Ischemic stroke rehabilitation through optogenetic modulation of parvalbumin neurons in the contralateral motor cortex.

BACKGROUND: Based on the theory of interhemispheric inhibition and the bimodal balance-recovery model in stroke, we explored the effects of excitation/inhibition (E/I) of parvalbumin (PV) neurons in the contralateral primary motor cortex (cM1) connecting the ipsilateral M1 (iM1) via the corpus callosum (cM1-CC-iM1) of ischemic stroke rats by optogenetic stimulation. METHODS: We tested this by injecting anterograde and retrograde virus in rats with middle cerebral artery occlusion (MCAO), and evaluated the neurological scores, motor behavior, volume of cerebral infarction and the E/I balance of the bilateral M1 two weeks after employing optogenetic treatment. RESULTS: We found that concentrations of Glu and GABA decreased and increased, respectively, in the iM1 of MCAO rats, and that the former increased in the cM1, suggesting E/I imbalance in bilateral M1 after ischemic stroke. Interestingly, optogenetic stimulation improved M1 E/I imbalance, as illustrated by the increase of Glu in the iM1 and the decrease of GABA in both iM1 and cM1, which were accompanied by an improvement in neurological deficit and motor dysfunction. In addition, we observed a reduced infarct volume, an increase in the expression of the NMDAR and AMPAR, and a decrease in GAD67 in the iM1 after intervention. CONCLUSIONS: Optogenetic modulation of PV neurons of the iM1-CC-cM1 improve E/I balance, leading to reduced neurological deficit and improved motor dysfunction following ischemic stroke in rats.

Neurogranin as an important regulator in swimming training to improve the spatial memory dysfunction of mice with chronic cerebral hypoperfusion.

BACKGROUND: Vascular cognitive impairment caused by chronic cerebral hypoperfusion (CCH) has become a hot issue worldwide. Aerobic exercise positively contributes to the preservation or restoration of cognitive abilities; however, the specific mechanism has remained inconclusive. And recent studies found that neurogranin (Ng) is a potential biomarker for cognitive impairment. This study aims to investigate the underlying role of Ng in swimming training to improve cognitive impairment. METHODS: To test this hypothesis, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system was utilized to construct a strain of Ng conditional knockout (Ng cKO) mice, and bilateral common carotid artery stenosis (BCAS) surgery was performed to prepare the model. In Experiment 1, 2-month-old male and female transgenic mice were divided into a control group (wild-type littermate, n = 9) and a Ng cKO group (n = 9). Then, 2-month-old male and female C57BL/6 mice were divided into a sham group (C57BL/6, n = 12) and a BCAS group (n = 12). In Experiment 2, 2-month-old male and female mice were divided into a sham group (wild-type littermate, n = 12), BCAS group (n = 12), swim group (n = 12), BCAS + Ng cKO group (n = 12), and swim + Ng cKO group (n = 12). Then, 7 days after BCAS, mice were given swimming training for 5 weeks (1 week for adaptation and 4 weeks for training, 5 days a week, 60 min a day). After intervention, laser speckle was used to detect cerebral blood perfusion in the mice, and the T maze and Morris water maze were adopted to test their spatial memory. Furthermore, electrophysiology and Western blotting were conducted to record long-term potential and observe the expressions of Ca2+ pathway-related proteins, respectively. Immunohistochemistry was applied to analyze the expression of relevant markers in neuronal damage, inflammation, and white matter injury. RESULTS: The figures showed that spatial memory impairment was detected in Ng cKO mice, and a sharp decline of cerebral blood flow and an impairment of progressive spatial memory were observed in BCAS mice. Regular swimming training improved the spatial memory impairment of BCAS mice. This was achieved by preventing long-term potential damage and reversing the decline of Ca2+ signal transduction pathway-related proteins. At the same time, the results suggested that swimming also led to improvements in neuronal death, inflammation, and white matter injury induced by CCH. Further study adopted the use of Ng cKO transgenic mice, and the results indicated that the positive effects of swimming training on cognitive impairments, synaptic plasticity, and related pathological changes caused by CCH could be abolished by the knockout of Ng. CONCLUSION: Swimming training can mediate the expression of Ng to enhance hippocampal synaptic plasticity and improve related pathological changes induced by CCH, thereby ameliorating the spatial memory impairment of vascular cognitive impairment.

