GAP-43 Protein Expression Research Articles

Lnc-Pim1 Promotes Neurite Outgrowth and Regeneration of Neuron-Like Cells Following ACR-Induced Neuronal Injury.

Decreased regenerative capacity of central nervous system neurons is the main cause for failure of damaged neuron regeneration and functional recovery. Long noncoding RNAs (lncRNAs) are abundant in mammalian transcriptomes, and many time- and tissue-specific lncRNAs are thought to be closely related to specific biological functions. The promoting effect of Pim-1 gene on neural differentiation and regeneration has been documented, but the effect and mechanism of its neighbor gene Lnc-Pim1 in regulating the response of central neurons to injury remain unclear. RT-PCR in this study demonstrated that the expression of Lnc-Pim1 was upregulated in acrylamide (ACR)-induced neuronal injury. FISH and nucleus-cytoplasmic assay demonstrated that Lnc-Pim1 was mainly expressed in the neuron cytoplasm, with a small amount in the nucleus. Western blot analysis proved that Lnc-Pim1 overexpression induced by the lentivirus vector could promote neurite outgrowth in Neuro-2a cells by activating the Erk1/2 signal pathway, and improve neurite regeneration of injured neurons by upregulating GAP-43 and β-Ⅲ tubulin protein expression. However, silencing Lnc-Pim1 expression by interfering RNA could effectively downregulate the GAP-43 and β-Ⅲ tubulin protein expression, and inhibit neurite growth of neurons. In addition, CHIRP-MS was performed to identify several potential targets of Lnc-Pim1 involved in the regulation of neurite regeneration of injured neurons. In conclusion, our study demonstrated that Lnc-Pim1 is a potential lnc-RNA, playing an important role in regulating central nerve regeneration.

CX3CL1-CX3CR1 axis protects retinal ganglion cells by inhibiting microglia activation in a distal optic nerve trauma model

BackgroundThe chemokine CX3CL1 has been reported to play an important role in optic nerve protection, but the underlying mechanism is still unclear. CX3CR1, the only receptor of CX3CL1, is specifically expressed on retinal microglia, whose activation plays a role in the pathological process of optic nerve injury. This study aimed to evaluate whether CX3CL1 exerts optic neuroprotection by affecting the activation of microglia by combining with CX3CR1.MethodsA mouse model of distal optic nerve trauma (ONT) was used to evaluate the effects of the CX3CL1-CX3CR1 axis on the activation of microglia and survival or axonal regeneration of retinal ganglion cells (RGCs). The activation of microglia, loss of RGCs, and damage to visual function were detected weekly till 4 weeks after modeling. CX3CL1 was injected intravitreally immediately or delayed after injury and the status of microglia and RGCs were examined.ResultsIncreases in microglia activation and optic nerve damage were accompanied by a reduced production of the CX3CL1-CX3CR1 axis after the distal ONT modeling. Both immediate and delayed intravitreal injection of CX3CL1 inhibited microglia activation, promoted survival of RGCs, and improved axonal regenerative capacity. Injection with CX3CL1 was no longer effective after 48 h post ONT. The CX3CL1-CX3CR1 axis promotes survival and axonal regeneration, as indicated by GAP43 protein and gene expression, of RGCs by inhibiting the microglial activation after ONT.ConclusionsThe CX3CL1-CX3CR1 axis could promote survival and axonal regeneration of RGCs by inhibiting the microglial activation after optic nerve injury. The CX3CL1-CX3CR1 axis may become a potential target for the treatment of optic nerve injury. Forty-eight hours is the longest time window for effective treatment after injury. The study is expected to provide new ideas for the development of targeted drugs for the repair of optic nerve.

Abstract TP30: Exercise Postconditioning After Ischemic Stroke: Neuroprotection Through NGF/TRPV1-Mediated Astrocyte-Neuron Interaction

Introduction: Astrocytes activation and interaction with neurons potentially affect stroke outcomes. Transient receptor potential vanilloid 1 (TRPV1) participated in reducing brain injury and nerve growth factor (NGF) affected TRPV1 activation. The present study investigated neuroprotection induced by exercise postconditioning (PostE) and elucidated the mechanistic regulation of astrocytes-neuron crosstalk via NGF/TRPV1 pathway. Methods: Adult Sprague-Dawley rats received 2 h middle cerebral artery (MCA) occlusion, followed by 24 h of reperfusion. The treadmill exercise was then initiated 24 h after reperfusion for 3 days (PostE). Brain damage was determined by infarct volume, edema and apoptotic proteins expression (Bcl-2, BAX and Caspase-3). Neuroplasticity was determined by dendritic spine staining and protein expression of BDNF, GAP-43, PSD-95 and synaptophysin. Neuronal TRPV1 expression, astrocyte proliferation and astrocytic NGF expression was assessed with immunofluorescence. Primary astrocyte-neuron Transwell cultivation were used to determine the secretion of astrocytic NGF activating neural TRPV1. Interaction between TRPV1 and NGF or TRPV1 and A-kinase anchoring protein150 (AKAP150) were detected by immunofluorescence and Co-IP. Results: PostE significantly decreased brain damage and induced neuroplasticity in ischemic rats. PostE induced astrocyte proliferation and astrocytic NGF secretion, which, in turn, activated TRPV1 expression in neuron. Either NGF or TRPV1 inhibitor eliminated the neuroplasticity, while NGF inhibitor decreased TRPV1 expression, suggesting the potential regulating role of astrocytic NGF on TRPV1. Primary cells cultivation demonstrated a NGF/TRPV1 and TRPV1/AKAP150 interplays showing astrocyte-neuron communication. Conclusion: PostE induced neuroprotection, which was mediated by the activation of astrocytes and their interaction with neurons through the NGF/TRPV1 pathway, highlighting the significance of astrocyte-neuron crosstalk in post-stroke recovery processes.

