Engineering personalized neural tissue using functionalized transcription factors

Diseases and disorders of the central nervous system often require significant interventions to restore lost function due to their com- plexity. Examples of such disorders include Parkinson＇s disease, Alzheimer＇s disease, multiple sclerosis, traumatic brain injury, and spinal cord in）ury. These diseases and disorders result trom healthy cells being destroyed, which in turn causes dysfunction in the cen- tral nervous system, The death of these cells can trigger a cascade of events that affect the rest of the body, causing symptoms that become progressively worse over time. Developing strategies for repairing the damage to the central nervous system remains chal- lenging, in part due to its inability to regenerate.

Expression change of stem cell-derived neural stem/progenitor cell sup-porting factor gene in injured spinal cord of rats

Objective To explore the expression change of stem cell-derived neural stem/progenitor cell supporting factor （SDNSF） gene in the injuried spinal cord tissues of rats, and the relation between the expressions of SDNSF and nestin. Methods The spinal cord contusion model of rat was established according to Allen＇s falling strike method. The expression of SDNSF was studied by RT-PCR and in situ hybridization （ISH）, and the expression of nestin was detected by immunochemistry. Results RT-PCR revealed that SDNSF mRNA was upregulated on day 4 after injury, peaked on day 8-12, and decreased to the sham operation level on day 16. ISH revealed that SDNSF mRNA was mainly expressed in the gray matter cells, probably neurons, of spinal cord. The immunohistochemistry showed that accompanied with SDNSF mRNA upregulation, the nestin-positive cells showed erupted roots, migrated peripherad and proliferation on the 8-day slice. However, the distribution pattern of these new cells was different from that of SDNSF-positive cells. Conclusion （1） SDNSF is expressed in the gray matter of spinal cord. The expression of SDNSF mRNA in the spinal cord varies with injured time. （2） The nestin-positive cells proliferate accompanied with spinal cord injury repair, but do not secrete SDNSF.

Denervated hippocampus provides a favorable microenvironment for neuronal differentiation of endogenous neural stem cells

Fimbria-fornix transection induces both exogenous and endogenous neural stem cells to differentiate into neurons in the hippocampus.This indicates that the denervated hippocampus provides an environment for neuronal differentiation of neural stem cells.However,the pathways and mechanisms in this process are still unclear.Seven days after fimbria fornix transection,our reverse transcription polymerase chain reaction,western blot assay,and enzyme linked immunosorbent assay results show a significant increase in ciliary neurotrophic factor m RNA and protein expression in the denervated hippocampus.Moreover,neural stem cells derived from hippocampi of fetal（embryonic day 17） Sprague-Dawley rats were treated with ciliary neurotrophic factor for 7 days,with an increased number of microtubule associated protein-2-positive cells and decreased number of glial fibrillary acidic protein-positive cells detected.Our results show that ciliary neurotrophic factor expression is up-regulated in the denervated hippocampus,which may promote neuronal differentiation of neural stem cells in the denervated hippocampus.

Panax notoginseng saponins improve recovery after spinal cord transection by upregulating neurotrophic factors

Saponins extracted from Panax notoginseng are neuroprotective, but the mechanisms underlying this effect remain unclear. In the present study, we established a rat model of thoracic（T10） spinal cord transection, and injected Panax notoginseng saponins（100 mg/kg） or saline 30 minutes after injury. Locomotor functions were assessed using the Basso, Beattie, and Bresnahan（BBB） scale from 1 to 30 days after injury, and immunohistochemistry was carried out in the ventral horn of the spinal cord at 1 and 7 days to determine expression of nerve growth factor（NGF） and brain-derived neurotrophic factor（BDNF）. Our results show that at 7–30 days post injury, the BBB score was higher in rats treated with Panax notoginseng saponins than in those that received saline. Furthermore, at 7 days, more NGF- and BDNF-immunoreactive neurons were observed in the ventral horn of the spinal cord of rats that had received Panax notoginseng saponins than in those that received saline. These results indicate that Panax notoginseng saponins caused an upregulation of NGF and BDNF in rats with spinal cord transection, and improved hindlimb motor function.

