BACKGROUND： Substantia nigra is deep in position and limited in range, the glial cell line-derived neurotrophic factor （GDNF） injection directly into substantia nigra has relatively greater damages with higher diff...BACKGROUND： Substantia nigra is deep in position and limited in range, the glial cell line-derived neurotrophic factor （GDNF） injection directly into substantia nigra has relatively greater damages with higher difficulty. GDNF injection into striatum, the target area of dopaminergic neuron, may protect the dopaminergic neurons in the compact part of substantia nigra through retrograde transport. OBJECTIVE： To investigate the protective effect of intrastriatal GDNF on dopaminergic neurons in the substantia nigra of mice with Parkinson disease （PD）, and analyze the action pathway. DESIGN： A controlled observation. SETTING： Neurobiological Laboratory of Xuzhou Medical College. MATERIALS： Twenty-four male Kunming mice of 7 - 8 weeks old were used. GDNF, 1-methy1-4-pheny1-1,2,3,6-tetrahydropyridine （MPTP） were purchased from Sigma Company （USA）; LEICAQWin image processing and analytical system. METHODS： The experiments were carded out in the Neurobiological Laboratory of Xuzhou Medical College from September 2005 to October 2006. The PD models were established in adult KunMing mice by intraperitoneal injection of MPTP. The model mice were were randomly divided into four groups with 6 mice in each group： GDNF 4-day group, phosphate buffer solution （PSB） 4-day group, GDNF 6-day group and PSB 6-day group. Mice in the GDNF 4 and 6-day groups were administrated with 1 μ L GDNF solution （20 μ g/L, dispensed with 0.01 mol/L PBS） injected into right striatum at 4 and 6 days after model establishment. Mice in the PSB 4 and 6-day groups were administrated with 0.01 mol/L PBS of the same volume to the same injection at corresponding time points. ② On the 12^th day after model establishment, the midbrain tissue section of each mice was divided into 3 areas from rostral to caudal sides. The positive neurons of tyroxine hydroxylase （TH） and calcium binding protein （CB） with obvious nucleolus and clear outline were randomly selected for the measurement, and the number of positive neurons in unit area was counted. MAIN OUTCOME MEASURES： Number of positive neurons of TH and CB in midbrain substantia nigra of mice in each group. RESULTS： All the 24 mice were involved in the analysis of results. The numbers of TH^＋ and CB^＋ neurons in the GDNF 4-day group （54.33±6.92, 46.33±5.54） were obviously more than those in the PBS 4-day group （27.67±5.01, 21.50±5.96, P 〈 0.01）. The numbers of TH^＋ and CB^＋ neurons in the GDNF 6-day group （75.67±5.39, 69.67±8.69） were obviously more than those in the PBS 6-day group （27.17±4.50, 21.33 ±5.72, P 〈 0.01） and those in the GDNF 4-day group （P 〈 0.01 ）. CONCLUSION： Intrastriatal GDNF can protect dopaminergic neurons in substantia nigra of PD mice, and it may be related to the increase of CB expression.展开更多
The efficacy of electroacupuncture in the treatment of peripheral facial paralysis is known, but the specific mechanism has not been clarified. Glial cell-derived neurotrophic factor(GDNF) has been shown to protect ne...The efficacy of electroacupuncture in the treatment of peripheral facial paralysis is known, but the specific mechanism has not been clarified. Glial cell-derived neurotrophic factor(GDNF) has been shown to protect neurons by binding to N-cadherin. Our previous results have shown that electroacupuncture could increase the expression of N-cadherin mRNA in facial neurons and promote facial nerve regeneration. In this study, the potential mechanisms by which electroacupuncture promotes nerve regeneration were elucidated through assessing the effects of electroacupuncture on GDNF and N-cadherin expression in facial motoneurons of rabbits with peripheral facial nerve crush injury. New Zealand rabbits were randomly divided into a normal group(normal control, n = 21), injury group(n = 45) and electroacupuncture group(n = 45). Model rabbits underwent facial nerve crush injury only. Rabbits in the electroacupuncture group received facial nerve injury, and then underwent electroacupuncture at Yifeng(TE17), Jiache(ST6), Sibai(ST2), Dicang(ST4), Yangbai(GB14), Quanliao(SI18), and Hegu(LI4; only acupuncture, no electrical stimulation). The results showed that in behavioral assessments, the total scores of blink reflex, vibrissae movement, and position of apex nasi, were markedly lower in the EA group than those in the injury group. Hematoxylin-eosin staining of the right buccinator muscle of each group showed that the cross-sectional area of buccinator was larger in the electroacupuncture group than in the injury group on days 1, 14 and 21 post-surgery. Toluidine blue staining of the right facial nerve tissue of each group revealed that on day 14 post-surgery, there was less axonal demyelination and fewer inflammatory cells in the electroacupuncture group compared with the injury group. Quantitative real