Dorsal root ganglion progenitors differentiate to gamma-aminobutyric acid- and choline acetyltransferase-positive neurons

This study examined the isolation and differentiation of dorsal root ganglion progenitor cells for therapeutic use in neurodegenerative diseases. Rat embryonic dorsal root ganglia progenitors were isolated and purified using the differential adhesion method combined with cytosine arabinoside treatment. After culture in serum-free medium supplemented with B27, basic fibroblast growth factor and epidermal growth factor, these cells remained viable and survived for more than 18 months in vitro. Most cells differentiated to neurons that were immunoreactive for gamma-aminobutyric acid and choline acetyltransferase as detected by immunohistochemical staining. In addition, nerve growth factor and neurotrophic tyrosine kinase receptor expression were also observed in dorsal root ganglion progenitors and differentiated cells. K252a, an inhibitor that blocks nerve growth factor-induced signaling, inhibited cell survival, suggesting the possible existence of a nerve growth factor autocrine loop in these proliferating cells.     

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

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.     

Enteric glia mediate neuronal outgrowth through release of neurotrophic factors

Previous studies have shown that transplanted enteric glia enhance axonal regeneration, reduce tissue damage, and promote functional recovery following spinal cord injury. However, the mechanisms by which enteric glia mediate these beneficial effects are unknown. Neurotrophic factors can promote neuronal differentiation, survival and neurite extension. We hypothesized that enteric glia may exert their protective effects against spinal cord injury partially through the secretion of neurotrophic factors. In the present study, we demonstrated that primary enteric glia cells release nerve growth factor, brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor over time with their concentrations reaching approximately 250, 100 and 50 pg/mL of culture medium respectively after 48 hours. The biological relevance of this secretion was assessed by incubating dissociated dorsal root ganglion neuronal cultures in enteric glia-conditioned medium with and/or without neutralizing antibodies to each of these proteins and evaluating the differences in neurite growth. We discovered that conditioned medium enhances neurite outgrowth in dorsal root ganglion neurons. Even though there was no detectable amount of neurotrophin-3 secretion using ELISA analysis, the neurite outgrowth effect can be attenuated by the antibody-mediated neutralization of each of the aforementioned neurotrophic factors. Therefore, enteric glia secrete nerve growth factor, brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor and neurotrophin-3 into their surrounding environment in concentrations that can cause a biological effect.     

Changes in compressed neurons from dogs with acute and severe cauda equina constrictions following intrathecal injection of brain-derived neurotrophic factor-conjugated polymer nanoparticles

This study established a dog model of acute multiple cauda equina constriction by experimental constriction injury (48 hours) of the lumbosacral central processes in dorsal root ganglia neurons. The repair effect of intrathecal injection of brain-derived neurotrophic factor with 15 mg encapsulated biodegradable poly(lactide-co-glycolide) nanoparticles on this injury was then analyzed. Dorsal root ganglion cells (L7) of all experimental dogs were analyzed using hematoxylin-eosin staining and immunohistochemistry at 1, 2 and 4 weeks following model induction. Intrathecal injection of brain-derived neurotrophic factor can relieve degeneration and inflammation, and elevate the expression of brain-derived neurotrophic factor in sensory neurons of compressed dorsal root ganglion. Simultaneously, intrathecal injection of brain-derived neurotrophic factor obviously improved neurological function in the dog model of acute multiple cauda equina constriction. Results verified that sustained intraspinal delivery of brain-derived neurotrophic factor encapsulated in biodegradable nanoparticles promoted the repair of histomorphology and function of neurons within the dorsal root ganglia in dogs with acute and severe cauda equina syndrome.     