Nerve growth factor promotes in vitro proliferation of neural stem cells from tree shrews

Neural stem cells promote neuronal regeneration and repair of brain tissue after injury,but have limited resources and proliferative ability in vivo.We hypothesized that nerve growth factor would promote in vitro proliferation of neural stem cells derived from the tree shrews,a primate-like mammal that has been proposed as an alternative to primates in biomedical translational research.We cultured neural stem cells from the hippocampus of tree shrews at embryonic day 38,and added nerve growth factor（100 μg/L） to the culture medium.Neural stem cells from the hippocampus of tree shrews cultured without nerve growth factor were used as controls.After 3 days,fluorescence microscopy after DAPI and nestin staining revealed that the number of neurospheres and DAPI/nestin-positive cells was markedly greater in the nerve growth factor-treated cells than in control cells.These findings demonstrate that nerve growth factor promotes the proliferation of neural stem cells derived from tree shrews.

Function of pioneer neurons specified by the basic helix-loop-helix transcription factor atonal in neural development

Basic helix-loop-helix （bHLH） transcription factors regulate the differentiation of various tissues in a vast diversity of species. The bHLH protein Atonal was first identified as a proneural gene involved in the formation of mechanosensory cells and photoreceptor cells in Drosophila （larman et al., 1993, 1994）. Atonal is expressed in sensory organ precursors and is required and sufficient for the development of chordotonal organs （Jar- man et al., 1993）. Moreover, Atonal expression is observed in the developing eye and is essential for the differentiation of R8 photoreceptors, which are the first photoreceptors that appear during development. Atonal is not involved in the formation of other photoreceptors （R1-R7） directly. However, R8 photore- ceptors recruit other photoreceptors from the surrounding cells （Jarman et al., 1994）.

社会交流中人际神经同步的认知机制

近年来,许多研究者开始关注社会交流中的人际神经同步机制,并将人际神经同步作为研究社会交流的一个神经指标,这对于揭示社会交流的本质和规律具有重要意义。本文从心理理论和镜像神经系统的角度,分析社会交流中神经同步的认知机制及其影响因素。未来的研究应关注这两套机制是否因交流目的、对象、形式或内容的不同,而在不同的脑区表现出神经同步,进而引发了不同认知机制的争议;以及这两套机制各自或协同工作适用的情景和任务。

Anti-inflammatory properties of lipoxin A4 protect against diabetes mellitus complicated by focal cerebral ischemia/reperfusion injury

Lipoxin A4 can alleviate cerebral ischemia/reperfusion injury by reducing the inflammatory reaction,but it is currently unclear whether it has a protective effect on diabetes mellitus complicated by focal cerebral ischemia/reperfusion injury.In this study,we established rat models of diabetes mellitus using an intraperitoneal injection of streptozotocin.We then induced focal cerebral ischemia/reperfusion injury by occlusion of the middle cerebral artery for 2 hours and reperfusion for 24 hours.After administration of lipoxin A4 via the lateral ventricle,infarction volume was reduced,the expression levels of pro-inflammatory factors tumor necrosis factor alpha and nuclear factor-kappa B in the cerebral cortex were decreased,and neurological functioning was improved.These findings suggest that lipoxin A4 has strong neuroprotective effects in diabetes mellitus complicated by focal cerebral ischemia/reperfusion injury and that the underlying mechanism is related to the anti-inflammatory action of lipoxin A4.

Human umbilical cord blood stem cells and brainderived neurotrophic factor for optic nerve injury: a biomechanical evaluation

Treatment for optic nerve injury by brain-derived neurotrophic factor or the transplantation of human umbilical cord blood stem cells has gained progress, but analysis by biomechanical indicators is rare. Rabbit models of optic nerve injury were established by a clamp. At 7 days after injury, the vitreous body received a one-time injection of 50 μg brain-derived neurotrophic factor or 1 × 10^6 human umbilical cord blood stem cells. After 30 days, the maximum load, maximum stress, maximum strain, elastic limit load, elastic limit stress, and elastic limit strain had clearly improved in rabbit models of optical nerve injury after treatment with brain-derived neurotrophic factor or human umbilical cord blood stem cells. The damage to the ultrastructure of the optic nerve had also been reduced. These findings suggest that human umbilical cord blood stem cells and brain-derived neurotrophic factor effectively repair the injured optical nerve, improve biomechanical properties, and contribute to the recovery after injury.

Inflammation and cutaneous nervous system involvement in hypertrophic scarring

This study aimed to use a mouse model of hypertrophic scarring by mechanical loading on the dorsum of mice to determine whether the nervous system of the skin and inflammation participates in hypertrophic scarring. Results of hematoxylin-eosin and immunohistochemical staining demonstrated that inflammation contributed to the formation of a hypertrophic scar and increased the nerve density in scar tissue.Western blot assay verified that interleukin-13 expression was increased in scar tissue. These findings suggest that inflammation and the cutaneous nervous system play a role in hypertrophic scar formation.