Effect of TRPM8 on pain symptom and neuronal proliferation in mice with interstitial cystitis/bladder pain syndrome

Objective: To explore the effects between transient receptor potential melastatin 8 (TRPM8) on pain symptom and neuronal proliferation in mice with interstitial cystitis/bladder pain syndrome (IC/BPS). Methods: Female wild-type C57BL/6 mice (8-10 weeks old) were divided into control group, IC/BPS model group, and IC/BPS model+menthol group (6 mice each) by random number table method; TRPM8 knockout mice were randomly divided into TRPM8 knockout group and TRPM8 knockout model group (6 mice each). The IC/BPS model group, the IC/BPS model+menthol group, and the TRPM8 knockout model group were injected subcutaneously with residues 65-84 of murine uroplakin 3A (UPK3A65-84). The IC/BPS model+menthol group continued to be injected with menthol. After successful modeling, the differences in pain thresholds between the groups were assessed by mechanosensitivity. The bladder wall was injected with a cell membrane red fluorescent probe (Dil), and the dorsal root ganglion (DRG) tissues were collected 10 days later. The differences in the protein and mRNA levels of TRPM8 and GAP43 in the groups were detected by Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. Immunofluorescence was used to detect the distribution of TRPM8 expression with GAP43 or Dil in DRG tissues. The relationship between TRPM8 and pain symptom and its role in neuronal proliferation in IC/BPS mice were analyzed. Results: The models were all constructed successfully. Compared with the control group, the pain thresholds of mice in the IC/BPS model group and IC/BPS model+menthol group were reduced [(8.50±1.22), (5.50±1.05) vs (11.67±2.16), respectively, all P<0.001]. Compared with the control group, the expression of TRPM8 mRNA was elevated in the IC/BPS model group and IC/BPS model+menthol group, while TRPM8 was not expressed in the TRPM8 knockout group [(3.16±0.05), (6.46±0.21), and 0 vs (1.00±0.06), respectively, all P<0.001]. The expression of TRPM8 protein and mRNA in each group had the same trend (P<0.001). Compared with the control group, the expression of GAP43 mRNA in the DRG of the IC/BPS model group and the IC/BPS model+menthol group was increased, whereas the expression of GAP43 mRNA in the TRPM8 knockout model group was decreased (all P<0.001). The trend of GAP43 protein expression was the same as that of mRNA expression (P<0.001). Immunofluorescence results showed an increase in the number of GAP43-positive neurons in the DRG of the IC/BPS model group and the IC/BPS model+menthol group, and a decrease in the TRPM8 knockout group compared with the control group (all P<0.001). Compared with the control group, the number of Dil-positive neurons in the DRG of the IC/BPS model group and the IC/BPS model+menthol group was increased, while the TRPM8 knockout group was decreased (all P<0.001). Conclusion: TRPM8 can exacerbate pain symptom in IC/BPS mice, and the mechanism may be related to the induction of sensory nerve proliferation at the DRG level.

Geniposide improves depression-like behavior in prenatal stress male offspring through restoring HPA axis- and glucocorticoid receptor-associated dysfunction

AimsPrenatal stress (PS) has an important impact on the brain development of offspring, which can lead to attention deficits, anxiety and depression in offspring. Geniposide (GE) is a kind of iridoid glycoside extracted from Gardenia jasminoides Ellis. It has various pharmacological effects and has been proved that have antidepressant effects. The aim of this study was to investigate the effect of GE on depression-like behavior in PS-induced male offspring mice and explore the possible molecular mechanisms. MethodsWe used a prenatal restraint stress model, focusing on male PS-induced offspring mice to study the effects of GE. Key findingsThe results showed that GE administration for 4 weeks significantly improved the depression-like behavior in PS offspring mice, which was manifested by markedly increasing the sucrose preference of PS offspring and the activity in the open field test, and reducing the immobility time in the forced swimming test. In addition, GE significantly reduced the levels of hypothalamic-pituitary-adrenal (HPA) axis-related hormones and exceedingly increased the protein expression of MAP2 and GAP43 in PS offspring. Furthermore, GE increased Glucocorticoid receptors (GR) nuclear translocation in the hippocampus of PS offspring, and enhanced the expression of synaptic plasticity-related proteins. ConclusionThe results of this study showed that GE exerts antidepressant effects in male PS offspring mice by regulating the HPA axis, GR function and proteins related to synaptic plasticity.