Extracellular matrix from human umbilical cord-derived mesenchymal stem cells as a scaffold for peripheral nerve regeneration

The extracellular matrix, which includes collagens, laminin, or fibronectin, plays an important role in peripheral nerve regeneration. Recently, a Schwann cell-derived extracellular matrix with classical biomaterial was used to mimic the neural niche. However, extensive clinical use of Schwann cells remains limited because of the limited origin, loss of an autologous nerve, and extended in vitro culture times. In the present study, human umbilical cord-derived mesenchymal stem cells (hUCMSCs), which are easily accessible and more proliferative than Schwann cells, were used to prepare an extracellular matrix. We identiifed the morphology and function of hUCMSCs and investi-gated their effect on peripheral nerve regeneration. Compared with a non-coated dish tissue culture, the hUCMSC-derived extracellular matrix enhanced Schwann cell proliferation, upregulated gene and protein expression levels of brain-derived neurotrophic factor, glial cell-derived neurotrophic factor, and vascular endothelial growth factor in Schwann cells, and enhanced neurite outgrowth from dorsal root ganglion neurons. These ifndings suggest that the hUCMSC-derived extracellular matrix promotes peripheral nerve repair and can be used as a basis for the rational design of engineered neural niches.     

Electroacupuncture upregulated platelet derived growth factor expression in spared dorsal root ganglion of cats

A bilateral spared dorsal root ganglion model was established in healthy adult cats by bilateral resection of L 1-5 and L 7 -S 2 dorsal root ganglia. L 6 dorsal root ganglia were spared. Zusanli (ST36) and Xuanzhong (BL39) or Futu (ST32) and Sanyinjiao (SP6) were alternatively electro-stimulated on the right leg. Immunohistochemical staining of anti-serum platelet-derived growth factor demonstrated that the number of total neurons and medium-small sized platelet-derived growth factor positive neurons was significantly decreased on the 7 th day following injury. After 7 days of acupuncture, the total number of positive and large neurons staining for platelet-derived growth factor on the acupuncture side significantly increased compared to the non-acupuncture side. After acupuncture for 14 days, the total positive and medium-small sized neurons significantly increased compared with the non-acupuncture side. Results indicate that acupuncture promoted the synthesis of platelet-derived growth factor in spared dorsal root ganglia.     

In vitro neuroprotective effects of ciliary neurotrophic factor on dorsal root ganglion neurons with glutamate-induced neurotoxicity

Ciliary neurotrophic factor has neuroprotective effects mediated through signal transducer and Janus kinase (JAK) 2/activator of transcrip-tion 3 (STAT3) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways. Whether ciliary neurotrophic factor is neuroprotective for glutamate-induced excitotoxicity of dorsal root ganglion neurons is poorly understood. In the present study, the in vitro neuroprotective effects of ciliary neurotrophic factor against glutamate-induced excitotoxicity were determined in a primary culture of dorsal root ganglion neurons from Wistar rat embryos at embryonic day 15. Whether the JAK2/STAT3 and PI3K/Akt signaling pathways were related to the protective effects of ciliary neurotrophic factor was also determined. Glutamate exposure inhibited neurite outgrowth, cell viability, and growth-associated protein 43 expression and promoted apoptotic neuronal cell death, all of which were reversed by the administration of exogenous ciliary neurotrophic factor. Additionally, preincubation with either JAK2 inhibitor AG490 or PI3K inhibitor LY294002 blocked the neuroprotective effect of ciliary neurotrophic factor. These data indicate that the two pathways JAK2/STAT3 and PI3K/Akt play major roles in mediating the in vitro neuroprotective effects of ciliary neurotrophic factor on dorsal root ganglion neurons with glutamate-in-duced neurotoxicity.     

Microencapsulated Schwann cell transplantation inhibits P2X3 receptor expression in dorsal root ganglia and neuropathic pain

Schwann cell transplantation is a promising method to promote neural repair, and can be used for peripheral nerve protection and myelination. Microcapsule technology largely mitigates immune rejection of transplanted cells. We previously showed that microencapsulated olfactory ensheathing cells can reduce neuropathic pain and we hypothesized that microencapsulated Schwann cells can also inhibit neuropathic pain. Rat Schwann cells were cultured by subculture and then microencapsulated and were tested using a rat chronic constriction injury(CCI) neuropathic pain model. CCI rats were treated with Schwann cells or microencapsulated Schwann cells and were compared with sham and CCI groups. Mechanical withdrawal threshold and thermal withdrawal latency were assessed preoperatively and at 1, 3, 5, 7, 9, 11 and 14 days postoperatively. The expression of P2X3 receptors in L4–5 dorsal root ganglia of the different groups was detected by double-label immunofluorescence on day 14 after surgery. Compared with the chronic constriction injury group, mechanical withdrawal threshold and thermal withdrawal latency were higher, but the expression of P2X3 receptors was remarkably decreased in rats treated with Schwann cells and microencapsulated Schwann cells, especially in the rats transplanted with microencapsulated Schwann cells. The above data show that microencapsulated Schwann cell transplantation inhibits P2X3 receptor expression in L4–5 dorsal root ganglia and neuropathic pain.     

