Effects of electroacupuncture preconditioning on microglial cells and serum inflammatory factors in the early stage of ischemic stroke. / ç”µé’ˆé¢„å¤„ç†å¯¹ç¼ºè¡€æ€§ä¸­é£Žå°é¼ æ—©æœŸå°èƒ¶è´¨ç»†èƒžåŠè¡€æ¸ ç‚Žæ€§å› å­çš„å½±å“.

OBJECTIVES: To investigate the effects on the motor function, cortex blood flow perfusion, microglial cells, and the contents of serum inflammatory factors, i.e. interleukin-1ß (IL-1ß), transforming growth factor-ß (TGF-ß), and interleukin-10 (IL-10) after electroacupuncture (EA) preconditioning at "Baihui" (GV20) and "Dazhui" (GV14) in the mice with ischemic stroke, so as to explore the mechanism of EA preconditioning for improving motor function after ischemic stroke. METHODS: C57BL/6 mice were randomly divided into sham-operation group, model group, and EA preconditioning group (EA group), with 15 mice in each group. A photothrombotic method was used to induce the model of unilateral ischemic stroke and motor impairment. The mice in the EA group received EA preconditioning, 20 min each time, once daily for 7 consecutive days before modeling. The motor function of mice was evaluated by the grid-walking test and cylinder test before and after modeling. Laser speckle blood flow video monitoring system was employed to assess the cerebral blood flow perfusion in the primary motor cortex of mice. The contents of IL-1ß, TGF-ß, and IL-10 in the serum were measured by ELISA, and the expressions of microglial cell and M2 subtype cell marker in the primary motor cortex were detected using immunofluorescence staining. RESULTS: After modeling, compared with the sham-operation group, the grid error rate and the dragging rate of the affected limb were increased (P<0.01)；the utilization rate of the affected limb and percentage of the blood perfusion in the affected cortex to healthy side were decreased (P<0.01)；the contents of serum IL-1ß, TGF-ß, and IL-10 were increased (P<0.01, P<0.05)；and the microglia in the primary motor cortex on the affected side showed ameboid, the fluorescence intensity of ionized calcium-binding adapter molecule 1 (IBA1) and CD206 was increased (P<0.01) in the model group. In the EA group, when compared with the model group, the grid error rate and the dragging rate of affected limb were decreased (P<0.01)；the utilization rate of affected limb and the percentage of blood perfusion were increased (P<0.05)；the content of serum IL-1ß was decreased (P<0.01), while the contents of TGF-ß and IL-10 were increased (P<0.01)；and the microglia in the primary motor cortex on the affected side got more round and were distributed more densely, the fluorescence intensity of IBA1 and CD206 was increased (P<0.01). CONCLUSIONS: Electroacupuncture preconditioning at "GV20" and "GV14" can up-regulate the expression of microglial cells, especially the M2 subtype cell marker, and increase the contents of the anti-inflammatory factors and decrease that of the pro-inflammatory factors in the serum, thereby alleviate the inflammatory reaction.

TNEA therapy promotes the autophagic degradation of NLRP3 inflammasome in a transgenic mouse model of Alzheimer's disease via TFEB/TFE3 activation.