Emerging blood exosome-based biomarkers for preclinical and clinical Alzheimer's disease: a meta-analysis and systematic review.

Blood exosomes, which are extracellular vesicles secreted by living cells into the circulating blood, are regarded as a relatively noninvasive novel tool for monitoring brain physiology and disease states. An increasing number of blood cargo-loaded exosomes are emerging as potential biomarkers for preclinical and clinical Alzheimer's disease. Therefore, we conducted a meta-analysis and systematic review of molecular biomarkers derived from blood exosomes to comprehensively analyze their diagnostic performance in preclinical Alzheimer's disease, mild cognitive impairment, and Alzheimer's disease. We performed a literature search in PubMed, Web of Science, Embase, and Cochrane Library from their inception to August 15, 2020. The research subjects mainly included Alzheimer's disease, mild cognitive impairment, and preclinical Alzheimer's disease. We identified 34 observational studies, of which 15 were included in the quantitative analysis (Newcastle-Ottawa Scale score 5.87 points) and 19 were used in the qualitative analysis. The meta-analysis results showed that core biomarkers including Aß1-42, P-T181-tau, P-S396-tau, and T-tau were increased in blood neuron-derived exosomes of preclinical Alzheimer's disease, mild cognitive impairment, and Alzheimer's disease patients. Molecules related to additional risk factors that are involved in neuroinflammation (C1q), metabolism disorder (P-S312-IRS-1), neurotrophic deficiency (HGF), vascular injury (VEGF-D), and autophagy-lysosomal system dysfunction (cathepsin D) were also increased. At the gene level, the differential expression of transcription-related factors (REST) and microRNAs (miR-132) also affects RNA splicing, transport, and translation. These pathological changes contribute to neural loss and synaptic dysfunction. The data confirm that the above-mentioned core molecules and additional risk-related factors in blood exosomes can serve as candidate biomarkers for preclinical and clinical Alzheimer's disease. These findings support further development of exosome biomarkers for a clinical blood test for Alzheimer's disease. This meta-analysis was registered at the International Prospective Register of Systematic Reviews (Registration No. CRD4200173498, 28/04/2020).

Enhanced Medial Prefrontal Cortex and Hippocampal Activity Improves Memory Generalization in APP/PS1 Mice: A Multimodal Animal MRI Study.

Memory generalization allows individuals to extend previously learned movement patterns to similar environments, contributing to cognitive flexibility. In Alzheimer's disease (AD), the disturbance of generalization is responsible for the deficits of episodic memory, causing patients with AD to forget or misplace things, even lose track of the way home. Cognitive training can effectively improve the cognition of patients with AD through changing thinking mode and memory flexibility. In this study, a T-shaped maze was utilized to simulate cognitive training in APP/PS1 mice to elucidate the potential mechanisms of beneficial effects after cognitive training. We found that cognitive training conducted by a T-shaped maze for 4 weeks can improve the memory generalization ability of APP/PS1 mice. The results of functional magnetic resonance imaging (fMRI) showed that the functional activity of the medial prefrontal cortex (mPFC) and hippocampus was enhanced after cognitive training, and the results of magnetic resonance spectroscopy (MRS) showed that the neurochemical metabolism of N-acetyl aspartate (NAA) and glutamic acid (Glu) in mPFC, hippocampus and reuniens (Re) thalamic nucleus were escalated. Furthermore, the functional activity of mPFC and hippocampus was negatively correlated with the escape latency in memory generalization test. Therefore, these results suggested that cognitive training might improve memory generalization through enhancing the functional activity of mPFC and hippocampus and increasing the metabolism of NAA and Glu in the brain regions of mPFC, hippocampus and Re nucleus.

Longitudinal tracing of neurochemical metabolic disorders in working memory neural circuit and optogenetics modulation in rats with vascular cognitive impairment.