Bestrophin-1 Participates in Neuropathic Pain Induced by Spinal Nerve Transection but not Spinal Nerve Ligation

Previous studies have reported that L5/L6 spinal nerve ligation (SNL), but not L5 spinal nerve transection (SNT), enhances anoctamin-1 in injured and uninjured dorsal root ganglia (DRG) of rats suggesting some differences in function of the type of nerve injury. The role of bestrophin-1 in these conditions is unknown. The aim of this study was to investigate the role of bestrophin-1 in rats subjected to L5 SNT and L5/L6 SNL. SNT up-regulated bestrophin-1 protein expression in injured L5 and uninjured L4 DRG at day 7, whereas it enhanced GAP43 mainly in injured, but also in uninjured DRG. In contrast, SNL enhanced GAP43 at day 1 and 7, while bestrophin-1 expression increased only at day 1 after nerve injury. Accordingly, intrathecal injection of the bestrophin-1 blocker CaCCinh-A01 (1-10 µg) reverted SNT- or SNL-induced tactile allodynia in a concentration-dependent manner. Intrathecal injection of CaCCinh-A01 (10 µg) prevented SNT-induced upregulation of bestrophin-1 and GAP43 at day 7. In contrast, CaCCinh-A01 did not affect SNL-induced up-regulation of GAP43 nor bestrophin-1. Bestrophin-1 was mainly expressed in small- and medium-size neurons in naïve rats, while SNT increased bestrophin-1 immunoreactivity in CGRP+, but not in IB4+ neuronal cells in DRG. Intrathecal injection of bestrophin-1 plasmid (pCMVBest) induced tactile allodynia and increased bestrophin-1 expression in DRG and spinal cord in naïve rats. CaCCinh-A01 reversed bestrophin-1 overexpression-induced tactile allodynia and restored bestrophin-1 expression. Our data suggest that bestrophin-1 plays a relevant role in neuropathic pain induced by SNT, but not by SNL. PerspectiveSNT, but not SNL, up-regulates bestrophin-1 and GAP43 protein expression in injured L5 and uninjured L4 DRG. SNT increases bestrophin-1 immunoreactivity in CGRP+ neurons in DRG. Bestrophin-1 overexpression induces allodynia. CaCCinh-A01 reduces allodynia and restores bestrophin-1 expression. Our data suggest bestrophin-1 is differentially regulated depending on the neuropathic pain model.

Expressions of NeuroD and GAP43 as diagnostic markers for olfactory neuroblastoma

ObjectiveOlfactory neuroblastoma (ONB) is often difficult to pathologically distinguish from other small round cell tumors (SRCTs) arising in the nasal cavities. Although there are several diagnostic markers used for differential diagnosis of ONB, these molecules are also expressed in various neuronal derived tumors. Here, we examined the expression of NeuroD, GAP43, and olfactory marker protein (OMP) in ONB and non-ONB SRCT to determine their utility in the differential diagnosis of ONB. MethodsTwenty-six patients diagnosed with and treated for ONB at Kobe University Hospital between 1997 and 2017 with formalin-fixed, paraffin-embedded biopsy or surgical resection specimens were included. The expressions of NeuroD, GAP43, and OMP were immunohistochemically examined in these 26 ONB specimens and specimens from 13 SRCTs arising in the nasal cavities for reference. ResultsAmong the 26 ONB samples, focal, patchy, and marked staining for NeuroD was observed in 4, 3, and 9 samples, respectively. Focal, patchy, and marked GAP43 staining was observed in 5, 3, and 11 samples, respectively. Consequently, marked positive staining for either NeuroD or GAP43 was observed in 54% (14/26) of ONBs. Among the 13 SRCTs, marked staining for NeuroD was observed in two small cell carcinomas, one undifferentiated carcinoma, and one neuroendocrine carcinoma, whereas marked positive staining for GAP43 was observed only in one undifferentiated carcinoma. No specimen in this study exhibited OMP staining. ConclusionsOur results suggest possible roles of GAP43 immunostaining in the differential diagnosis of ONB.

A Single Administration of Riluzole Applied Acutely After Spinal Cord Injury Attenuates Pro-inflammatory Activity and Improves Long-Term Functional Recovery in Rats.