BACKGROUND: The impairment in the autophagy-lysosomal pathway (ALP) and the activation of NLR family pyrin domain containing 3 (NLRP3) inflammasome represent two molecular events leading to neurodegeneration and neuroinflammation in Alzheimer's disease (AD), a devastating neurodegenerative disorder without a cure. Previously we demonstrated the cognitive-enhancing effect of a combined electroacupuncture (EA) therapy termed TNEA in a transgenic mouse model of AD, involving activation of transcription factor EB (TFEB), a master regulator of ALP. However, whether and how TNEA inhibits NLRP3 inflammasome via TFEB-mediated ALP in AD remains to be investigated. METHODS: 5xFAD mice overexpressing amyloid-ß (Aß) were treated with TNEA or EA on its composing acupoints (GB13 and GV24). The changes in the signaling pathways regulating NLRP3 inflammasome, the association of NLRP3 inflammasome with ALP, and the roles of TFEB/TFE3 in mice brains were determined by immunoblots, immunohistochemistry and AAV-mediated knockdown assays. RESULTS: TNEA inhibits the activation of NLRP3 inflammasome and the release of active interleukin 1ß (IL1B) in the hippocampi of 5xFAD mice. Mechanistically, TNEA promoted the autophagic degradation of inflammasome components via activating both TFEB and TFE3 by modulating kinases including AMPK and AKT. The composing acupoints in TNEA showed synergistic effects on regulating these molecular events and memory improvement. CONCLUSION: Our findings suggest that TNEA attenuates AD-associated memory impairment via promoting TFEB/TFE3-mediated autophagic clearance of Aß and NLRP3 inflammasome, and partially reveal the molecular basis of combined acupoints therapy originated from ancient wisdom.

Minocycline Ameliorates Chronic Unpredictable Mild Stress-Induced Neuroinflammation and Abnormal mPFC-HIPP Oscillations in Mice.

Stress-induced neuroinflammation is a hallmark of modern society and has been linked to various emotional disorders, including anxiety. However, how microglia-associated neuroinflammation under chronic unpredictable mild stress (CUMS) alters mitochondrial function and subsequent medial prefrontal cortex-hippocampus (mPFC-HIPP) connectivity remains obscure. We speculated that CUMS might induce neuroinflammation, which involves altered mitochondrial protein levels, blockade of neuroinflammation by a microglial modulator, minocycline, protects against CUMS-induced alterations. Mice were exposed to CUMS for 3 weeks and received minocycline (50 mg/kg) intraperitoneally for 7 consecutive days during the 3rd week of CUMS. Novelty-suppressed feeding test and contextual anxiety test assessed anxiety-like behavior. Western blotting and immunofluorescent staining were employed to evaluate levels of proteins involved in neuroinflammation and mitochondrial function. In vivo dual-site extracellular recordings of local field potential (LFP) were conducted to evaluate the oscillatory activity and brain connectivity in mPFC-HIPP circuitry. We show that CUMS results in excessive microglial activation accompanied by aberrant levels of mitochondrial proteins, such as ATP-5A and the fission protein, Drp-1, increased oxidative stress indicated by elevated levels of nitrotyrosine, and decreased Nrf-2 levels. Furthermore, CUMS causes downregulation of α1 subunit of GABAAR, vesicular GABA transporter (Vgat), and glutamine synthetase (GS), leading to impaired LFP and connectivity of the mPFC-HIPP circuitry. Strikingly, blockage of microglial activation by minocycline ameliorates CUMS-induced aberrant levels of mitochondrial and GABAergic signaling proteins and prevents CUMS-induced anxiety-like behavior in mice. To the end, the study revealed that microglia is critically involved in stress-induced neuroinflammation, which may underlie the molecular mechanism of CUMS-induced anxiety behavior.

Systemic LPS-induced microglial activation results in increased GABAergic tone: A mechanism of protection against neuroinflammation in the medial prefrontal cortex in mice.

