsatellite markers in a five-generation Chinese pedigree with 55 members. We also used array-comparative genomic hybridisation (CGH) and next-generation sequencing (NGS) technologies (whole-exome sequencing, capture region deep sequencing and whole-genome sequencing) to identify the causative mutations in the disease locus. Recently, we used low-coverage (~10×) long-read genome sequencing (LRS) on the PacBio Sequel and Oxford Nanopore platforms to identify the causative mutations, and used repeat-primed PCR for validation of the repeat expansions. RESULTS: Linkage analysis mapped the disease locus to 8q23.3-24.23. Array-CGH and NGS failed to identify causative mutations in this locus. LRS identified the intronic TTTCA and TTTTA repeat expansions in SAMD12 as the causative mutations, thus corroborating the recently published results in Japanese pedigrees. CONCLUSIONS: We identified the pentanucleotide repeat expansion in SAMD12 as the causative mutation in Chinese FCMTE pedigrees. Our study also suggested that LRS is an effective tool for molecular diagnosis of genetic disorders, especially for neurological diseases that cannot be positively diagnosed by conventional clinical microarray and NGS technologies.

Overexpression of Egr2 and Egr4 protects rat brains against ischemic stroke by downregulating JNK signaling pathway.

OBJECTIVE: The purpose of this study was to investigate the effect of Egr2 and Egr4 upregulation on ischemic stroke recovery of rats. METHODS: In this study, Sprague Dawley (SD) rats assigned at random into control, sham and MCAO (middle cerebral artery occlusion) group were treated accordingly to build MCAO models. The neurological severity scores (NSS) test was applied to assess rats' behavior. Triphenyltetrazolium chloride (TTC) staining reflected infarct areas while Nissl staining revealed the number of neurons. Levels of pro-inflammatory cytokines (interleukin [IL]-1ß, IL-6 and tumor necrosis factor [TNF]-α) were judged by enzyme-linked immunosorbent assay (ELISA) in brain and serum tissues. We applied western blot to check the expression of Egr2, Egr4 and JNK/c-JUN (c-Jun N-terminal kinase) pathway. Further grouping of rats were based on various transfection, requiring control, sham, MCAO, MCAO + Egr2 cDNA (complementary DNA), MCAO + Egr4 cDNA, MCAO + Egr2 cDNA + Egr4 cDNA group to observe difference in MCAO recovery and JNK/c-JUN-pathway-related protein expression. RESULTS: Under successful modeling of MCAO, western blot results suggested down-regulation of Egr2 and Egr4 and overexpression of pro-inflammatory cytokines. The JNK/c-JUN pathway was activated. On upregulation of Egr2 and Egr4 in infarct areas, neurological function of SD rats recovered along with repressed JNK/c-JUN pathway activation and increased neuron number. CONCLUSION: Upregulation of Egr2 and Egr4 could demote the activation of JNK/c-JUN pathway and the expression of pro-inflammatory cytokines in MCAO rats, so that Egr2 and Egr4 might be potential targets for ischemic stroke in future.

Clinical study of Butylphthalide combined with Xue Shuan Tong on serum inflammatory factors and prognosis effect of patients with cerebral infarction.