Chronic cerebral ischemia leads to vascular cognitive impairment (VCI) that exacerbates along with ischemia time and eventually develops into dementia. Recent advances in molecular neuroimaging contribute to understand its pathological characteristics. We previously traced the anisotropic diffusion of water molecules suggests that chronic cerebral ischemia leads to irreversible progressive damage to white matter integrity. However, the abnormalities of gray matter activity following chronic cerebral ischemia remains not entirely understood. In this study, in vivo hydrogen proton magnetic resonance spectroscopy (1H-MRS) was applied to longitudinally track the neurochemical metabolic disorder of gray matter associated with working memory, and optogenetics modulation of neurochemical metabolism was performed for targeted treatment of VCI. The results showed that the concentration of N-acetylaspartate (NAA) in the right hippocampus, left hippocampus, right medial prefrontal cortex (mPFC) and mediodorsal thalamus was decreased as early as 7 days after chronic cerebral ischemia, subsequently gamma-aminobutyric acid (GABA) declined whereas myo-inositol (mI) and glutamate (Glu) increased at 14 days, as well as choline (Cho) lost at 28 days, concurrently the change of Glu and GABA in the mPFC and hippocampus was ischemia time-dependent manner within 1 month. Behaviorally, working memory and object recognition memory were impaired at 14 days, 28 days that significantly correlated with neurochemical metabolic disorders. Interestingly, using optogenetics modulation of PV neurons in the mPFC, the metabolic abnormalities of NAA and GABA in working memory neural circuit could be repaired after chronic cerebral ischemia, together with behavior improvements. These findings suggested that as early as 1∼4 weeks after chronic cerebral ischemia, the metabolism of NAA, Glu, mI and Cho was synchronously impaired in neural circuit of hippocampus-mediodorsal thalamus-mPFC, and the loss of GABA delayed in the hippocampus, and optogenetics modulation of parvalbumin (PV) neurons in the mPFC can improve the neurochemical metabolism of working memory neural circuit and enhance working memory.

Neurogranin as a cognitive biomarker in cerebrospinal fluid and blood exosomes for Alzheimer's disease and mild cognitive impairment.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder with clinical, biological, and pathological features occurring along a continuum from normal to end-stage disease. Currently, the diagnosis of AD depends on clinical assessments and post-mortem neuropathology, which is unbenefited early diagnosis and progressive monitoring. In recent years, clinical studies have reported that the level of cerebrospinal fluid (CSF) and blood neurogranin (Ng) are closely related to the occurrence and subsequent progression of AD. Therefore, the study used meta-analysis to identify the CSF and blood Ng levels for the development of diagnosis biomarker of patients with AD and mild cognitive impairment (MCI). We searched the Pubmed, Embase, Cochrane Library, and Web of Science databases. A total of 24 articles eligible for inclusion and exclusion criteria were assessed, including 4661 individuals, consisting of 1518 AD patients, 1501 MCI patients, and 1642 healthy control subjects. The level of CSF Ng significantly increased in patients with AD and MCI compared with healthy control subjects (SMD: 0.84 [95% CI: 0.70-0.98], P < 0.001; SMD: 0.53 [95% CI: 0.40-0.66], P = 0.008), and higher in AD patients than in MCI patients (SMD: 0.18 [95% CI: 0.07-0.30], P = 0.002), and CSF Ng level of patients with MCI-AD who progressed from MCI to AD was significantly higher than that of patients with stable MCI (sMCI) (SMD: 0.71 [95% CI: 0.25-1.16], P = 0.002). Moreover, the concentration of Ng in blood plasma exosomes of patients with AD and MCI was lower than that of healthy control subjects (SMD: -6.657 [95% CI: -10.558 to -2.755], P = 0.001; and SMD: -3.64 [95% CI: -6.50 to -0.78], P = 0.013), and which in patients with AD and MCI-AD were also lower than those in patients with sMCI (P < 0.001). Furthermore, regression analysis showed a negative relationship between MMSE scores and CSF Ng levels in MCI patients (slope = -0.249 [95% CI: -0.003 to -0.495], P = 0.047). Therefore, the Ng levels increased in CSF, but decreased in blood plasma exosomes of patients with AD and MCI-AD, and highly associated with cognitive declines. These findings provide the clinical evidence that CSF and blood exosomes Ng can be used as a cognitive biomarker for AD and MCI-AD, and further studies are needed to define the specific range of Ng values for diagnosis at the different stages of AD.

Longitudinal tracing of white matter integrity on diffusion tensor imaging in the chronic cerebral ischemia and acute cerebral ischemia.