Beta-nerve growth factor promotes neurogenesis and angiogenesis during the repair of bone defects

We previously showed that the repair of bone defects is regulated by neural and vascular signals. In the present study, we examined the effect of topically applied β-nerve growth factor（β-NGF） on neurogenesis and angiogenesis in critical-sized bone defects filled with collagen bone substitute. We created two symmetrical defects, 2.5 mm in diameter, on either side of the parietal bone of the skull, and filled them with bone substitute. Subcutaneously implanted osmotic pumps were used to infuse 10 μgβ-NGF in PBS（β-NGF ＋ PBS） into the right-hand side defect, and PBS into the left（control） defect, over the 7 days following surgery. Immunohistochemical staining and hematoxylin-eosin staining were carried out at 3, 7, 14, 21 and 28 days postoperatively. On day 7, expression of β III-tubulin was lower on the β-NGF ＋ PBS side than on the control side, and that of neurofilament 160 was greater. On day 14, β III-tubulin and protein gene product 9.5 were greater on the β-NGF ＋ PBS side than on the control side. Vascular endothelial growth factor expression was greater on the experimental side than the control side at 7 days, and vascular endothelial growth factor receptor 2 expression was elevated on days 14 and 21, but lower than control levels on day 28. However, no difference in the number of blood vessels was observed between sides. Our results indicate that topical application of β-NGF promoted neurogenesis, and may modulate angiogenesis by promoting nerve regeneration in collagen bone substitute-filled defects.

Brain-derived neurotropic factor and GABAergic transmission in neurodegeneration and neuroregeneration

Neurotoxicity induced by stress,radiation,chemicals,or metabolic diseases,is commonly associated with excitotoxicity,oxidative stress,and neuroinflammation.The pathological process of neurotoxicity induces neuronal death,interrupts synaptic plasticity in the brain,and is similar to that of diverse neurodegenerative diseases.Animal models of neurotoxicity have revealed that clinical symptoms and brain lesions can recover over time via neuroregenerative processes.Specifically,brain-derived neurotropic factor（BDNF） and gamma-aminobutyric acid（GABA）-ergic transmission are related to both neurodegeneration and neuroregeneration.This review summarizes the accumulating evidences that suggest a pathogenic role of BDNF and GABAergic transmission,their underlying mechanisms,and the relationship between BDNF and GABA in neurodegeneration and neuroregeneration.This review will provide a comprehensive overview of the underlying mechanisms of neuroregeneration that may help in developing potential strategies for pharmacotherapeutic approaches to treat neurotoxicity and neurodegenerative disease.

New antigens involved in membranous nephropathy beyond phospholipase A2 receptor

When the physiopathology of membranous nephropathy was first described,almost 30%of cases were recognized to be secondary to well-known diseases such as autoimmune diseases,tumors or infections.The remaining 70%cases were called primary membranous nephropathy as the exact mechanism or pathogenic factor involved was unknown.The discovery of the M type phospholipase A2 receptor and thrombospondin type 1 domain containing 7A as causative antigens in these“so called”primary membranous nephropathies provided new insights into the effective causes of a large proportion of these cases.Novel techniques such as laser microdissection and tandem mass spectrometry as well as immunochemistry with antibodies directed against novel proteins allowed the confirmation of new involved antigens.Finally,using confocal microscopy to localize these new antigens and immunoglobulin G and Western blot analysis of serum samples,these new antigens were detected on the glomerular membrane,and the related antibodies were detected in serum samples.The same antigens have been recognized in some cases of secondary membranous disease due to autoimmune diseases,tumors and infections.This has allowed examination of the relationship between antigens in primary membranous nephropathy and their presence in some secondary nephropathies.The aim of this study is to describe the characteristics of the new antigens discovered and their association with other diseases.

Axonal remodeling in the corticospinal tract after stroke： how does rehabilitative training modulate it？