Neuroinflammation with excess microglial activation and synaptic dysfunction are early symptoms of most neurological diseases. However, how microglia-associated neuroinflammation regulates synaptic activity remains obscure. We report here that acute neuroinflammation induced by intraperitoneal injection of lipopolysaccharide (LPS) results in cell-type-specific increases in inhibitory postsynaptic currents in the glutamatergic, but not the GABAergic, neurons of medial prefrontal cortex (mPFC), coinciding with excessive microglial activation. LPS causes upregulation in levels of GABAAR subunits, glutamine synthetase and vesicular GABA transporter, and downregulation in brain-derived neurotrophic factor (BDNF) and its receptor, pTrkB. Blockage of microglial activation by minocycline ameliorates LPS-induced abnormal expression of GABA signaling-related proteins and activity of synaptic and network. Moreover, minocycline prevents the mice from LPS-induced aberrant behavior, such as a reduction in total distance and time spent in the centre in the open field test; decreases in entries into the open arm of elevated-plus maze and in consumption of sucrose; increased immobility in the tail suspension test. Furthermore, upregulation of GABA signaling by tiagabine also prevents LPS-induced microglial activation and aberrant behavior. This study illustrates a mode of bidirectional constitutive signaling between the neural and immune compartments of the brain, and suggests that the mPFC is an important area for brain-immune system communication. Moreover, the present study highlights GABAergic signaling as a key therapeutic target for mitigating neuroinflammation-induced abnormal synaptic activity in the mPFC, together with the associated behavioral abnormalities.

Mitochondrial Deficits With Neural and Social Damage in Early-Stage Alzheimer's Disease Model Mice.

Alzheimer's disease (AD) is the most common neurodegenerative disorder worldwide. Mitochondrial dysfunction is thought to be an early event in the onset and progression of AD; however, the precise underlying mechanisms remain unclear. In this study, we investigated mitochondrial proteins involved in organelle dynamics, morphology and energy production in the medial prefrontal cortex (mPFC) and hippocampus (HIPP) of young (1∼2 months), adult (4∼5 months) and aged (9∼10, 12∼18 months) APP/PS1 mice. We observed increased levels of mitochondrial fission protein, Drp1, and decreased levels of ATP synthase subunit, ATP5A, leading to abnormal mitochondrial morphology, increased oxidative stress, glial activation, apoptosis, and altered neuronal morphology as early as 4∼5 months of age in APP/PS1 mice. Electrophysiological recordings revealed abnormal miniature excitatory postsynaptic current in the mPFC together with a minor connectivity change between the mPFC and HIPP, correlating with social deficits. These results suggest that abnormal mitochondrial dynamics, which worsen with disease progression, could be a biomarker of early-stage AD. Therapeutic interventions that improve mitochondrial function thus represent a promising approach for slowing the progression or delaying the onset of AD.

Clinical therapeutic effects of probiotics in patients with constipation associated with Parkinson disease: A protocol for systematic review and meta-analysis.

BACKGROUND: Constipation is the most predominant symptom of Parkinson disease (PD), preceding the occurrence of motor symptoms in some patients, leading to reduced quality of life (QOL). The general approaches for the treatment have some side effects, but probiotics are live or attenuated microorganisms attributed to ameliorating constipation effects. Moreover, as treatments are generally well tolerated and side effects are scarce, there is room for further research. Therefore this work aims at investigating the clinical effectiveness and safety of probiotics for constipation in PD. METHODS: Published RCTs will be retrieved by searching Medline, Embase, Cochrane Library, Web of Science, China National Knowledge Infrastructure (CNKI), VIP, Wan Fang database, and China Biology Medicine Database (complete bowel movement), which will be searched from establishment of the database to October 10, 2021. Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P) guidelines are used to design this protocol. RevMan V.5.3 software will be used for meta-analysis, risk of bias will be assessed by the Cochrane Collaboration tool and the collected evidence will be narratively synthesized. We will also perform a meta-analysis to pool estimates from studies considered to be homogenous. Subgroup analyses will be based on intervention or overall bias. CONCLUSION: The meta-analysis will assess the effectiveness and safety of using probiotics to treat and heal the constipation of PD. ETHICS AND DISSEMINATION: Ethics approval is unrequired. REGISTRATION NUMBER: CRD42021276215.

Amyloid-ß Protein Precursor Deficiency Changes Neuronal Electrical Activity and Levels of Mitochondrial Proteins in the Medial Prefrontal Cortex.