To investigate the effect of Butylphthalide and Xue Shuan Tong on serum inflammatory factors and prognosis of patients with cerebral infarction. One hundred and twenty patients with acute cerebral infarction were randomly divided into control group, Butylphthalide group and Xue Shuan Tong group, with 40 patients in each group. Conventional therapy was performed in the control group; On the basis of conventional therapy, 100ml Butylphthalide intravenously twice a day was administrated among patients in Butylphthalide group; On the basis of conventional therapy, 250ml 0.9% NaCl intravenously once a day was conducted among patients in Xue Shuan Tong group. A treatment course of continuous 7 days was taken in the three groups. The serum levels of IL-2 and CGRP were detected for patients in the three groups before and after treatment. Carotid plaque thickness and size as well as intima-media thickness were detected by ultrasonic testing for patients in three groups before treatment and 90 days after follow-up. The NIHSS, Barthel and MRS scoring were performed for all the patients after 90-day follow-up to evaluate the prognosis. After treatment, differences in the levels of IL-2 and CGRP for patients in the three groups showed statistical significance (P<0.05), while the levels of IL-2 and CGRP in Xue Shuan Tong group were significantly higher than those in the other two groups (P<0.05). After 7-day treatment, plaque size and thickness in Xue Shuan Tong group and Butylphthalide group were significantly reduced, compared with those before treatment (P<0.05), but no significant differences was shown in the plaque size and thickness between Xue Shuan Tong group and Butylphthalide group (P>0.05) .The CA-IMT in Xue Shuan Tong group and Butylphthalide group was significantly reduced after treatment, and that in Butylphthalide group was significantly larger than that in Xue Shuan Tong group (P<0.05). After 90-day follow-up, NIHSS scores in Butylphthalide group were significantly less than those in the other two groups (P<0.05). After 90-day follow-up, Barthel scores in Butylphthalide group and Xue Shuan Tong group were significantly larger than those in control group (P<0.05), while differences between Butylphthalide group and Xue Shuan Tong group indicated no statistical significance (P>0.05). There were significant differences in MRS scores among patients in the three groups after 90-day follow-up (P<0.05). Butylphthalide and Xue Shuan Tong are clinically effective in treating acute cerebral infarction. Butylphthalide has significant efficacy in inhibiting inflammation and improving prognosis of neurological function, while Xue Shuan Tong has obvious effect in improving carotid intima-media thickness.

Study on the clinical efficacy of Human Urinary Kalllikrein in the treatment of acute cerebral infarction according to TOAST classification.

To observe and evaluate the clinical efficacy of Human Urinary Kallikrein in the treatment of acute cerebral infarction (ACT) according to TOAST (The Trial of Org 10172 in Acute Stroke Treatment) classification. In accordance with randomized controlled trial, 110 patients with acute cerebral infarction were randomly assigned to kallikrein treatment group (55 cases) and control group (55 cases). TOAST classification and basic treatment were administrated on patients between two groups respectively. 0.15 PNA unit of Human Urinary Kallikrein injection plus 100 mL saline in intravenous infusion was performed in the kallikrein group, with once a day for 14 consecutive days. The National Institutes of Health Stroke Scale (NIHSS) scores in two groups were analyzed before and after the treatment. No difference was shown in the NIHSS scores before treatment among patients between two groups (P>0.05). While after the treatment, the NIHSS scores in both groups were reduced (P<0.05) and the NIHSS scores in the kallikrein treatment group were less than those in control group (P<0.05). Moreover, after the treatment, the NIHSS scores for large-artery atherosclerosis subtype (L) and small-artery occlusion lacunar subtype (S) as two subtypes of TOAST classification in the two groups were both reduced (P<0.05). After the treatment, NIHSS scores for L subtype in the kallikrein treatment were less than those in the control group (P<0.05). After the treatment, NIHSS scores for S subtype in the kallikrein treatment were less than those in the control group, without statistically significant difference. Comparisons on clinical efficacy indicated differences on the S subtype between two groups (P<0.05). The standardization effective rate was calculated, indicating 81.82% in the kallikrein treatment group and 54.55% in the control group, respectively. In TOAST classification, Human Urinary Kallikrein is able to remarkably improve the NIHSS scores for L subtype and S subtype patients with acute cerebral infarction and help to enhance the clinical efficacy.

Relationship between procalcitonin serum levels and functional outcome in stroke patients.