Distribution and localization of fibroblast growth factor-8 in rat brain and nerve cells during neural stem/progenitor cell differentiation

The present study explored the distribution and localization of fibroblast growth factor-8 and its potential receptor,fibroblast growth factor receptor-3,in adult rat brain in vivo and in nerve cells during differentiation of neural stem/progenitor cells in vitro.Immunohistochemistry was used to examine the distribution of fibroblast growth factor-8 in adult rat brain in vivo.Localization of fibroblast growth factor-8 and fibroblast growth factor receptor-3 in cells during neural stem/progenitor cell differentiation in vitro was detected by immunofluorescence.Flow cytometry and immunofluorescence were used to evaluate the effect of an anti-fibroblast growth factor-8 antibody on neural stem/progenitor cell differentiation and expansion in vitro.Results from this study confirmed that fibroblast growth factor-8 was mainly distributed in adult midbrain,namely the substantia nigra,compact part,dorsal tier,substantia nigra and reticular part,but was not detected in the forebrain comprising the caudate putamen and striatum.Unusual results were obtained in retrosplenial locations of adult rat brain.We found that fibroblast growth factor-8 and fibroblast growth factor receptor-3 were distributed on the cell membrane and in the cytoplasm of nerve cells using immunohistochemistry and immunofluorescence analyses.We considered that the distribution of fibroblast growth factor-8 and fibroblast growth factor receptor-3 in neural cells corresponded to the characteristics of fibroblast growth factor-8,a secretory factor.Addition of an anti-fibroblast growth factor-8 antibody to cultures significantly affected the rate of expansion and differentiation of neural stem/progenitor cells.In contrast,addition of recombinant fibroblast growth factor-8 to differentiation medium promoted neural stem/progenitor cell differentiation and increased the final yields of dopaminergic neurons and total neurons.Our study may help delineate the important roles of fibroblast growth factor-8 in brain activities and neural stem/progenitor cell differentiation.     

Beta-nerve growth factor gene therapy alleviates pyridoxine-induced neuropathic damage by increasing doublecortin and tyrosine kinase A in the dorsal root ganglion

Beta-nerve growth factor(β-NGF)is known to be a major leading cause of neuronal plasticity.To identify the possible action mechanisms ofβ-NGF gene therapy for sciatic nerve recovery,experimental dogs were randomly divided into control,pyridoxine,and pyridoxine+β-NGF groups.We observed chronological changes of morphology in the dorsal root ganglia in response to pyridoxine toxicity based on cresyl violet staining.The number of large neurons positive for cresyl violet was dramatically decreased after pyridoxine intoxication for 7 days in the dorsal root ganglia and the neuron number was gradually increased after pyridoxine withdrawal.In addition,we also investigated the effects ofβ-NGF gene therapy on neuronal plasticity in pyridoxine-induced neuropathic dogs.To accomplish this,tyrosine kinase receptor A(TrkA),βIII-tubulin and doublecortin(DCX)immunohistochemical staining was performed at 3 days after the last pyridoxine treatment.TrkA-immunoreactive neurons were dramatically decreased in the pyridoxine group compared to the control group,but strong TrkA immunoreactivity was observed in the small-sized dorsal root ganglia in this group.TrkA immunoreactivity in the dorsal root ganglia was similar betweenβ-NGF and control groups.The numbers ofβⅢ-tubulin-and DCX-immunoreactive cells decreased significantly in the pyridoxine group compared to the control group.However,the reduction ofβⅢ-tubulin-and DCX-immunoreactive cells in the dorsal root ganglia in theβ-NGF group was significantly ameliorated than that in the pyridoxine group.These results indicate thatβ-NGF gene therapy is a powerful treatment of pyridoxine-induced neuropathic damage by increasing the TrkA and DCX levels in the dorsal root ganglia.The experimental protocol was approved by the Institutional Animal Care and Use Committee(IACUC)of Seoul National University,South Korea(approval No.SNU-060623-1,SNU-091009-1)on June 23,2006 and October 9,2009,respectively.     

