Neurotrophins differentially stimulate the growth of cochlear neurites on collagen surfaces and in gels

The electrodes of a cochlear implant are located far from the surviving neurons of the spiral ganglion, which results in decreased precision of neural activation compared to the normal ear. If the neurons could be induced to extend neurites toward the implant, it might be possible to stimulate more discrete subpopulations of neurons, and to increase the resolution of the device. However, a major barrier to neurite growth toward a cochlear implant is the fluid filling the scala tympani, which separates the neurons from the electrodes. The goal of this study was to evaluate the growth of cochlear neurites in three-dimensional extracellular matrix molecule gels, and to increase biocompatibility by using fibroblasts stably transfected to produce neurotrophin-3 and brain-derived neurotrophic factor. Spiral ganglion explants from neonatal rats were evaluated in cultures. They were exposed to soluble neurotrophins, cells transfected to secrete neurotrophins, and/or collagen gels. We found that cochlear neurites grew readily on collagen surfaces and in three-dimensional collagen gels. Co-culture with cells producing neurotrophin-3 resulted in increased numbers of neurites, and neurites that were longer than when explants were cultured with control fibroblasts stably transfected with green fluorescent protein. Brain-derived neurotrophic factor-producing cells resulted in a more dramatic increase in the number of neurites, but there was no significant effect on neurite length. It is suggested that extracellular matrix molecule gels and cells transfected to produce neurotrophins offer an opportunity to attract spiral ganglion neurites toward a cochlear implant.

Rbm8a regulates neurogenesis and reduces Alzheimer's disease-associated pathology in the dentate gyrus of 5×FAD mice

Alzheimer’s disease is a prevalent and debilitating neurodegenerative condition that profoundly affects a patient’s daily functioning with progressive cognitive decline,which can be partly attributed to impaired hippocampal neurogenesis.Neurogenesis in the hippocampal dentate gyrus is likely to persist throughout life but declines with aging,especially in Alzheimer’s disease.Recent evidence indicated that RNA-binding protein 8A(Rbm8a)promotes the proliferation of neural progenitor cells,with lower expression levels observed in Alzheimer’s disease patients compared with healthy people.This study investigated the hypothesis that Rbm8a overexpression may enhance neurogenesis by promoting the proliferation of neural progenitor cells to improve memory impairment in Alzheimer’s disease.Therefore,Rbm8a overexpression was induced in the dentate gyrus of 5×FAD mice to validate this hypothesis.Elevated Rbm8a levels in the dentate gyrus triggered neurogenesis and abated pathological phenotypes(such as plaque formation,gliosis reaction,and dystrophic neurites),leading to ameliorated memory performance in 5×FAD mice.RNA sequencing data further substantiated these findings,showing the enrichment of differentially expressed genes involved in biological processes including neurogenesis,cell proliferation,and amyloid protein formation.In conclusion,overexpressing Rbm8a in the dentate gyrus of 5×FAD mouse brains improved cognitive function by ameliorating amyloid-beta-associated pathological phenotypes and enhancing neurogenesis.

Activation of cerebral Ras-related C3 botulinum toxin substrate(Rac) 1 promotes post-ischemic stroke functional recovery in aged mice