After spinal cord injury (SCI), emergency treatment intervention can minimize tissue damage, which is closely related to the recovery of long-term function. Here, we examined whether the administration of a single dose of riluzole (6mg/kg) immediately after SCI was a critical window for the drug to exert its regulatory effect and limit long-term neurological deficits. The animals were sacrificed 1day after administration for investigation of neuronal survival and a potential neuroinflammatory response, and sacrificed in the 6th week for assessment of neurological function. Riluzole applied in a single dose immediately post-SCI decreased the mRNA level of interleukin-1β at 6h, reduced the destruction of neurons, and reduced the activation of microglia/macrophage M1 expression at day 1 post-SCI. Additionally, riluzole-treated rats showed higher expressions of interleukin-33 and its receptor ST2 in microglia/macrophages of the spinal cord than vehicle-treated rats, suggesting that this signaling pathway might be involved in microglia/macrophage-mediated inflammation. At 6weeks, riluzole-treated rats exhibited higher motor function scores than vehicle-treated controls. In addition, riluzole-treated rats exhibited higher expression of GAP43 protein and shorter N1 peak latency and larger N1-P1 amplitude in motor-evoked potentials, compared to vehicle-treated rats. Together, these data suggested that early application of riluzole after SCI could be crucial for long-term functional recovery, so it may represent a promising therapeutic candidate within the critical therapeutic window for acute SCI.

Proteomics Reveals the Effect of Low-Intensity Focused Ultrasound on Spasticity After Spinal Cord Injury.

To explore the efficacy and possible mechanisms of Low-intensity focused ultrasound (LIFU) in alleviating spasticity caused by Spinal cord injury (SCI). We selected male Sprague?Dawley rats as subjects and performed transverse injuries on the T9 vertebra of their spinal cord (SC) to build SCI. On the 7th day after SCI, LIFU treatment was performed below the SCI segment once a day for 20 min, for 4 consecutive weeks. During treatment, a pressure sensor was used to assess the degree of spasticity. After treatment, the SC tissues from the treatment sites of the SCI+LIFU(-) and SCI+LIFU(+) groups were extracted, and high-throughput sequencing was performed to identify the changes in proteomics. In addition, expression of the growth associated protein 43 (Gap43) was validated by western blotting. The behavioral results suggested that after 2 weeks of SCI, the rats were significantly induced to have a spastic reaction (p < 0.05), while after 4 weeks of LIFU treatment, the spastic response of rats was significantly improved (p < 0.05). Western blot analysis showed a significant increase in Gap43 expression in the SCI+LIFU(-) group compared with the sham group, whereas after 4 weeks of LIFU treatment, Gap43 protein expression was significantly decreased (p < 0.05). The results of this study showed that LIFU is an alternative treatment that can effectively relieve spastic reactions caused by SCI, possibly by reducing abnormal neuroplasticity or axon regeneration below the SCI segment.

Expression of Slit and Robo during remodeling of corticospinal tract in cervical spinal cord in middle cerebral artery occlusion rats.

Slits and Robos were associated with the generation of axons of corticospinal tract during the corticospinal tract (CST) remodeling after the cerebral ischemic stroke (CIS). However, little is known about the mechanism of CST remodeling. In this study, we detected the expression of Slits and Robos in middle cerebral artery occlusion (MCAO) rats to investigate the roles of Slits and Robos in the CIS. MCAO model was established using modified Zea Longa method. Beam walking test (BWT) was conducted to evaluate the motor function. The images of the track of cortical spinal cord beam on day 7, 14 and 21 were observed by anterograde CST tracing. Biopinylated dextan amine (BDA) was used to mark CST anterogradely. Expression of GAP-43 mRNA and GAP-43 protein in cervical spinal cord was detected by Real-Time PCR and Western blot analysis, respectively. The expression of Slit1, Slit2 and Robo1 in cervical spinal cord was detected by immunofluorescence staining. The scores in the model group were significantly reduced compared to sham-operation group on day 7 (P < 0.001), 14 (P < 0.001) and 21 (P < 0.001), respectively. There was no significant difference in the score on day 7, 14 and 21 of the sham-operation groups (P > 0.05). In contrast, significant increase was noticed in the scores in model group, presenting a time-dependent manner. More CST staining fibers could be observed at the degenerative side in the model group compared with that of the sham-operation group on day 21. GAP-43 mRNA expression in the model group showed significant increase compared to that of sham-operation group on day 14 (P = 0.015) and 21days (P = 0.002). The expression of GAP-43 protein in model group showed significant increase compared to that of sham-operation group on day 14 (P = 0.022) and day 21 (P = 0.008), respectively. The expression of Slit1 and Slit2 showed increase on day 14 and day 21, while the expression of Robo1 showed significant decrease in MCAO rats. Up-regulation of Slit1 and Slit2 and the downregulation of Robo1 may be related to the axons of CST midline crossing in spinal cord of MCAO rat during the spontaneous recovery of impaired motor function.