人胚胎背根神经节雪旺氏细胞的培养

BACKGROUND： Previous studies have used many methods for in vitro Schwann cells （SCs） cultures and purification, such as single cell suspension and cytosine arabinoside. However, it has been difficult to obtain sufficient cellular density, and the procedures have been quite tedious. OBJECTIVE： To investigate the feasibility of culturing high-density SCs using fetal human dorsal root ganglion tissue explants. DESIGN, TIME AND SETTING： Cell culture and irnmunohistochernistry were performed at the Central Laboratory of Kunrning General Hospital of Chinese PLA between March 2001 and October 2008. MATERIALS： Culture media containing 10% fetal bovine serum, as well as 0.2% collagenase and 0.25% trypsin were purchased from Gibco, USA; mouse anti-human S-100 monoclonal antibody and goat anti-mouse IgG labeled with horseradish peroxidase were provided by Beijing Institute of Biological Products, China. METHODS： Primarily cultured SCs were dissociated from dorsal root ganglia of human aborted fetuses at 4 6 months pregnancy. Following removal of the dorsal root ganglion perineurium, the ganglia were dissected into tiny pieces and digested with 0.2% collagenase and 0.25% trypsin （volume ratio 1：1）, then explanted and cultured. SC purification was performed with 5 rnL 10% fetal bovine serum added to the culture media, followed by differential adhesion. MAIN OUTCOME MEASURES： SCs morphology was observed under inverted phase contrast light microscopy. SC purity was evaluated according to percentage of S-100 immunostained cells. RESULTS： SCs were primarily cultured for 5 6 days and then subcultured for 4 5 passages. The highly enriched SC population reached 〉 95% purity and presented with normal morphology. CONCLUSION： A high purity of SCs was obtained with culture methods using human fetal dorsal root ganglion tissue explants.     

Role of neurotrophic factors in enhancing linear axonal growth of ganglionic sensory neurons in vitro

Neurotrophins play a major role in the regulation of neuronal growth such as neurite sprouting or regeneration in response to nerve injuries. The role of nerve growth factor, neurotrophin-3, and brain-derived neurotrophic factor in maintaining the survival of peripheral neurons remains poorly understood. In regenerative medicine, different modalities have been investigated for the delivery of growth factors to the injured neurons, in search of a suitable system for clinical applications. This study was to investigate the influence of nerve growth factor, neurotrophin-3 and brain-derived neurotrophic factor on the growth of neurites using two in vitro models of dorsal root ganglia explants and dorsal root ganglia-derived primary cell dissociated cultures. Quantitative data showed that the total neurite length and tortuosity were differently influenced by trophic factors. Nerve growth factor and, indirectly, brain-derived neurotrophic factor stimulate the tortuous growth of sensory fibers and the formation of cell clusters. Neurotrophin-3, however, enhances neurite growth in terms of length and linearity allowing for a more organized and directed axonal elongation towards a peripheral target compared to the other growth factors. These findings could be of considerable importance for any clinical application of neurotrophic factors in peripheral nerve regeneration. Ethical approval was obtained from the Regione Piemonte Animal Ethics Committee ASLTO1(file # 864/2016-PR) on September 14, 2016.     