Brain functional impairment after stroke is common;however,the molecular mechanisms of post-stroke recovery remain unclear.It is well-recognized that age is the most important independent predictor of poor outcomes after stroke as older patients show poorer functional outcomes following stroke.Mounting evidence suggests that axonal regeneration and angiogenesis,the major forms of brain plasticity responsible for post-stroke recovery,diminished with advanced age.Previous studies suggest that Ras-related C3 botulinum toxin substrate(Rac)1 enhances stroke recovery as activation of Rac1 improved behavior recovery in a young mice stroke model.Here,we investigated the role of Rac1 signaling in long-term functional recovery and brain plasticity in an aged(male,18 to 22 months old C57BL/6J)brain after ischemic stroke.We found that as mice aged,Rac1 expression declined in the brain.Delayed overexpression of Rac1,using lentivirus encoding Rac1 injected day 1 after ischemic stroke,promoted cognitive(assessed using novel object recognition test)and sensorimotor(assessed using adhesive removal tests)recovery on days 14–28.This was accompanied by the increase of neurite and proliferative endothelial cells in the periinfarct zone assessed by immunostaining.In a reverse approach,pharmacological inhibition of Rac1 by intraperitoneal injection of Rac1 inhibitor NSC23766 for 14 successive days after ischemic stroke worsened the outcome with the reduction of neurite and proliferative endothelial cells.Furthermore,Rac1 inhibition reduced the activation of p21-activated kinase 1,the protein level of brain-derived neurotrophic factor,and increased the protein level of glial fibrillary acidic protein in the ischemic brain on day 28 after stroke.Our work provided insight into the mechanisms behind the diminished plasticity after cerebral ischemia in aged brains and identified Rac1 as a potential therapeutic target for improving functional recovery in the older adults after stroke.

Role of transforming growth factor-βin peripheral nerve regeneration

Injuries caused by trauma and neurodegenerative diseases can damage the peripheral nervous system and cause functional deficits.Unlike in the central nervous system,damaged axons in peripheral nerves can be induced to regenerate in response to intrinsic cues after reprogramming or in a growth-promoting microenvironment created by Schwann cells.However,axon regeneration and repair do not automatically result in the restoration of function,which is the ultimate therapeutic goal but also a major clinical challenge.Transforming growth factor(TGF)is a multifunctional cytokine that regulates various biological processes including tissue repair,embryo development,and cell growth and differentiation.There is accumulating evidence that TGF-βfamily proteins participate in peripheral nerve repair through various factors and signaling pathways by regulating the growth and transformation of Schwann cells;recruiting specific immune cells;controlling the permeability of the blood-nerve barrier,thereby stimulating axon growth;and inhibiting remyelination of regenerated axons.TGF-βhas been applied to the treatment of peripheral nerve injury in animal models.In this context,we review the functions of TGF-βin peripheral nerve regeneration and potential clinical applications.

Axonal growth inhibitors and their receptors in spinal cord injury:from biology to clinical translation

Axonal growth inhibitors are released during traumatic injuries to the adult mammalian central nervous system, including after spinal cord injury. These molecules accumulate at the injury site and form a highly inhibitory environment for axonal regeneration. Among these inhibitory molecules, myelinassociated inhibitors, including neurite outgrowth inhibitor A, oligodendrocyte myelin glycoprotein, myelin-associated glycoprotein, chondroitin sulfate proteoglycans and repulsive guidance molecule A are of particular importance. Due to their inhibitory nature, they represent exciting molecular targets to study axonal inhibition and regeneration after central injuries. These molecules are mainly produced by neurons, oligodendrocytes, and astrocytes within the scar and in its immediate vicinity. They exert their effects by binding to specific receptors, localized in the membranes of neurons. Receptors for these inhibitory cues include Nogo receptor 1, leucine-rich repeat, and Ig domain containing 1 and p75 neurotrophin receptor/tumor necrosis factor receptor superfamily member 19(that form a receptor complex that binds all myelin-associated inhibitors), and also paired immunoglobulin-like receptor B. Chondroitin sulfate proteoglycans and repulsive guidance molecule A bind to Nogo receptor 1, Nogo receptor 3, receptor protein tyrosine phosphatase σ and leucocyte common antigen related phosphatase, and neogenin, respectively. Once activated, these receptors initiate downstream signaling pathways, the most common amongst them being the Rho A/ROCK signaling pathway. These signaling cascades result in actin depolymerization, neurite outgrowth inhibition, and failure to regenerate after spinal cord injury. Currently, there are no approved pharmacological treatments to overcome spinal cord injuries other than physical rehabilitation and management of the array of symptoms brought on by spinal cord injuries. However, several novel therapies aiming to modulate these inhibitory proteins and/or their receptors are under investigation in ongoing clinical trials. Investigation has also been demonstrating that combinatorial therapies of growth inhibitors with other therapies, such as growth factors or stem-cell therapies, produce stronger results and their potential application in the clinics opens new venues in spinal cord injury treatment.