Mesenchymal stem cells act as stimulators of neurogenesis and synaptic function in a rat model of Alzheimer's disease

BackgroundAlzheimer's disease (AD) is one of the most common NDs leading to cognitive dysfunctions and dementia which are progressively worsen with age. Cell therapy is currently of particular interest in treatment of neurodegenerative disease (ND) such as AD. However, the effective treatment for AD is yet to be found. ObjectiveIn this study, the possible roles of human umbilical mesnchymal stromal cord (hUMSCs) and adipose mesenchymal stem cells (hAD-MSCs) in neurogenesis and synaptic function were investigated using a β-amyloid 1–42 (β A42)-induced AD rat model. MethodshUMSCs and hAD-MSCs were isolated from umbilical cord stroma and adipose tissue, respectively. The expression of Mesenchymal (CD73, CD90 and CD105) and hematopoietic (CD45 and CD133) markers of hUMSCs and hAD-MSCs were confirmed by flow cytometry. Alzheimer's rat model was created by β-amyloid 1–42 injection into the hippocampus and confirmed by Morris Water Maze and immunohistochemical staining. hUMSCs and hAD-MSCs were injected in Alzheimer's rat model, intravenously. Deposition of β-amyloid in the CA1 of hippocampus was assayed 3 months after cell administration. The expression of synaptophysin and GAP43 proteins was assessed by Western blot. Neural death was assessed by Nissl staining. ResultsThe data obtained from flow cytometry showed that surface mesenchymal and hematopoteic markers of the fibroblastic like cells isolated from adipose tissue and umbilical cord were expressed highly in hUMSCs and mostly in hAD-SCs. Transplantation of MSCs reduced β-amyloid deposition in the hippocampus of the AD rats compared to the β-amyloid group. The rate of neuronal cell death in the hippocampus of the β-amyloid-treated rats was significantly increased compared to that of the control group. The percentage of apoptotic cells in this group was 72.98 ± 1.25, which was significantly increased compared to the control group. Transplantation of either hUMSCs or hAD-SCs, respectively, resulted in a significant reduction in the apoptotic rate of the neuronal cells in the hippocampus by 39.47 ± 0.01 (p = 0.0001) and 43.23 ± 0.577 (p = 0.001) compared to the β-amyloid group. MSC transplantation resulted in a significant up-regulation in the expression levels of both synaptogenic (synaptophysin) and neurogenic markers (GAP43) by 1.289 ± 0.112 (P = 0.02) and 1.112 ± 0.106 (P = 0.005) fold in the hUMSCs-treated group and 1.174 ± 0.105 (P = 0.04) and 0.978 ± 0.167 (P = 0.008) fold in the hAD-SCs-treated group, respectively. ConclusionIntravenous injection of hUMSCs and hAD-MSCs is a safe approach that improves synaptic function and neurogenesis via up-regulation of synaptophysin and GAP43 protein expression levels, respectively, in Alzheimer's model. Intravenous injection of both applied SCs could improve learning and cognitive impairment induced by β A42 injection.

In vitro differentiation of human bone marrow stromal cells into neural precursor cells using small molecules

BackgroundNeurogenic differentiation of human marrow stromal stem cells (hMSCs) into neural precursor cells (NPCs) offers new hope in many neurological diseases. Stromal cells can be differentiated into NPCs using small molecules acting as chemical inducers. The aim of this study is to formulate an efficient, direct, fast and safe protocol to differentiate hMSCs into NPCs using different inducers: b-mercaptoethanol (BME), triiodothyronine (T3), and curcumin (CUR). New methodhMSCs were subjected to either 1 mM BME, 0.5 µM T3, or 5 µM CUR. Neurogenic differentiation was determined by assessing the protein expression of PAX6, SOX2, DLX2, and GAP-43 with flow cytometry and immunofluorescence, along with Nissl staining of differentiated cells. Results and Comparison with Existing MethodIt was revealed that T3 and CUR are 70–80% better than BME in terms of efficiency and safety, and surprisingly BME was a good promoting factor for cell preconditioning with limited effects on neural trans-differentiation related to its toxic effects on cell viability. ConclusionReprogramming of bone marrow stromal cells into neural cells gives hope for treating different neurological disorders. Our study shows that T3 and CUR were effective in generation of NPCs from hMSCs with preservation of cell viability. BME was a good promoting factor for cell preconditioning with limited effects on neural transdifferentiation related to its toxic effects on cell viability.

Electroacupuncture improves cardiac function and reduces infarct size by modulating cardiac autonomic remodeling in a mouse model of myocardial ischemia.

Sympathetic and parasympathetic nerve remodeling play an important role in cardiac function after myocardial ischemia (MI) injury. Increasing evidence indicates that electroacupuncture (EA) can regulate cardiac function by modulating the autonomic nervous system (ANS), but little is known about its effectiveness on neural remodeling post-MI. To investigate the role of EA in ANS remodeling post-MI. Adult male C57/BL6 mice were equally divided into the Control (Ctrl), MI and EA groups after generating the MI model by ligating the left anterior descending (LAD) coronary artery. Echocardiography and 2,3,5-triphenyltetrazolium (TTC) staining were employed to evaluate cardiac function and infarct size after EA treatment for five consecutive days. Serum norepinephrine (NE) levels were measured by ELISA to quantify sympathetic activation. Then, ANS remodeling was detected by immunohistochemistry (IHC), RT-qPCR, and Western blotting. Our preliminary findings showed that EA increased ejection fraction and fractional shortening and reduced infarct area after MI injury. Serum NE levels in the EA group were significantly decreased compared with those in the MI group. IHC staining results demonstrated that the density of growth associated protein (GAP)43 and tyrosine hydroxylase (TH) positive nerve fibers in the EA group were decreased with increased choline acetyltransferase (CHAT) and vesicular acetylcholine transporter (VACHT). Meanwhile, the results verified that mRNA and protein expression of GAP43 and TH were significantly inhibited by EA treatment in the MI mice, accompanied by elevated CHAT and VACHT. EA treatment could improve cardiac function and reduce infarct size by modulating sympathetic and parasympathetic nerve remodeling post-MI, thus helping the cardiac ANS reach a new balance to try to protect the heart from further possible injury.