Transfer of mitochondria from mesenchymal stem cells derived from induced pluripotent stem cells attenuates hypoxia-ischemia-induced mitochondrial dysfunction in PC12 cells

Mitochondrial dysfunction in neurons has been implicated in hypoxia-ischemia-induced brain injury. Although mesenchymal stem cell therapy has emerged as a novel treatment for this pathology, the mechanisms are not fully understood. To address this issue, we first co-cultured 1.5 × 105 PC12 cells with mesenchymal stem cells that were derived from induced pluripotent stem cells at a ratio of 1:1, and then intervened with cobalt chloride(CoCl2) for 24 hours. Reactive oxygen species in PC12 cells was measured by Mito-sox. Mitochondrial membrane potential(?Ψm) in PC12 cells was determined by JC-1 staining. Apoptosis of PC12 cells was detected by terminal deoxynucleotidal transferase-mediated dUTP nick end-labeling staining. Mitochondrial morphology in PC12 cells was examined by transmission electron microscopy. Transfer of mitochondria from the mesenchymal stem cells derived from induced pluripotent stem cells to damaged PC12 cells was measured by flow cytometry. Mesenchymal stem cells were induced from pluripotent stem cells by lentivirus infection containing green fluorescent protein in mitochondria. Then they were co-cultured with PC12 cells in Transwell chambers and treated with CoCl_2 for 24 hours to detect adenosine triphosphate level in PC12 cells. CoCl_2-induced PC12 cell damage was dose-dependent. Co-culture with mesenchymal stem cells significantly reduced apoptosis and restored ?Ψm in the injured PC12 cells under CoCl_2 challenge. Co-culture with mesenchymal stem cells ameliorated mitochondrial swelling, the disappearance of cristae, and chromatin margination in the injured PC12 cells. After direct co-culture, mitochondrial transfer from the mesenchymal stem cells stem cells to PC12 cells was detected via formed tunneling nanotubes between these two types of cells. The transfer efficiency was greatly enhanced in the presence of CoCl_2. More importantly, inhibition of tunneling nanotubes partially abrogated the beneficial effects of mesenchymal stem cells on CoCl_2-induced PC12 cell injury. Mesenchymal stem cells reduced CoCl2-induced PC12 cell injury and these effects were in part due to efficacious mitochondrial transfer.     

Emodin prevents hypoxic-ischemic neuronal injury Involvement of the activin A pathway

Emodin, an extract of dried rhizomes and the root of the Rhizoma Polygoni Cuspidati, can protect neurons from hypoxic-ischemic brain damage. This study aimed to verify the underlying mechanism. After PC12 cells had differentiated into neuron-like cells under the induction of mouse nerve growth factor, cells were subjected to oxygen-glucose deprivation and treated with emodin. Results showed that the viability of neuron-like cells cultured under an ischemia-hypoxia environment decreased, while the expression of activin A and caspase-3 in cells increased. Emodin raised the survival rate of oxygen-glucose deprived neuron-like cells, increased activin A expression, and decreased caspase-3 expression. Experimental findings indicate that emodin can inhibit neuronal apoptosis and alleviate the injury of nerve cells after oxygen-glucose deprivation through the activin A pathway.     

Molecular mechanism of panaxydol on promoting axonal growth in PC12 cells

Nerve growth factor(NGF) promotes axonal growth in PC12 cells primarily by regulating the RTK-RAS-MEK-ERK pathway. Panaxydol, a polyacetylene isolated from Panax notoginseng, can mimic the effects of NGF. Panaxydol promotes neurite outgrowth in PC12 cells, but its molecular mechanism remains unclear. Indeed, although alkynol compounds such as panaxydol can increase intracellular cyclic adenosine 3′,5′-monophosphate(cAMP) levels and the ERK inhibitor U0126 inhibits alkynol-induced axonal growth, how pathways downstream of cAMP activate ERK have not been investigated. This study observed the molecular mechanism of panaxydol-, NGF-and forskolin-induced PC12 cell axon growth using specific signaling pathway inhibitors. The results demonstrated that although the RTK inhibitor SU5416 obviously inhibited the growth-promoting effect of NGF, it could not inhibit the promoting effect of panaxydol on axonal growth of PC12 cells. The adenylate cyclase inhibitor SQ22536 and cAMP-dependent protein kinase inhibitor RpcAMPS could suppress the promoting effect of forskolin and panaxydol on axonal growth. The ERK inhibitor U0126 inhibited axonal growth induced by all three factors. However, the PKA inhibitor H89 inhibited the promoting effect of forskolin on axonal growth but could not suppress the promoting effect of panaxydol. A western blot assay was used to determine the effects of stimulating factors and inhibitors on ERK phosphorylation levels. The results revealed that NGF activates the ERK pathway through tyrosine receptors to induce axonal growth of PC12 cells. In contrast, panaxydol and forskolin increased cellular cAMP levels and were inhibited by adenylyl cyclase inhibitors. The protein kinase A inhibitor H89 completely inhibited forskolin-induced axonal outgrowth and ERK phosphorylation, but could not inhibit panaxydol-induced axonal growth and ERK phosphorylation. These results indicated that panaxydol promoted axonal growth of PC12 cells through different pathways downstream of cAMP. Considering that exchange protein directly activated by cAMP 1(Epac1) plays an important role in mediating cAMP signaling pathways, RNA interference experiments targeting the Epac1 gene were employed. The results verified that Epac1 could mediate the axonal growth signaling pathway induced by panaxydol. These findings suggest that compared with NGF and forskolin, panaxydol elicits axonal growth through the cAMP-Epac1-Rap1-MEK-ERK-CREB pathway, which is independent of PKA.     