Novel insights into the mechanism of reactive oxygen species-mediated neurodegeneration

Neurite degeneration,a major component of many neurodegenerative diseases,such as Parkinson’s disease,Alzheimer’s disease,and amyotrophic lateral sclerosis,is not part of the typical apoptosis signaling mechanism,but rather it appears that a self-destructive process is in action.Oxidative stress is a well-known inducer of neurodegenerative pathways:neuronal cell death and neurite degeneration.Although oxidative stress exerts cytotoxic effects leading to neuronal loss,the pathogenic mechanisms and precise signaling pathways by which oxidative stress causes neurite degeneration have remained entirely unknown.We previously reported that reactive oxygen species generated by NADPH oxidases induce activation of the E3 ubiquitin ligase ZNRF1 in neurons,which promotes neurite degeneration.In this process,the phosphorylation of an NADPH oxidase subunit p47-phox at the 345serine residue serves as an important checkpoint to initiate the ZNRF1-dependent neurite degeneration.Evidence provides new insights into the mechanism of reactive oxygen species-mediated neurodegeneration.In this review,we focus specifically on reactive oxygen species-induced neurite degeneration by highlighting a phosphorylation-dependent regulation of the molecular interaction between ZNRF1 and the NADPH oxidase complex.

Microcurvature landscapes induce neural stem cell polarity and enhance neural differentiation

Tissue curvature has long been recognized as an important anatomical parameter that affects intracellular behaviors,and there is emerging interest in applying cell-scale curvature as a designer property to drive cell fates for tissue engineering purposes.Although neural cells are known to undergo dramatic and terminal morphological changes during development and curvature-limiting behaviors have been demonstrated in neurite outgrowth studies,there are still crucial gaps in understanding neural cell behaviors,particularly in the context of a three-dimensional(3D)curvature landscape similar to an actual tissue engineering scaffold.In this study,we fabricated two substrates of microcurvature(curvature-substrates)that present a smooth and repeating landscape with focuses of either a concave or a convex pattern.Using these curvature-substrates,we studied the properties of morphological differentiation in N2a neuroblastoma cells.In contrast to other studies where two-dimensional(2D)curvature was demonstrated to limit neurite outgrowth,we found that both the concave and convex substrates acted as continuous and uniform mechanical protrusions that significantly enhanced neural polarity and differentiation with few morphological changes in the main cell body.This enhanced differentiation was manifested in various properties,including increased neurite length,increased nuclear displacement,and upregulation of various neural markers.By demonstrating how the micron-scale curvature landscape induces neuronal polarity,we provide further insights into the design of biomaterials utilizing the influence of surface curvature in neural tissue engineering.

Tissue Microstructure Estimation of SANDI Based on Deep Network

Diffusion magnetic resonance imaging(dMRI)is a noninvasive method to capture the anisotropic pattern of water displacement in the neuronal tissue.The soma and neurite density imaging(SANDI)model introduced soma size and density to biophysical model for the first time.In addition to neurite density,it can achieve their joint estimation non-invasively using dMRI.In the traditional method,parameters of the SANDI are estimated in a maximum likelihood frame-work,where the nonlinear model fitting is computationally intensive.Also,the present methods require a large number of diffusion gradients.Efficient and accurate algorithms for tissue microstructure estimation of SANDI is still a challenge currently.Consequently,we introduce deep learning method for tissue microstructure estimation of the SANDI model.The model comprises two functional components.The first component produces the sparse representation of diffusion sig-nals of input patches.The second component computes tissue microstructure from the sparse repre-sentation given by the first component.The deep network can produce not only tissue microstruc-ture estimates but also the uncertainty of the estimates with a reduced number of diffusion gradi-ents.Then,multiple deep networks are trained and their results are fused for the final prediction of tissue microstructure and uncertainty quantification.The deep network was evaluated on the MGH Connectome Diffusion Microstructure Dataset.Results indicate that our approach outperforms the traditional methods in terms of estimation accuracy.