LncRNA Neat1 Promotes Regeneration after Spinal Cord Injury by Targeting miR-29b.

Previous studies have shown that lncRNA NEAT1 and miR-29b are closely associated with repair of the injured spinal cord. However, the mechanism by which lncRNA NEAT1 promotes regeneration after spinal cord injury by regulating miR-29b has not been reported. Toexplore this mechanism, we established a rat model of spinalcord injury(SCI). The experimental rats were randomly assigned to one of six groups:the sham,model, si-NEAT1, miR-29b, si-NEAT1 + negative control and si-NEAT1 + si-miR-29b groups. Thehind limb motor functionof the rats was evaluatedondays 1,3, 7, 14,and 21 aftermodellingusingtheBBBratingscale. Seven days after theoperation, attenuation of pathological changes ininjured spinal cord tissues wasevaluated by HE staining. Anteriorhornneuronsandcavitiesinthe injuredareawerecounted by Nissl staining. In addition, the TUNEL assay was employed to study neuronal apoptosis in the anterior horn, and the expression of the apoptotic proteins Bcl-2 and Bax was analysed by western blotting. Finally,the protein expression of GFAP,NCAM,GAP43, and SCG10 wasmeasured byimmunohistochemistryand western blotting. BBBscoresrevealedthat decreasing the level of NEAT1 improvedthehind limb motor function ofthe rats by increasing miR-29b expression. H&Eand Nissl staining showedthatsilencingNEAT1attenuatedlesions in thespinal cord anddecreasedthenumber of cavities inthe injured spinal cord by upregulatingmiR-29b. Immunohistochemistryandwesternblottingsuggestedthatsilencing NEAT1significantly downregulatedGFAP expressionand upregulatedGAP43, SCG10 andNCAM expression byinducing overexpression ofmiR-29b. The TUNEL assay and western blotting also showed that silencing NEAT1 attenuated neuronal apoptosis.

The Regenerative Potential of Facial Nerve Motoneurons following Chronic Axotomy in Rats.

Background The precise mechanisms of nerve regeneration remain unclear. The potential of facial nerve regeneration and probable mechanisms involved following chronic facial nerve injury should be further studied. Methods Adult male Wistar rats were used to model either (i) facial nerve injury (axotomy) or (ii) reinjury (chronic axotomy followed by a second axotomy within 5 months). The rats were housed in the animal facility of the Eye and ENT Hospital of Shanghai Medical School, Fudan University (Shanghai, China). Expression of Shh (sonic hedgehog) and growth-associated protein 43 (GAP43, a neuronal marker) was detected in bilateral facial nuclei using reverse transcriptase PCR, western blotting analysis, and immunohistochemistry. The number of surviving motoneurons was quantified, and facial nerve regeneration was examined using transmission electron microscopy. Results Reinjury of the facial nerve 12 weeks after the first axotomy resulted in upregulation of GAP43 mRNA and protein expression in neurons ipsilateral to the axotomy; immunohistochemistry revealed that Shh expression was higher compared with control side facial nuclei at the same time point. GAP43 expression subsequently decreased. Conclusion The greatest regeneration potential of the facial nerve occurred within 5 months following chronic axotomy in rats, and regeneration may involve the Shh signaling pathway.

RETRACTED ARTICLE: microRNA-195 attenuates neuronal apoptosis in rats with ischemic stroke through inhibiting KLF5-mediated activation of the JNK signaling pathway

BackgroundAccumulating evidence has implicated the regulation of microRNAs (miRs) in ischemia stroke. The current study aimed to elucidate the role of microRNA-195 (miR-195) in neuronal apoptosis and brain plasticity in rats with ischemic stroke via the JNK signaling pathway/KLF5 axis.MethodsIschemic stroke rat models were established by middle cerebral artery occlusion (MCAO), and oxygen deprivation (OGD) models were constructed in rat neuronal cells, followed by gain- or loss-of-function of miR-195 and/or KLF5 in rats and cells. Infarct volume, neuronal loss and ultrastructure, the expression of GAP-43, SYP and KLF5 protein as well as cell apoptosis were determined in the rats. Caspase-3 activity as well as the expression of miR-195, KLF5, GAP-43, SYP, JNK, phosphorylated JNK, Bax and Bcl-2 was measured in the cells.ResultsThe infarct size, expression of GAP-43 and SYP protein and apoptotic cells were increased in the miR-195−/− MCAO rats, while reductions were detected in the miR-195 mimic MCAO and KLF5−/− MCAO rats. Bcl-2 expression was increased, Bax and Caspase-3 expression as well as the ratio of phosphorylated JNK/JNK was decreased in response to miR-195 overexpression or KLF5 knockdown. Interestingly, the silencing of KLF5 reversed the effects exerted by the miR-195 inhibitor on the expression of Bcl-2, phosphorylated JNK/JNK, Bax and Caspase-3.ConclusionsCollectively, our study unraveled that miR-195 could down-regulate KLF5 and block the JNK signaling pathway, ultimately inhibiting neuronal apoptosis in rats with ischemic stroke.