Stroke causes long-term disability, and rehabilitative training is commonly used to improve the consecutive functional recovery. Following brain damage, surviving neurons undergo morphological alterations to reconstruct the remaining neural network. In the motor system, such neural network remodeling is observed as a motor map reorganization. Because of its significant correlation with functional recovery, motor map reorganization has been regarded as a key phenomenon for functional recovery after stroke. Although the mechanism underlying motor map reorganization remains unclear, increasing evidence has shown a critical role for axonal remodeling in the corticospinal tract. In this study, we review previous studies investigating axonal remodeling in the corticospinal tract after stroke and discuss which mechanisms may underlie the stimulatory effect of rehabilitative training. Axonal remodeling in the corticospinal tract can be classified into three types based on the location and the original targets of corticospinal neurons, and it seems that all the surviving corticospinal neurons in both ipsilesional and contralesional hemisphere can participate in axonal remodeling and motor map reorganization. Through axonal remodeling, corticospinal neurons alter their output selectivity from a single to multiple areas to compensate for the lost function. The remodeling of the corticospinal axon is influenced by the extent of tissue destruction and promoted by various therapeutic interventions, including rehabilitative training. Although the precise molecular mechanism underlying rehabilitation-promoted axonal remodeling remains elusive, previous data suggest that rehabilitative training promotes axonal remodeling by upregulating growth-promoting and downregulating growth-inhibiting signals.

Buyanghuanwu decoction promotes angiogenesis after cerebral ischemia/reperfusion injury：mechanisms of brain tissue repair

Buyanghuanwu decoction has been shown to protect against cerebral ischemia/reperfusion injury,but the underlying mechanisms remain unclear.In this study,rats were intragastrically given Buyanghuanwu decoction,15 m L/kg,for 3 days.A rat model of cerebral ischemia/reperfusion injury was established by middle cerebral artery occlusion.In rats administered Buyanghuanwu decoction,infarct volume was reduced,serum vascular endothelial growth factor and integrin αvβ3 levels were increased,and brain tissue vascular endothelial growth factor and CD34 expression levels were increased compared with untreated animals.These effects of Buyanghuanwu decoction were partially suppressed by an angiogenesis inhibitor（administered through the lateral ventricle for 7 consecutive days）.These data suggest that Buyanghuanwu decoction promotes angiogenesis,improves cerebral circulation,and enhances brain tissue repair after cerebral ischemia/reperfusion injury.

Human umbilical cord blood-derived stem cells and brain-derived neurotrophic factor protect injured optic nerve:viscoelasticity characterization

The optic nerve is a viscoelastic solid-like biomaterial.Its normal stress relaxation and creep properties enable the nerve to resist constant strain and protect it from injury.We hypothesized that stress relaxation and creep properties of the optic nerve change after injury.Moreover,human brain-derived neurotrophic factor or umbilical cord blood-derived stem cells may restore these changes to normal.To validate this hypothesis,a rabbit model of optic nerve injury was established using a clamp approach.At 7 days after injury,the vitreous body received a one-time injection of 50 μg human brain-derived neurotrophic factor or 1 × 106 human umbilical cord blood-derived stem cells.At 30 days after injury,stress relaxation and creep properties of the optic nerve that received treatment had recovered greatly,with pathological changes in the injured optic nerve also noticeably improved.These results suggest that human brain-derived neurotrophic factor or umbilical cord blood-derived stem cell intervention promotes viscoelasticity recovery of injured optic nerves,and thereby contributes to nerve recovery.

Effect of ciliary neurotrophic factor on bcl-2 and c-jun gene and protein expression in cultured retinal nerve cells

BACKGROUND：In various retinal neurodegenerative animal models,ciliary neurotrophic factor （CNTF） exhibits prominent neuroprotective effects on retinal nerve cells.Bcl-2 is an anti-apoptotic protein.c-Jun is upregulated and phosphorylated in the activated c-Jun N-terminal kinase pathway,which subsequently mediates apoptosis.However,the effect of CNTF on Bcl-2 and c-Jun expression in retinal nerve cells remains unclear.OBJECTIVE：To determine the dynamic changes in retinal nerve cell apoptosis,as well as bcl-2 and c-jun gene and protein expression,following a single dose of CNTF in a short period of time.DESIGN,TIME AND SETTING：A single-blind,randomized,controlled,in vitro experiment was performed at the Central Laboratory of Beijing Tongren Hospital from May 2008 to April 2009.MATERIALS：Neonatal bovine retinal nerve cells （Chinese Holstein）,recombinant human CNTF （PeproTech,Rocky Hill,NJ,USA）,rabbit polyclonal anti-Bcl-2 and c-Jun antibodies （Abeam,Cambridge,UK）,fluorescein isothiocyanate-conjugated annexin V/propidium iodide kit （BioVision,Mountain View,CA,USA）,real time polymerase chain reaction instrument （ABI,Foster City,CA,USA）,and flow cytometer （BD FACSCalibur,Franklin Lakes,NJ,USA）.METHODS：Neonatal bovine retinal cells from passage 2 were cultured for 3 days and incubated with,or without,50 ng/mL CNTF （control）.MAIN OUTCOME MEASURES：Cell apoptosis was detected via Annexin V-FITC/PI double-staining and flow cytometry.bcl-2 and c-jun mRNA and protein expression were detected by quantitative real time polymerase chain reaction and western blot analysis.RESULTS：The proportion of late-stage apoptotic cells was significantly decreased at 2,4,and 6 days after CNTF treatment compared with the control group （P 〈 0.01）.CNTF did not alter bcl-2 mRNA expression at the three time points,but significantly increased Bcl-2 protein expression at 2 and 4 days （P 〈 0.01）.c-jun mRNA expression was significantly decreased 4 days after CNTF treatment （P〈 0.01）.In addition,c-Jun protein expression was slightly increased at 4 days （P〈 0.01）,but decreased at 6 days,compared with the control group （P〈 0.05）.CONCLUSION：A single dose of CNTF （50 ng/mL） upregulated Bcl-2 protein and downregulated c-jun mRNA expression,followed by a parallel,but lagged,change in c-Jun protein production in cultured neonatal bovine retinal nerve cells.These results suggested that CNTF reduces retinal nerve cell apoptosis by modifying Bcl-2 and c-Jun expression.