time-polymerase chain reaction showed that compared with the injury group, N-cadherin mRNA levels on days 4, 7, 14 and 21 and GDNF mRNA levels on days 4, 7 and 14 were significantly higher in the electroacupuncture group. Western blot assay displayed that compared with the injury group, the expression of GDNF protein levels on days 7, 14 and 21 were significantly upregulated in the electroacupuncture group. The histology with hematoxylin-eosin staining and Nissl staining of brainstem tissues containing facial neurons in the middle and lower part of the pons exhibited that on day 7 post-surgery, there were significantly fewer apoptotic neurons in the electroacupuncture group than in the injury group. By day 21, there was no significantly difference in the number of neurons between the electroacupuncture and normal groups. Taken together, these results have confirmed that electroacupuncture promotes regeneration of peripheral facial nerve injury in rabbits, inhibits neuronal apoptosis, and reduces peripheral inflammatory response, resulting in the recovery of facial muscle function. This is achieved by up-regulating the expression of GDNF and N-cadherin in central facial neurons.展开更多
To evaluate the effects of glial cell line-derived neurotrophic factor transplantation combined with adipose-derived stem cells-transdifferentiated motoneuron delivery on spinal cord con-tusion injury, we developed ra...To evaluate the effects of glial cell line-derived neurotrophic factor transplantation combined with adipose-derived stem cells-transdifferentiated motoneuron delivery on spinal cord con-tusion injury, we developed rat models of spinal cord contusion injury, 7 days later, injected adipose-derived stem cells-transdifferentiated motoneurons into the epicenter, rostral and caudal regions of the impact site and simultaneously transplanted glial cell line-derived neuro-trophic factor-gelfoam complex into the myelin sheath. Motoneuron-like cell transplantation combined with glial cell line-derived neurotrophic factor delivery reduced cavity formations and increased cell density in the transplantation site. The combined therapy exhibited superior promoting effects on recovery of motor function to transplantation of glial cell line-derived neurotrophic factor, adipose-derived stem cells or motoneurons alone. These ifndings suggest that motoneuron-like cell transplantation combined with glial cell line-derived neurotrophic factor delivery holds a great promise for repair of spinal cord injury.展开更多
Objective:To investigate the interference and expression of human glial cell line-derived neurotrophic factor(hCDNF) and soluble TNF alpha(sTMFRⅠ) receptor genes in neural stem cells and to evaluate the roles of thes...Objective:To investigate the interference and expression of human glial cell line-derived neurotrophic factor(hCDNF) and soluble TNF alpha(sTMFRⅠ) receptor genes in neural stem cells and to evaluate the roles of these proteins in the genetic treatment of spinal cord injury.Methods:Full-length of GDNF cDNA(538 bp) and sTMFRⅠcDNA(504 bp) were inserted into the early 1 region of adenovirus genomic DNA respectively and were immediated by the human cytomegalovirus(gene promoter/enhancer). These adenoviruses were propagated in HEK293 cells via homologous recombination for 7-10 days in vivo,then they were used to infect human neural stem ceils.The infection and expression of gene were tested under immunofluorescence.ELISA and Westem-blot after 48 hours.Results:Almost all the cultured cells showed the nestin immunofluorescence positive staining,which was the characteristics of neural stem cell.A great quantity of EGFP and KFP were observed in neural stem cells,which indicated the expression of GDNF and sTMFRⅠ.After transfection of GDNF and sTMFRⅠgenes,many neural stem cells show GFAP and tubulin immunofluorescence positive staining,which meant that most neural stem cells differentiated into neuron at that condition.Conclusions:The infective efficiency of adenovirus is greatly acceptable to neural stem cell,thus adenovirus provide a useful vector for exogenous GDNF and sTMFRⅠgenes expressing in neural stem cells,which is useful for differentiation of neural stem cell.展开更多
The neurotrophin and glial cell line-derived neurotrophic factor(GDNF) family of growth factors have been extensively studied because of their proven ability to regulate development of the peripheral nervous system.Th...The neurotrophin and glial cell line-derived neurotrophic factor(GDNF) family of growth factors have been extensively studied because of their proven ability to regulate development of the peripheral nervous system.The neurotrophin family,which includes nerve growth factor(NGF),NT-3,NT4/5 and BDNF,is also known for its ability to regulate the function of adult sensory neurons.Until recently,little was known concerning the role of the GNDF-family(that includes GDNF,artemin,neurturin and persephin) in adult sensory neuron function.Here we describe recent data that indicates that the GDNF family can regulate sensory neuron function,that some of its members are elevated in inflammatory pain models and that application of these growth factors produces pain in vivo.Finally we discuss how these two families of growth factors may converge on a single membrane receptor,TRPV1,to produce long-lasting hyperalgesia.展开更多