BACKGROUND: Neuropathological features of Alzheimer's disease are characterized by the deposition of amyloid-ß (Aß) plaques and impairments in synaptic activity and memory. However, we know little about the physiological role of amyloid-ß protein precursor (AßPP) from which Aß derives. OBJECTIVE: Evaluate APP deficiency induced alterations in neuronal electrical activity and mitochondrial protein expression. METHODS: Utilizing electrophysiological, biochemical, pharmacological, and behavioral tests, we revealed aberrant local field potential (LFP), extracellular neuronal firing and levels of mitochondrial proteins. RESULT: We show that APP knockout (APP-/-) leads to increased gamma oscillations in the medial prefrontal cortex (mPFC) at 1-2 months old, which can be restored by baclofen (Bac), a γ-aminobutyric acid type B receptor (GABABR) agonist. A higher dose and longer exposure time is required for Bac to suppress neuronal firing in APP-/- mice than in wild type animals, indicating enhanced GABABR mediated activity in the mPFC of APP-/- mice. In line with increased GABABR function, the glutamine synthetase inhibitor, L-methionine sulfonate, significantly increases GABABR levels in the mPFC of APP-/- mice and this is associated with a significantly lower incidence of death. The results suggest that APP-/- mice developed stronger GABABR mediated inhibition. Using HEK 293 as an expression system, we uncover that AßPP functions to suppress GABABR expression. Furthermore, APP-/- mice show abnormal expression of several mitochondrial proteins. CONCLUSION: APP deficiency leads to both abnormal network activity involving defected GABABR and mitochondrial dysfunction, suggesting critical role of AßPP in synaptic and network function.

Enhancing GABAergic signaling ameliorates aberrant gamma oscillations of olfactory bulb in AD mouse models.

BACKGROUND: Before the deposition of amyloid-beta plaques and the onset of learning memory deficits, patients with Alzheimer's disease (AD) experience olfactory dysfunction, typified by a reduced ability to detect, discriminate, and identify odors. Rodent models of AD, such as the Tg2576 and APP/PS1 mice, also display impaired olfaction, accompanied by aberrant in vivo or in vitro gamma rhythms in the olfactory pathway. However, the mechanistic relationships between the electrophysiological, biochemical and behavioral phenomena remain unclear. METHODS: To address the above issues in AD models, we conducted in vivo measurement of local field potential (LFP) with a combination of in vitro electro-olfactogram (EOG), whole-cell patch and field recordings to evaluate oscillatory and synaptic function and pharmacological regulation in the olfactory pathway, particularly in the olfactory bulb (OB). Levels of protein involved in excitation and inhibition of the OB were investigated by western blotting and fluorescence staining, while behavioral studies assessed olfaction and memory function. RESULTS: LFP measurements demonstrated an increase in gamma oscillations in the OB accompanied by altered olfactory behavior in both APP/PS1 and 3xTg mice at 3-5 months old, i.e. an age before the onset of plaque formation. Fewer olfactory sensory neurons (OSNs) and a reduced EOG contributed to a decrease in the excitatory responses of M/T cells, suggesting a decreased ability of M/T cells to trigger interneuron GABA release indicated by altered paired-pulse ratio (PPR), a presynaptic parameter. Postsynaptically, there was a compensatory increase in levels of GABAAR α1 and ß3 subunits and subsequent higher amplitude of inhibitory responses. Strikingly, the GABA uptake inhibitor tiagabine (TGB) ameliorated abnormal gamma oscillations and levels of GABAAR subunits, suggesting a potential therapeutic strategy for early AD symptoms. These findings reveal increased gamma oscillations in the OB as a core indicator prior to onset of AD and uncover mechanisms underlying aberrant gamma activity in the OB. CONCLUSIONS: This study suggests that the concomitant dysfunction of both olfactory behavior and gamma oscillations have important implications for early AD diagnosis: in particular, awareness of aberrant GABAergic signaling mechanisms might both aid diagnosis and suggest therapeutic strategies for olfactory damage in AD.