Advanced diffusion magnetic resonance imaging in patients with Alzheimer’s and Parkinson’s diseases

The prevalence of neurodegenerative diseases is increasing as human longevity increases. The objective biomarkers that enable the staging and early diagnosis of neurodegenerative diseases are eagerly anticipated. It has recently become possible to determine pathological changes in the brain without autopsy with the advancement of diffusion magnetic resonance imaging techniques. Diffusion magnetic resonance imaging is a robust tool used to evaluate brain microstructural complexity and integrity, axonal order, density, and myelination via the micron-scale displacement of water molecules diffusing in tissues. Diffusion tensor imaging, a type of diffusion magnetic resonance imaging technique is widely utilized in clinical and research settings;however, it has several limitations. To overcome these limitations, cutting-edge diffusion magnetic resonance imaging techniques, such as diffusional kurtosis imaging, neurite orientation dispersion and density imaging, and free water imaging, have been recently proposed and applied to evaluate the pathology of neurodegenerative diseases. This review focused on the main applications, findings, and future directions of advanced diffusion magnetic resonance imaging techniques in patients with Alzheimer's and Parkinson's diseases, the first and second most common neurodegenerative diseases, respectively.

Effect of Rho-kinase pathway on neurite outgrowth of rat hippocampal neurons under atomic force microscopy

Hippocampal neurons of neonatal rats were cultured in serum-free culture medium for 5 days in vitro, and treated with the Rho-kinase inducer lysophosphatidic acid. Atomic force microscopy revealed that the numbers of level-1, -2 and -3 neurites protruding from rat hippocampal neurons was significantly reduced. After treatment with the Rho kinase inhibitor Y27632, a significant increase in the numbers of these neurites was observed. Our experimental findings indicate that the Rho-kinase pathway is closely associated with the neurites of hippocampal neurons.

Effect of type-2 astrocytes on the viability of dorsal root ganglion neurons and length of neuronal processes

The role of type-2 astrocytes in the repair of central nervous system injury remains poorly un- derstood. In this study, using a relatively simple culture condition in vitro, type-2 astrocytes, differentiated from oligodendrocyte precursor cells by induction with bone morphogenetic pro- tein-4, were co-cultured with dorsal root ganglion neurons. We examined the effects of type-2 astrocytes differentiated from oligodendrocyte precursor cells on the survival and growth of dorsal root ganglion neurons. Results demonstrated that the number of dorsal root ganglion neurons was higher following co-culture of oligodendrocyte precursor cells and type-2 astrocytes than when cultured alone, but lower than that of neurons co-cultured with type-1 astrocytes. The length of the longest process and the length of all processes of a single neuron were shortest in neurons cultured alone, followed by neurons co-cultured with type-2 astroc^es, then neurons co-cultured with oligodendrocyte precursor cells, and longest in neurons co-cultured with type-1 astrocytes. These results indicate that co-culture with type-2 astrocytes can increase neuronal survival rate and process length. However, compared with type-1 astrocytes and oligodendrocyte precursor cells, the promotion effects of type-2 astrocytes on the growth of dorsal root ganglion neurons were weaker.