Does glioblastoma cyst fluid promote sciatic nerve regeneration?

Glioblastoma cyst fluid contains growth factors and extracellular matrix proteins which are known as neurotrophic and neurite-promoting agents. Therefore, we hypothesized that glioblastoma cyst fluid can promote the regeneration of injured peripheral nerves. To validate this hypothesis, we transected rat sciatic nerve, performed epineural anastomosis, and wrapped the injured sciatic nerve with glioblastoma cyst fluid- or saline-soaked gelatin sponges. Neurological function and histomorphological examinations showed that compared with the rats receiving local saline treatment, those receiving local glioblastoma cyst fluid treatment had better sciatic nerve function, fewer scars, greater axon area, counts and diameter as well as fiber diameter. These findings suggest that glioblastoma cyst fluid can promote the regeneration of injured sciatic nerve and has the potential for future clinical application in patients with peripheral nerve injury.

Specific effects of c-Jun NH2-terminal kinaseinteracting protein 1 in neuronal axons

c-Jun NH2-terminal kinase（JNK）-interacting protein 3 plays an important role in brain-derived neurotrophic factor/tropomyosin-related kinase B（Trk B） anterograde axonal transport. It remains unclear whether JNK-interacting protein 1 mediates similar effects, or whether JNK-interacting protein 1 affects the regulation of Trk B anterograde axonal transport. In this study, we isolated rat embryonic hippocampus and cultured hippocampal neurons in vitro. Coimmunoprecipitation results demonstrated that JNK-interacting protein 1 formed Trk B complexes in vitro and in vivo. Immunocytochemistry results showed that when JNK-interacting protein 1 was highly expressed, the distribution of Trk B gradually increased in axon terminals. However, the distribution of Trk B reduced in axon terminals after knocking out JNK-interacting protein 1. In addition, there were differences in distribution of Trk B after JNK-interacting protein 1 was knocked out compared with not. However, knockout of JNK-interacting protein 1 did not affect the distribution of Trk B in dendrites. These findings confirm that JNK-interacting protein 1 can interact with Trk B in neuronal cells, and can regulate the transport of Trk B in axons, but not in dendrites.

Biodegradable magnesium wire promotes regeneration of compressed sciatic nerves

Magnesium（Mg） wire has been shown to be biodegradable and have anti-inflammatory properties. It can induce Schwann cells to secrete nerve growth factor and promote the regeneration of nerve axons after central nervous system injury. We hypothesized that biodegradable Mg wire may enhance compressed peripheral nerve regeneration. A rat acute sciatic nerve compression model was made, and AZ31 Mg wire（3 mm diameter; 8 mm length） bridged at both ends of the nerve. Our results demonstrate that sciatic functional index, nerve growth factor, p75 neurotrophin receptor, and tyrosine receptor kinase A m RNA expression are increased by Mg wire in Mg model. The numbers of cross section nerve fibers and regenerating axons were also increased. Sciatic nerve function was improved and the myelinated axon number was increased in injured sciatic nerve following Mg treatment. Immunofluorescence histopathology showed that there were increased vigorous axonal regeneration and myelin sheath coverage in injured sciatic nerve after Mg treatment. Our findings confirm that biodegradable Mg wire can promote the regeneration of acute compressed sciatic nerves.