Electroacupuncture ameliorates beta-amyloid pathology and cognitive impairment in Alzheimer disease via a novel mechanism involving activation of TFEB (transcription factor EB).

Alzheimer disease (AD) is the most prevalent neurodegenerative disorder leading to dementia in the elderly. Unfortunately, no cure for AD is available to date. Increasing evidence has proved the roles of misfolded protein aggregation due to impairment of the macroautophagy/autophagy-lysosomal pathway (ALP) in the pathogenesis of AD, and thus making TFEB (transcription factor EB), which orchestrates ALP, as a promising target for treating AD. As a complementary therapy, acupuncture or electroacupuncture (EA) has been commonly used for treating human diseases. Although the beneficial effects of acupuncture for AD have been primarily studied both pre-clinically and clinically, the real efficacy of acupuncture on AD remains inconclusive and the underlying mechanisms are largely unexplored. In this study, we demonstrated the cognitive-enhancing effect of three-needle EA (TNEA) in an animal model of AD with beta-amyloid (Aß) pathology (5xFAD). TNEA reduced APP (amyloid beta (A4) precursor protein), C-terminal fragments (CTFs) of APP and Aß load, and inhibited glial cell activation in the prefrontal cortex and hippocampus of 5xFAD. Mechanistically, TNEA activated TFEB via inhibiting the AKT-MAPK1-MTORC1 pathway, thus promoting ALP in the brains. Therefore, TNEA represents a promising acupuncture therapy for AD, via a novel mechanism involving TFEB activation.Abbreviations Aß: ß-amyloid; AD: Alzheimer disease; AIF1/IBA1: allograft inflammatory factor 1; AKT1: thymoma viral proto-oncogene 1; ALP: autophagy-lysosomal pathway; APP: amyloid beta (A4) precursor protein; BACE1: beta-site APP cleaving enzyme 1; CQ: chloroquine; CTFs: C-terminal fragments; CTSD: cathepsin D; EA: electroacupuncture; FC: fear conditioning; GFAP: glial fibrillary acidic protein; HI: hippocampus; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MAPK1/ERK2: mitogen-activated protein kinase 1; MAPT: microtubule-associated protein tau; MTORC1: mechanistic target of rapamycin kinase complex 1; MWM: Morris water maze; NFT: neurofibrillary tangles; PFC: prefrontal cortex; PSEN1: presenilin 1; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; TNEA: three-needle electroacupuncture.

Immobilization of Fibronectin-Loaded Polyelectrolyte Nanoparticles on Cardiovascular Material Surface to Improve the Biocompatibility.

Vascular stent interventional therapy is the main method for clinical treatment of coronary artery diseases. However, due to the insufficient biocompatibility of cardiovascular materials, the implantation of stents often leads to serious adverse cardiac events. Surface biofunctional modification to improve the biocompatibility of vascular stents has been the focus of current research. In this study, based on the structure and function of extracellular matrix on vascular injury healing, a novel fibronectin-loaded poly-l-lysine/heparin nanoparticles was constructed for stent surface modification. In vitro blood compatibility evaluation results showed that the nanoparticles-modified surface could effectively reduce platelet adhesion and activation. In vitro cellular compatibility evaluation results indicated that the nanocoating may provide adequate efficacy in promoting the adhesion and proliferation of endothelial cells and thereby accelerate endothelialization. This study provides a new approach for the surface biological function modification of vascular stents.

Gluconate suppresses seizure activity in developing brains by inhibiting CLC-3 chloride channels.