Effect of Y-27632 on the cultured retinal neurocytes of rats

AIM:To investigate the effect of Y-27632 on the survival and neurite outgrowth of the cultured retinal neurocytes. METHODS:After the postnatal day 2-3, Sprague-Dawley retinal neurocytes were cultured for 48 hours, the culture media was replaced with serum-free media (control group) and serum-free media contained 30μmol/L Y-27632 (Y-27632 group), and the cells were continually cultured another 48 hours. The cultured retinal neurocytes were identified with anti-neuron specific enolase (NSE) immunocytochemistry. The survival state of those cells was estimated by MTT assay, and the neurite outgrowth of those cells was evaluated by the computerized image-analysis system. RESULTS:Compared with the control group, the absorbance values of cells survival in Y -27632 group increased 12.90% and 33.33% respectively after 72 and 96 hours culture. Y-27632 had no significant effect on the diameter of cultured retinal neurocytes. Compared with the control group, Y-27632 induced a stable improvement of neurite outgrowth of retinal neurocytes after 72 and 96 hours culture (P =0.001). CONCLUSION:Y-27632 could promote the survival and neurite outgrowth of the early postnatal cultured retinal neurocytes.

Stereotactic Injection of shRNA GSK-3β-AAV Promotes Axonal Regeneration after Spinal Cord Injury

Evidence suggested that glycogen synthase kinase-3β（GSK-3β） is involved in Nogo-66 inhibiting axonal regeneration in vitro, but its effect in vivo was poorly understood. We showed that stereotactic injection of sh RNA GSK-3β-adeno associated virus（GSK-3β-AAV） diminished syringomyelia and promoted axonal regeneration after spinal cord injury（SCI）, using stereotactic injection of sh RNA GSK-3β-AAV（tested with Western blotting and RT-PCR） into the sensorimotor cortex of rats with SCI and by the detection of biotin dextran amine（BDA）-labeled axonal regeneration. We also determined the right position to inject into the sensorimotor cortex. Our findings consolidate the hypothesis that downregulation of GSK-3β promotes axonal regeneration after SCI.

急性一氧化碳中毒致迟发性脑病大鼠胼胝体损伤及神经生长抑制因子A的表达

目的：研究急性一氧化碳（ carbon monoxide ， CO ）中毒致迟发性脑病（ delayed neuropsychologic sequelae ， DNS ）大鼠胼胝体的损伤及神经生长抑制因子 A （ neurite outgrowth inhibitor-A， Nogo-A）的表达情况。方法腹腔内分次注射CO气体造成DNS模型，于不同时间点取胼胝体。HE染色观察细胞形态，变色酸染色观察脱髓鞘改变，免疫组化检测观察Nogo-A蛋白表达，荧光定量聚合酶链式反应（ real time polymerase chain reaction ， RT-PCR）检测Nogo-A mRNA表达，电镜观察胼胝体超微结构变化。结果中毒组光镜下胼胝体出现脱髓鞘等变化，以造模成功后0 h变化较明显。免疫组化显示，Nogo-A在各时间点均高表达（P＜0．05）。 RT-PCR于造模成功后0、12、24 h高表达（P＜0．05），0 h为高峰，其后逐渐下降，第3天与对照组比较差异无统计学意义，中毒组第7天表达再次升高（P＜0．05）。电镜下，胼胝体出现相应改变。结论 CO中毒后大鼠胼胝体出现脱髓鞘损伤，且与Nogo-A表达相关。

Midkine promotes perineural invasion in human pancreatic cancer

AIM:To investigate midkine(MK)and syndecan-3protein expression in pancreatic cancer by immunohistochemistry,and to analyze their correlation with clinicopathological features,perineural invasion,and prognosis.METHODS:Pancreatic cancer tissues(including adequately sized tumor tissue samples and tissue samples taken from areas less than 2.0 cm around the tumor)were taken from 42 patients who were undergoing a partial duodenopancreatectomy.MK and syndecan-3proteins were detected by immunohistochemistry using a standardized streptavidin-peroxidase method,and analyzed for their correlation with clinicopathological features,perineural invasion,and prognosis.Associations of neural invasion with aggressive characteristics of pancreatic cancer and the presence of perineural invasion were assessed by two independent observers blinded to the patient status.RESULTS:MK and syndecan-3 were found in 26(61.9%)and 24(57.1%)specimens,respectively.MK and syndecan-3 expression was associated with perineural invasion(P=0.018 and 0.031,respectively).High MK expression was closely associated with advanced tumor,node and metastasis stage(P=0.008),lymph node metastasis(P=0.042),and decreased postoperative survival at 3years(51.0%vs 21.8%,P=0.001).Syndecan-3 levels were correlated with tumor size(P=0.028).Patients who were syndecan-3 negative had a higher cumulative survival rate than those who were positive,but the difference was not significant(44.0%vs 23.0%,P>0.05).CONCLUSION:MK and syndecan-3 are frequently expressed in pancreatic cancer and associated with perineural invasion.High expression of MK and syndecan-3may contribute to the highly perineural invasion and poor prognosis of human pancreatic cancer.