Expression and effect of sodium-potassium-chloride cotransporter on dorsal root ganglion neurons in a rat model of chronic constriction injury

Sodium-potassium-chloride cotransporter 1 (NKCC1) and potassium-chloride cotransporter 2 (KCC2) are associated with the transmission of peripheral pain.We investigated whether the increase of NKCC1 and KCC2 is associated with peripheral pain transmission in dorsal root ganglion neurons.To this aim,rats with persistent hyperalgesia were randomly divided into four groups.Rats in the control group received no treatment,and the rat sciatic nerve was only exposed in the sham group.Rats in the chronic constriction injury group were established into chronic constriction injury models by ligating sciatic nerve and rats were given bumetanide,an inhibitor of NKCC1,based on chronic constriction injury modeling in the chronic constriction injury + bumetanide group.In the experiment measuring thermal withdrawal latency,bumetanide (15 mg/kg) was intravenously administered.In the patch clamp experiment,bumetanide (10 μg/μL) and acutely isolated dorsal root ganglion neurons (on day 14) were incubated for 1 hour,or bumetanide (5 μg/μL) was intrathecally injected.The Hargreaves test was conducted to detect changes in thermal hyperalgesia in rats.We found that the thermal withdrawal latency of rats was significantly decreased on days 7,14,and 21 after model establishment.After intravenous injection of bumetanide,the reduction in thermal retraction latency caused by model establishment was significantly inhibited.Immunohistochemistry and western blot assay results revealed that the immune response and protein expression of NKCC1 in dorsal root ganglion neurons of the chronic constriction injury group increased significantly on days 7,14,and 21 after model establishment.No immune response or protein expression of KCC2 was observed in dorsal root ganglion neurons before and after model establishment.The Cl^– (chloride ion) fluorescent probe technique was used to evaluate the change of Cl^– concentration in dorsal root ganglion neurons of chronic constriction injury model rats.We found that the relative optical density of N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (a Cl^– fluorescent probe whose fluorescence Cenintensity decreases as Cl– concentration increases) in the dorsal root ganglion neurons of the chronic constriction injury group was significantly decreased on days 7 and 14 after model establishment.The whole-cell patch clamp technique revealed that the resting potential and action potential frequency of dorsal root ganglion neurons increased,and the threshold and rheobase of action potentials decreased in the chronic constriction injury group on day 14 after model establishment.After bumetanide administration,the above indicators were significantly suppressed.These results confirm that CCI can induce abnormal overexpression of NKCC1,thereby increasing the Cl^– concentration in dorsal root ganglion neurons;this then enhances the excitability of dorsal root ganglion neurons and ultimately promotes hyperalgesia and allodynia.In addition,bumetanide can achieve analgesic effects.All experiments were approved by the Institutional Ethics Review Board at the First Affiliated Hospital,College of Medicine,Shihezi University,China on February 22,2017 (approval No.A2017-169-01).     