Use of a Combination Strategy to Improve Morphological and Functional Recovery in Rats With Chronic Spinal Cord Injury.

Immunization with neural derived peptides (INDP), as well as scar removal (SR) and the use of matrices with bone marrow-mesenchymal stem cells (MSCs), have been studied separately and proven to induce a functional and morphological improvement after spinal cord injury (SCI). Herein, we evaluated the therapeutic effects of INDP combined with SR and a fibrin glue matrix (FGM) with MSCs (FGM-MSCs), on motor recovery, axonal regeneration-associated molecules and cytokine expression, axonal regeneration (catecholaminergic and serotonergic fibers), and the induction of neurogenesis after a chronic SCI. For this purpose, female adult Sprague-Dawley rats were subjected to SCI, 60 days after lesion, rats were randomly distributed in four groups: (1) Rats immunized with complete Freund's adjuvant + PBS (vehicle; PBS-I); (2) Rats with SR+ FGM-MSCs; (3) Rats with SR+ INDP + FGM-MSCs; (4) Rats only with INDP. Afterwards, we evaluated motor recovery using the BBB locomotor test. Sixty days after the therapy, protein expression of TNFα, IL-4, IL-10, BDNF, and GAP-43 were evaluated using ELISA assay. The number of catecholaminergic and serotonergic fibers were also determined. Neurogenesis was evaluated through immunofluorescence. The results show that treatment with INDP alone significantly increased motor recovery, anti-inflammatory cytokines, regeneration-associated molecules, axonal regeneration, and neurogenesis when compared to the rest of the groups. Our findings suggest that the combination therapy (SR + INDP + FGM-MSCs) modifies the non-permissive microenvironment post SCI, but it is not capable of inducing an appropriate axonal regeneration or neurogenesis when compared to the treatment with INDP alone.

The Bu Shen Yi Sui Formula Promotes Axonal Regeneration via Regulating the Neurotrophic Factor BDNF/TrkB and the Downstream PI3K/Akt Signaling Pathway.

Axonal damage is recognized as an important pathological feature in the chronic progressive neurological disorder multiple sclerosis (MS). Promoting axonal regeneration is a critical strategy for the treatment of MS. Our clinical and experimental studies have shown that the Bu Shen Yi Sui formula (BSYS) promotes axonal regeneration in MS and experimental autoimmune encephalomyelitis (EAE), an animal model of MS, but the exact mechanism has not been thoroughly elucidated to date. In this study, we investigated the effects of BSYS and its two decomposed formulas—the Bu Shen formula (BS) and the Hua Tan Huo Xue formula (HTHX)—on brain-derived neurotrophic factor (BDNF)/TrkB and related signaling pathways to explore the mechanism by which axonal regeneration is promoted in vitro and in vivo. Damaged SH-SY5Y cells incubated with low serum were treated with BSYS-, BS-, and HTHX-containing serum, and EAE mice induced by the myelin oligodendrocyte glycoprotein (MOG)35–55 peptide were treated with BSYS. The results showed that the BSYS-containing serum markedly increased cell viability and increased the levels of growth associated protein (GAP)-43, phosphorylated (p)-cAMP-response element binding protein (CREB), BDNF, TrkB, and p-PI3K. The BS and HTHX treatments also induced the protein expression of GAP-43 and p-extracellular signal-regulated kinase (ERK) in the cells. Furthermore, the effects of BSYS on cell viability, GAP-43, p-CREB, and neurite outgrowth were clearly inhibited by LY294002, a specific antagonist of the PI3K signaling pathways. The addition of U0126 and U73122, antagonists of the ERK and PLCγ pathway, respectively, significantly inhibited cell viability and GAP-43 protein expression. Moreover, BSYS treatment significantly increased the expression of the 68-, 160-, and 200-kDa neurofilaments (NFs) of proteins and the BDNF, TrkB, PI3K, and Akt mRNA and proteins in the brain or spinal cord of mice at different stages. These results indicated that BSYS promotes nerve regeneration, and its mechanism is mainly related to the upregulation of the BDNF/TrkB and PI3K/Akt signaling pathways. BS and HTHX also promoted nerve regeneration, and this effect involved the ERK pathway.