Pleiotrophin promotes perineural invasion in pancreatic cancer

Perineural invasion(PNI)in pancreatic cancer is an important cause of local recurrence,but little is known about its mechanism.Pleiotrophin(PTN)is an important neurotrophic factor.It is of interest that our recent experimental data showed its involvement in PNI of pancreatic cancer.PTN strongly presents in the cytoplasm of pancreatic cancer cells,and high expression of PTN and its receptor may contribute to the high PNI of pancreatic cancer.Correspondingly,PNI is prone to happen in PTN-positive tumors.We thus hypothesize that,as a neurite growth-promoting factor,PTN may promote PNI in pancreatic cancer.PTN is released at the time of tumor cell necrosis,and binds with its highaffinity receptor,N-syndecan on pancreatic nerves,to promote neural growth in pancreatic cancer.Furthermore,neural destruction leads to a distorted neural homeostasis.Neurons and Schwann cells produce more N-syndecan in an effort to repair the pancreatic nerves.However,the abundance of N-syndecan attracts further PTN-positive cancer cells to the site of injury,creating a vicious cycle.Ultimately,increased PTN and N-syndecan levels,due to the continuous nerve injury,may promote cancer invasion and propagation along the neural structures.Therefore,it is meaningful to discuss the relationship between PTN/N-syndecan signaling and PNI in pancreatic cancer,which may lead to a better understanding of the mechanism of PNI in pancreatic cancer.

Electroacupuncture treatment improves motor function and neurological outcomes after cerebral ischemia/reperfusion injury

Electroacupuncture(EA)has been widely used for functional restoration after stroke.However,its role in post-stroke rehabilitation and the associated regulatory mechanisms remain poorly understood.In this study,we applied EA to the Zusanli(ST36)and Quchi(LI11)acupoints in rats with middle cerebral artery occlusion and reperfusion.We found that EA effectively increased the expression of brain-derived neurotrophic factor and its receptor tyrosine kinase B,synapsin-1,postsynaptic dense protein 95,and microtubule-associated protein 2 in the ischemic penumbra of rats with middle cerebral artery occlusion and reperfusion.Moreover,EA greatly reduced the expression of myelin-related inhibitors Nogo-A and NgR in the ischemic penumbra.Tyrosine kinase B inhibitor ANA-12 weakened the therapeutic effects of EA.These findings suggest that EA can improve neurological function after middle cerebral artery occlusion and reperfusion,possibly through regulating the activity of the brain-derived neurotrophic factor/tyrosine kinase B signal pathway.All procedures and experiments were approved by the Animal Research Committee of Shanghai University of Traditional Chinese Medicine,China(approval No.PZSHUTCM200110002)on January 10,2020.

The role of exosomes in peripheral nerve regeneration

Peripheral nerve injuries remain problematic to treat, with poor functional recovery commonly observed. Injuries resulting in a nerve gap create specific difficulties for axonal regeneration. Approaches to address these difficulties include autologous nerve grafts （which are currently the gold standard treatment） and synthetic conduits, with the latter option being able to be im- pregnated with Schwann cells or stem cells which provide an appropriate micro-environment for neuronal regeneration to occur. Transplanting stem cells, however, infers additional risk of malignant transformation as well as manufacturing difficulties and ethical concerns, and the use of autologous nerve grafts and Schwann ceils requires the sacrifice of a functioning nerve. A new approach utilizing exosomes, secreted extracellular vesicles, could avoid these complications. In this review, we summarize the current literature on exosomes, and suggest how they could help to improve axonal regeneration following peripheral nerve injury.