Glial cell line-derived neurotrophic factor recombinant adenovirus vector-transfected bone marrow mesenchymal stem cells were induced to differentiate into neuron-like cells using inductive medium containing retinoic ...Glial cell line-derived neurotrophic factor recombinant adenovirus vector-transfected bone marrow mesenchymal stem cells were induced to differentiate into neuron-like cells using inductive medium containing retinoic acid and epidermal growth factor. Cell viability, micro- tubule-associated protein 2-positive cell ratio, and the expression levels of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43 protein in the su- pernatant were significantly higher in glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells compared with empty virus plasmid-transfected bone marrow mes- enchymal stem cells. Furthermore, microtubule-associated protein 2, glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein743 mRNA levels in cell pellets were statistically higher in glial cell line-derived neurotrophic factor/bone marrow mesen- chymal stem cells compared with empty virus plasmid-transfected bone marrow mesenchymal stem cells. These results suggest that glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells have a higher rate of induction into neuron-like cells, and this enhanced differentiation into neuron-like cells may be associated with up-regulated expression of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43.展开更多
Glial cell line-derived neurotrophic factor(GDNF) plays a critical role in neuronal survival and function. GDNF has two major splice variants in the brain,α-pro-GDNF and β-pro-GDNF, and both isoforms have strong neu...Glial cell line-derived neurotrophic factor(GDNF) plays a critical role in neuronal survival and function. GDNF has two major splice variants in the brain,α-pro-GDNF and β-pro-GDNF, and both isoforms have strong neuroprotective effects on dopamine neurons. However, the expression of the GDNF splice variants in dopaminergic neurons in the brain remains unclear. Therefore, in this study, we investigated the mRNA and protein expression of α-and β-pro-GDNF in the mouse brain by real-time quantitative polymerase chain reaction, using splice variant-specific primers, and western blot analysis. At the mRNA level,β-pro-GDNF expression was significantly greater than that of α-pro-GDNF in the mouse brain. In contrast, at the protein level,α-pro-GDNF expression was markedly greater than that of β-pro-GDNF. To clarify the mechanism underlying this inverse relationship in mRNA and protein expression levels of the GDNF splice variants, we analyzed the expression of sorting protein-related receptor with A-type repeats(SorLA) by real-time quantitative polymerase chain reaction. At the mRNA level, SorLA was positively associated with β-pro-GDNF expression, but not with α-pro-GDNF expression. This suggests that the differential expression of α-and β-pro-GDNF in the mouse brain is related to SorLA expression. As a sorting protein, SorLA could contribute to the inverse relationship among the mRNA and protein levels of the GDNF isoforms. This study was approved by the Animal Ethics Committee of Xuzhou Medical University, China on July 14, 2016.展开更多
The present study co-cultured human embryonic olfactory ensheathJng cells, human Schwann cells, human amniotic epithelial cells and human vascular endothelial cells in complete culture medium- containing cerebrospinal...The present study co-cultured human embryonic olfactory ensheathJng 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.展开更多
Several studies have suggested that exogenous glial cell line-derived neurotrophic factor may pro-tect neurons from cerebral ischemic injury. However, the mechanisms underlying the neuroprotec-tive effects of endogeno...Several studies have suggested that exogenous glial cell line-derived neurotrophic factor may pro-tect neurons from cerebral ischemic injury. However, the mechanisms underlying the neuroprotec-tive effects of endogenous glial cell line-derived neurotrophic factor remain unclear. The present experiments sought to elucidate the influence of various conditioned media on neuronal apoptosis, using a normal culture medium for astrocytes, an astrocyte medium highly expressing glial cell line-derived neurotrophic factor, and an astrocyte medium in which glial cell line-derived neurotro-phic factor expression was silenced using RNAi technology. The results confirmed that the use of RNAi silencing to target pretreated glial cell line-derived neurotrophic factor expression promoted neuronal apoptosis. In addition, oxygen and glucose deprivation preconditioning was found to upregulate glial cell line-derived neurotrophic factor expression, and significantly reduce neuronal apoptosis.展开更多