To determine whether serum procalcitonin (PCT) levels at admission were associated with short-term functional outcome after acute ischemic stroke (AIS) in a cohort Chinese sample. We prospectively studied 378 patients with AIS who were admitted within 24 h after the onset of symptoms. PCT and NIH stroke scale (NIHSS) were measured at the time of admission. Short-term functional outcome was measured by modified Rankin scale (mRS) 90 days after admission. The results indicated that the serum PCT levels were significantly higher in AIS patients as compared to normal controls (P < 0.0001). In the 114 patients with an unfavorable functional outcome, serum PCT levels were higher compared with those in patients with a favorable outcome (2.40 (IQR, 1.10-3.69) ng/mL and 0.42 (IQR, 0.10-1.05) ng/mL, respectively, P < 0.001). PCT was an independent prognostic marker of functional outcome [odds ratio (OR) 3.45 (2.29-4.77), adjusted for the NIHSS and other possible confounders] in patients with ischemic stroke, added significant additional predictive value to the clinical NIHSS score. In receiver operating characteristic curve analysis, the prognostic accuracy of PCT was higher compared to Hs-CRP and NIHSS score. PCT is an independent predictor of short-term functional outcome after ischemic stroke in Chinese sample even after correcting for possible confounding factors.

[A preliminary study of plasma microRNA levels in children with methylmalonic acidemia].

OBJECTIVE: To screen out differentially expressed microRNAs (miRNAs) in the plasma of children with methylmalonic acidemia (MMA), to determine the expression of miR-9-1 in plasma and to preliminarily evaluate the significance of miR-9-1 as a biomarker in MMA. METHODS: Plasma was obtained from 17 MMA children, 10 hyperhomocysteinemia (HHcy) children without MMA (HHcy group), and 10 normal controls. Of 17 MMA children, 12 had HHcy (MMA+HHcy group), and 5 had no HHcy (MMA group). The differentially expressed miRNAs were screened out by miRNA microarray. Differentially expressed miR-9-1 was selected, and plasma miR-9-1 levels were determined by RT-PCR. Urine was collected from MMA patients who received vitamin B12 treatment, and plasma miR-9-1 levels were determined by RT-PCR after treatment. RESULTS: The miRNA microarray analysis showed that 26 miRNAs were differentially expressed, among which 16 miRNAs (including miR-9-1) were down-regulated over 2 times, while 10 miRNAs were up-regulated over 2 times. The MMA+HHcy , MMA and HHcy groups had significantly down-regulated miR-9-1 compared with the normal control group (P<0.01). The patients who showed a good response to vitamin B12 treatment had significantly increased plasma miR-9-1 levels, without significant difference compared with the normal control group. CONCLUSIONS: Plasma miR-9-1 is significantly down-regulated in MMA patients, but it is significantly up-regulated after vitamin B12 treatment, suggesting that miR-9-1 may act as a biomarker in monitoring the progression of MMA.

MicroRNA-195 downregulates Alzheimer's disease amyloid-ß production by targeting BACE1.

Alzheimer's disease (AD) is a progressive neurodegenerative disease, characterized by amyloid-beta (Aß) deposition and neurofibrillary tangles. Numerous microRNAs have been found to play crucial roles in regulating Aß production in the process of AD. Previous investigations have reported lower levels of many microRNAs in AD patients and animal models. Here, we examined the role of miR-195 in the process of Aß formation. Bioinformatics' algorithms predicted miR-195 binding sites within the beta-site APP cleaving enzyme 1 (BACE1) 3'-untranslated region (3'-UTR), and we found the level of miR-195 to be negatively related to the protein level of BACE1 in SAMP8 mice. We confirmed the target site in HEK293 cells by luciferase assay. Overexpression of miR-195 in N2a/WT cells decreased the BACE1 protein level, and inhibition of miR-195 resulted in increase of BACE1 protein level. Furthermore, overexpression of miR-195 in N2a/APP decreased the level of Aß, while inhibition of miR-195 resulted in an increase of Aß. Thus, we demonstrated that miR-195 could downregulate the level of Aß by inhibiting the translation of BACE1. We conclude that miR-195 might provide a therapeutic strategy for AD.