Neonatal seizures are different from adult seizures, and many antiepileptic drugs that are effective in adults often fail to treat neonates. Here, we report that gluconate inhibits neonatal seizure by inhibiting CLC-3 chloride channels. We detect a voltage-dependent outward rectifying Cl- current mediated by CLC-3 Cl- channels in early developing brains but not adult mouse brains. Blocking CLC-3 Cl- channels by gluconate inhibits seizure activity both in neonatal brain slices and in neonatal animals with in vivo EEG recordings. Consistently, neonatal neurons of CLC-3 knockout mice lack the outward rectifying Cl- current and show reduced epileptiform activity upon stimulation. Mechanistically, we demonstrate that activation of CLC-3 Cl- channels alters intracellular Cl- homeostasis and enhances GABA excitatory activity. Our studies suggest that gluconate can suppress neonatal seizure activities through inhibiting CLC-3 Cl- channels in developing brains.

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method.

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such as epilepsy and neurodegenerative disorders. However, it is technically difficult to obtain EEG recordings in neonates as it requires specialized handling and great care. Here, we present a novel method to record EEG in neonatal rat pups (P8-P15). We designed a simple and reliable electrode using computer pin loci; it can be easily implanted into the skull of a rat pup to record high-quality EEG signals in the normal and epileptic brain. Pups were given an intraperitoneal (i.p.) injection of the neurotoxin kainic acid (KA) to induce epileptic seizures. The surgical implantation performed in this procedure is less expensive than other EEG procedures for neonates. This method allows one to record high-quality and stable EEG signals for more than 1 week. Furthermore, this procedure can also be applied to adult rats and mice to study epilepsy or other neurological disorders.

APP modulates KCC2 expression and function in hippocampal GABAergic inhibition.

Amyloid precursor protein (APP) is enriched at the synapse, but its synaptic function is still poorly understood. We previously showed that GABAergic short-term plasticity is impaired in App knock-out (App-/-) animals, but the precise mechanism by which APP regulates GABAergic synaptic transmission has remained elusive. Using electrophysiological, biochemical, moleculobiological, and pharmacological analysis, here we show that APP can physically interact with KCC2, a neuron-specific K+-Cl- cotransporter that is essential for Cl- homeostasis and fast GABAergic inhibition. APP deficiency results in significant reductions in both total and membrane KCC2 levels, leading to a depolarizing shift in the GABA reversal potential (EGABA). Simultaneous measurement of presynaptic action potentials and inhibitory postsynaptic currents (IPSCs) in hippocampal neurons reveals impaired unitary IPSC amplitudes attributable to a reduction in α1 subunit levels of GABAAR. Importantly, restoration of normal KCC2 expression and function in App-/- mice rescues EGABA, GABAAR α1 levels and GABAAR mediated phasic inhibition. We show that APP functions to limit tyrosine-phosphorylation and ubiquitination and thus subsequent degradation of KCC2, providing a mechanism by which APP influences KCC2 abundance. Together, these experiments elucidate a novel molecular pathway in which APP regulates, via protein-protein interaction with KCC2, GABAAR mediated inhibition in the hippocampus.

Prefrontal Cortical GABAergic Dysfunction Contributes to Aberrant UP-State Duration in APP Knockout Mice.

Genetic and biochemical studies have focused on the role of amyloid ß protein in the pathogenesis of Alzheimer's disease. In comparison, the physiological roles of its precursor protein, amyloid precursor protein (APP), in synaptic and network activity is less well studied. Using an APP knockout (APP-/-) mouse model, we show that the duration of UP state, which is a key feature of cortical synaptic integration occurring predominantly during slow-wave sleep, is significantly increased in the prefrontal cortex (PFC) in the absence of APP. This was accompanied by a specific reduction in the glutamine synthetase and tissue GABA content and sequential upregulation in the levels of GABABR expression. Pharmacological reinforcement of GABA signaling by application of either a GABA uptake inhibitor or an agonist of GABABR rescued the abnormality of UP-state duration and the former rescues altered GABABR expression as well. In addition to revealing an essential role of APP in the regulation of PFC network function, this study evidences the viability of GABA signaling pathway and its receptors, especially GABABRs, as a target for the treatment of aberrant neural network activity and thus information processing.