Dorsal root ganglion neurons promote proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells

Preliminary animal experiments have confirmed that sensory nerve fibers promote osteoblast differentiation, but motor nerve fibers have no promotion effect. Whether sensory neurons promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells remains unclear. No results at the cellular level have been reported. In this study, dorsal root ganglion neurons(sensory neurons) from Sprague-Dawley fetal rats were co-cultured with bone marrow mesenchymal stem cells transfected with green fluorescent protein 3 weeks after osteogenic differentiation in vitro, while osteoblasts derived from bone marrow mesenchymal stem cells served as the control group. The rat dorsal root ganglion neurons promoted the proliferation of bone marrow mesenchymal stem cell-derived osteoblasts at 3 and 5 days of co-culture, as observed by fluorescence microscopy. The levels of m RNAs for osteogenic differentiation-related factors(including alkaline phosphatase, osteocalcin, osteopontin and bone morphogenetic protein 2) in the co-culture group were higher than those in the control group, as detected by real-time quantitative PCR. Our findings indicate that dorsal root ganglion neurons promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells, which provides a theoretical basis for in vitro experiments aimed at constructing tissue-engineered bone.     

Transplantation of human adipose tissue-derived stem cells for repair of injured spiral ganglion neurons in deaf guinea pigs

Excessive noise, ototoxic drugs, infections, autoimmune diseases, and aging can cause loss of spiral gangli-on neurons, leading to permanent sensorineural hearing loss in mammals. Stem cells have been conifrmed to be able to differentiate into spiral ganglion neurons. Little has been reported on adipose tissue-derived stem cells (ADSCs) for repair of injured spiral ganglion neurons. In this study, we hypothesized that trans-plantation of neural induced-human ADSCs (NI-hADSCs) can repair the injured spiral ganglion neurons in guinea pigs with neomycin-induced sensorineural hearing loss. NI-hADSCs were induced with culture medium containing basic ifbroblast growth factor and forskolin and then injected to the injured cochleae. Guinea pigs that received injection of Hanks’ balanced salt solution into the cochleae were used as controls. Hematoxylin-eosin staining showed that at 8 weeks after cell transplantation, the number of surviving spiral ganglion neurons in the cell transplantation group was significantly increased than that in the control group. Also at 8 weeks after cell transplantation, immunohistochemical staining showed that a greater number of NI-hADSCs in the spiral ganglions were detected in the cell transplantation group than in the control group, and these NI-hADSCs expressed neuronal markers neuroiflament protein and microtubule-associated protein 2. Within 8 weeks after cell transplantation, the guinea pigs in the cell transplantation group had a gradually decreased auditory brainstem response threshold, while those in the control group had almost no response to 80 dB of clicks or pure tone burst. These ifndings suggest that a large amount of NI-hADSCs migrated to the spiral ganglions, survived for a period of time, re-paired the injured spiral ganglion cells, and thereby contributed to the recovery of sensorineural hearing loss in guinea pigs.     

Secretion of nerve growth factor,brain-derived neurotrophic factor,and glial cell-line derived neurotrophic factor in co-culture of four cell types in cerebrospinal fluid-containing medium

The present study co-cultured human embryonic olfactory ensheathing cells, human Schwann cells, human amniotic epithelial cells and human vascular endothelial cells in complete culture medium- containing cerebrospinal fluid. Enzyme linked immunosorbent assay was used to detect nerve growth factor, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor secretion in the supernatant of co-cultured cells. Results showed that the number of all cell types reached a peak at 7-10 days, and the expression of nerve growth factor, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor peaked at 9 days. Levels of secreted nerve growth factor were four-fold higher than brain-derived neurotrophic factor, which was three-fold higher than glial cell line-derived neurotrophic factor. Increasing concentrations of cerebrospinal fluid (10%, 20% and 30%) in the growth medium caused a decrease of neurotrophic factor secretion Results indicated co-culture of human embryonic olfactory ensheathing cells, human Schwann cells human amniotic epithelial cells and human vascular endothelial cells improved the expression of nerve growth factor, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor. The reduction of cerebrospinal fluid extravasation at the transplant site after spinal cord injury is beneficial for the survival and secretion of neurotrophic factors from transplanted cells.