Brain ischemia leads to insufficient oxygen supply or hypoxia and thus to ischemic stroke or chronic hypoperfusion, which results in neuronal death and white matter damage is irreversible or partially recoverable under the interruption of blood flow more than a few minutes. The present study investigated the abnormal characteristics of white matter integrity after the chronic cerebral ischemia in a mouse model of bilateral carotid artery stenosis and the acute cerebral ischemia in a mouse model of middle cerebral artery occlusion via longitudinal diffusion tensor imaging, which revealed that the mean diffusivity of corpus callosum (cc) was decreased as early as the 1 st day after chronic cerebral ischemia, and then the damage gradually aggravated and lasted to the 28th day. Moreover, the brain regions, including cingulum (cg), dorsal hippocampal commissure (dhc), forceps major of the corpus callosum (fmj), alveus of the hippocampus (alv) and medial lemniscus (ml), were damaged in duration of 7∼28 days after chronic cerebral ischemia. Oppositely, white matter signals in the contralateral hemisphere appeared compensatory increase in the internal capsule (ic) at the 1 st day after acute cerebral ischemia, simultaneously the ipsilateral hemisphere signals were decreased in alv, cerebral peduncle (cp), external capsule (ec), ml, fimbria of the hippocampus (fi), ic, forceps minor of the corpus callosum (fmi) and dhc. While these regional white matter signals were decreased in the bilateral hemisphere at the 7th day after acute cerebral ischemia. In addition, the motor function was impaired after acute cerebral ischemia, and cognitive function were impaired after chronic and acute cerebral ischemia. Furthermore, voxel-wise analysis revealed the obvious differences of white matter integrity in these two models of ischemia. The chronic cerebral ischemia showed better white matter integrity in the ipsilateral hemisphere of acute cerebral ischemic model at the 1 st day after surgery, but worse in the contralateral hemisphere. Subsequently, these differences were reduced significantly and just only the ipsilateral cp and bilateral ml signals were higher in chronic cerebral ischemia. Taken together, the present study demonstrates that the chronic and acute cerebral ischemia cause progressive damages of white matter that are irreversible in a relatively long time, of which the contralateral white matter present transient compensation in acute cerebral ischemia but not in chronic cerebral ischemia. These might be helpful to better diagnosis of clinical different cerebral ischemia.

Conditional knockout of tissue factor pathway inhibitor 2 in vascular endothelial cells accelerates atherosclerotic plaque development in mice.

BACKGROUND: Tissue factor pathway inhibitor-2 (TFPI-2) regulates matrix metalloproteinases activation and extracellular matrix degradation. Over-expression of TFPI-2 enhances atherosclerotic plaque stability. The aim of this study is to investigate the effect of conditional knockout (KO) of TFPI-2 in vascular endothelial cells on the initiation and development of atherosclerotic plaque. METHODS: A Cre/mloxP conditional KO system and Tek-Cre mice were used to generate offsprings with monoallelic deletion of the TFPI-2 gene in endothelial cells. TFPI-2(fl/+)/Tek-Cre mice, TFPI-2(fl/+) mice and ApoE(-/-) mice (n=6 for each group) were included. Arteries were obtained. HE, EVG and anti-α-SMA staining were used to examine the morphology of vessel and plaque. Protein expression and phosphorylation were detected by Western blot or immunohistochemistry. RESULTS: TFPI-2(fl/+)/Tek-Cre mice were generated. TFPI-2 level decreased to 40.68% in TFPI-2(fl/+)/Tek-Cre group. TFPI-2(fl/+)/Tek-Cre developed plaques when no plaque was found in TFPI-2(fl/+) mice. Compared with ApoE(-/-) group, TFPI-2(fl/+)/Tek-Cre group has smaller plaque area, decreased lipid content and less buried fibrous cap layers. MMP-2 and MMP-9 in TFPI-2(fl/+)/Tek-Cre group was higher than in TFPI-2(fl/+)group. The phosphorylation of PPAR-α and PPAR-γ was decreased in TFPI-2(fl/+)/Tek-Cre group. CONCLUSIONS: A novel mouse model is presented and can be used to investigate the role of TFPI-2 in the process of atherosclerosis. Our findings suggest that monoallelic deletion of TFPI-2 gene in vascular endothelial cells leads to significant downregulation of TFPI-2. TFPI-2 deficiency may accelerate initiation of atherosclerotic lesion in mice. Elevated MMP-2 and 9 and decreased phosphorylation of PPAR-α and PPAR-γ may contribute to this phenotype.