Role of microRNA-92b in the pathophysiology of acute transverse myelitis in children

Objective To explore microRNAs that play key regulatory roles in the pathophysiology of acute transverse myelitis in children, and to find therapeutic targets. Methods Twelve patients with acute transverse myelitis were enrolled as ATM group and three children with normal cerebrospinal fluid as the control group.MicroRNA in cerebrospinal fluid of children with acute transverse myelitis was detected by using microarray4.0 chip.Bioinformatics was used to demonstrate microRNA, which plays a key regulatory role, and to predict target genes.Real-time quantitative polymerase chain reaction (qPCR) technique was adopted for in biology and technology duplication.Enzyme-linked immunosorbent assay (ELISA) and Western blot technique were used to detect the expression of key miRNA target protein. The key candidate microRNA was inhibited/overexpressed in dorsal root ganglion neurons, and the function was verified in vitro.Flavopiridol was used to inhibit the activity of CDKs to verify that miR-92b worked through p57-CDKs-GAP-43 pathway. Results The characteristic elevation of miR-92b in cerebrospinal fluid samples of acute transverse myelitis was significant. Bioinformatics analysis showed that p57 was the target gene of miR-92b.The expression of miR-92b was contrary to the p57 protein.In vitro experiments showed that the length of axons in miR-92b mimics group was significantly shorter than that in the blank group.The axons of neurons in antimiR-92b group were obviously prolonged. In the miR-92b mimics+ Flavopiridol groups, the axons of neurons were still significantly prolonged compared with that in the blank group.Western blot showed that p57 and GAP-43 protein expression in miR-92b mimics group was lower than that in blank group.The expression of p57 and GAP-43 protein in antimiR-92b group was significantly higher than that in blank group.But in miR-92b mimics+ Flavopiridol group, the expression of p57 was lower compared with that in blank group, and the expression of GAP-43 protein was higher compared with that in blank group. Conclusions Up-regulation of miR-92b in children with acute transverse myelitis leads to a down-regulation of p57.The activity of CDKs is enhanced, which inhibits the expression of GAP-43 protein and the regeneration of axons in spinal cord injury region. MiR-92b is one of the key targets in the treatment of children with acute transverse myelitis. Key words: Acute transverse myelitis; Child; Cerebrospinal fluid; MicroRNA; Therapeutic target

The effects of paired associative stimulation on sensorimotor function and the expression of MAP-2 and GAP-43 after focal cerebral ischemia and reperfasion

Objective To observe the effect of paired associative stimulation (PAS) on the recovery of sensorimotor function and to explore the mechanism in terms of neural plasticity. Methods Ninety male adult Sprague-Dawley rats were randomly divided into a sham operation group (Sham group), a model group (Model group) and a paired associative stimulation group (PAS group), each of 30. Each group was then subdivided into 7-, 14- and 28-day subgroups with 10 rats in each. A model of focal cerebral ischemia and reperfusion was established using the Longa suture method in the Model and PAS groups. The rats in the Sham group underwent the same surgical procedure except for the occlusion of the middle cerebral artery. The rats received 30 minutes of paired peripheral nerve stimulation and transcranial magnetic stimulation comprising 90 pairs at 0.05 Hz beginning 24 h after the occlusion. The impulse wave width of the peripheral nerve stimulation was 200 μs and the intensity was 6 mA. The intensity of the transcranial magnetic stimulation was 120% of the resting motor threshold. The other two groups weren′t given any intervention. Neurological function was tested using Garcia scores on the 1st, 7th, 14th and 28th day after surgery. The rats were then sacrificed and the expression of MAP-2 and GAP-43 in the ischemic penumbra were detected using western blotting and immunohistochemistry. Results No neurological dysfunction was observed in the Sham group at any time. Compared with the Sham group at the same time points, the average Garcia scores of the Model and PAS groups were significantly lower (P≤0.05). However, the average Garcia scores on the 7th, 14th and 28th day were significantly higher in the PAS group compared with the Model group at the same time points (P≤0.05). The average Garcia scores of the Model and PAS groups on the 28th day after surgery were significantly higher than those on the 1st day (P≤0.05), but only the PAS group′s average Garcia score on the 28th day was significantly higher than that on the 7th day. Compared with the Sham group at the same time points, the expression of MAP-2 and GAP-43 protein in the Model and PAS groups was significantly higher, but with that of the Model group significantly lower than that of the PAS group (all P≤0.05). The protein expression of MAP-2 and GAP-43 protein in the PAS group on the 14th day was significantly higher than on the 7th and 28th day (P≤0.05 for both). Conclusions PAS can promote the recovery of sensorimotor function after cerebral thrombosis, at least in rats. That may be due to its promoting the expression of the neuroplasticity-associated proteins MAP-2 and GAP-43 in the ischemic penumbra. Key words: Cerebral ischemia; Paired associative stimulation; Sensorimotor functioning; MAP-2; GAP-43; Peripheral nerve stimulation; Transcranial magnetic stimulation