Ginsenoside Rg1 protects against neurodegeneration by inducing neurite outgrowth in cultured hippocampal neurons

Ginsenoside Rg1（Rg1） has anti-aging and anti-neurodegenerative effects. However, the mechanisms underlying these actions remain unclear. The aim of the present study was to determine whether Rg1 affects hippocampal survival and neurite outgrowth in vitro after exposure to amyloid-beta peptide fragment 25–35（Aβ_（25–35））, and to explore whether the extracellular signal-regulated kinase（ERK） and Akt signaling pathways are involved in these biological processes. We cultured hippocampal neurons from newborn rats for 24 hours, then added Rg1 to the medium for another 24 hours, with or without pharmacological inhibitors of the mitogen-activated protein kinase（MAPK） family or Akt signaling pathways for a further 24 hours. We then immunostained the neurons for growth associated protein-43, and measured neurite length. In a separate experiment, we exposed cultured hippocampal neurons to Aβ_（25–35） for 30 minutes, before adding Rg1 for 48 hours, with or without Akt or MAPK inhibitors, and assessed neuronal survival using Hoechst 33258 staining, and phosphorylation of ERK1/2 and Akt by western blot analysis. Rg1 induced neurite outgrowth, and this effect was blocked by API-2（Akt inhibitor） and PD98059（MAPK/ERK kinase inhibitor）, but not by SP600125 or SB203580（inhibitors of c-Jun N-terminal kinase and p38 MAPK, respectively）. Consistent with this effect, Rg1 upregulated the phosphorylation of Akt and ERK1/2; these effects were reversed by API-2 and PD98059, respectively. In addition, Rg1 significantly reversed Aβ_（25–35）-induced apoptosis; this effect was blocked by API-2 and PD98059, but not by SP600125 or SB203580. Finally, Rg1 significantly reversed the Aβ_（25–35）-induced decrease in Akt and ERK1/2 phosphorylation, but API-2 prevented this reversal. Our results indicate that Rg1 enhances neurite outgrowth and protects against Aβ_（25–35）-induced damage, and that its mechanism may involve the activation of Akt and ERK1/2 signaling.

Neuroprotective effects of ginsenoside Rb1 on hippocampal neuronal injury and neurite outgrowth

Ginsenoside Rb1 has been reported to exert anti-aging and anti-neurodegenerative effects. In the present study, we investigate whether ginsenoside Rb1 is involved in neurite outgrowth and neuroprotection against damage induced by amyloid beta（25–35） in cultured hippocampal neurons, and explore the underlying mechanisms. Ginsenoside Rb1 significantly increased neurite outgrowth in hippocampal neurons, and increased the expression of phosphorylated-Akt and phosphorylated extracellular signal-regulated kinase 1/2. These effects were abrogated by API-2 and PD98059, inhibitors of the signaling proteins Akt and MEK. Additionally, cultured hippocampal neurons were exposed to amyloid beta（25–35） for 30 minutes; ginsenoside Rb1 prevented apoptosis induced by amyloid beta（25–35）, and this effect was blocked by API-2 and PD98059. Furthermore, ginsenoside Rb1 significantly reversed the reduction in phosphorylated-Akt and phosphorylated extracellular signal-regulated kinase 1/2 levels induced by amyloid beta（25–35）, and API-2 neutralized the effect of ginsenoside Rb1. The present results indicate that ginsenoside Rb1 enhances neurite outgrowth and protects against neurotoxicity induced by amyloid beta（25–35） via a mechanism involving Akt and extracellular signal-regulated kinase 1/2 signaling.