Pitx3 is strongly associated with the phenotype, differentiation, and survival of dopaminergic neurons. The relationship between Pitx3 and glial cell line-derived neurotrophic factor（GDNF） in dopaminergic neurons re...Pitx3 is strongly associated with the phenotype, differentiation, and survival of dopaminergic neurons. The relationship between Pitx3 and glial cell line-derived neurotrophic factor（GDNF） in dopaminergic neurons remains poorly understood. The present investigation sought to construct and screen a lentivirus expression plasmid carrying a rat Pitx3 short hairpin（sh）RNA and to assess the impact of Pitx3 gene knockdown on GDNF transcriptional activity in MES23.5 dopaminergic neurons. Three pairs of interference sequences were designed and separately ligated into GV102 expression vectors. These recombinant plasmids were transfected into MES23.5 cells and western blot assays were performed to detect Pitx3 protein expression. Finally, the most effective Pitx3 sh RNA and a dual-luciferase reporter gene plasmid carrying the GDNF promoter region（GDNF-luciferase） were cotransfected into MES23.5 cells. Sequencing showed that the synthesized sequences were identical to the three Pitx3 interference sequences. Inverted fluorescence microscopy revealed that the lentivirus expression plasmids carrying Pitx3-sh RNA had 40-50% transfection efficiency. Western blot assay confirmed that the corresponding Pitx3 of the third knockdown sequence had the lowest expression level. Dual-luciferase reporter gene results showed that the GDNF transcriptional activity in dopaminergic cells cotransfected with both plasmids was decreased compared with those transfected with GDNF-luciferase alone. Together, the results showed that the designed Pitx3-sh RNA interference sequence decreased Pitx3 protein expression, which decreased GDNF transcriptional activity.展开更多
The purpose of this study was to evaluate the effect of poly（lactide-co-glycolic acid） delayed-release microspheres,which were prepared using glial cell line-derived neurotrophic factor（GDNF）,on the delayed-releas...The purpose of this study was to evaluate the effect of poly（lactide-co-glycolic acid） delayed-release microspheres,which were prepared using glial cell line-derived neurotrophic factor（GDNF）,on the delayed-release,controllability,and protection of GDNF activity.The present study is the first to combine chondroitinase ABC,GDNF,and Nogo A antibody delayed-release microspheres for the treatment of spinal cord injury.Results show that the combined therapy of chondroitinase ABC,GDNF,and Nogo A antibody microspheres can increase the immunoreaction of neurofilament 200 in the injured spinal cord,and this therapeutic effect was better than chondroitinase ABC,GDNF,or Nogo A antibody microspheres administered singularly.展开更多
We have been previously reported that disuse muscle atrophy exacerbates both motor neuron (MN) degeneration in caudal regions remote from a spinal cord injury, and decrease in glial cell line-derived neurotrophic fact...We have been previously reported that disuse muscle atrophy exacerbates both motor neuron (MN) degeneration in caudal regions remote from a spinal cord injury, and decrease in glial cell line-derived neurotrophic factor (GDNF) protein level in paralyzed muscle. In this study we found that disuse muscle atrophy exacerbated the decrease in GDNF protein level in the L4/5 spinal cord, which was not immunopositive for GDNF. Our results were consistent with the fact that in the lumbar spinal cord of rats with mid-thoracic contusion, GDNF expression was not detected, while expression of GDNF receptors (GFRα1 and RET) was. Our study showed that administration of exogenous recombinant GDNF into the atrophic muscle partially rescued α-MN degeneration in the L4/5 spinal cord. These results suggest that the depletion of GDNF protein by muscle atrophy exacerbates α-MN degeneration in caudal regions remote from the injury.展开更多
Brain-derived neurotrophic factor（BDNF） plays an important role in the repair of central nervous system injury,but cannot directly traverse the blood-brain barrier.Liposomes are a new type of non-viral vector,able t...Brain-derived neurotrophic factor（BDNF） plays an important role in the repair of central nervous system injury,but cannot directly traverse the blood-brain barrier.Liposomes are a new type of non-viral vector,able to carry macromolecules across the blood-brain barrier and into the brain.Here,we investigate whether BDNF could be transported across the blood-brain barrier by tail-vein injection of liposomes conjugated to transferrin（Tf） and polyethylene glycol（PEG）,and carrying BDNF modified with cytomegalovirus promoter（pC MV） or glial fibrillary acidic protein promoter（p GFAP）（Tf-p CMV-BDNF-PEG and Tf-p GFAP-BDNF-PEG,respectively）.Both liposomes were able to traverse the blood-brain barrier,and BDNF was mainly expressed in the cerebral cortex.BDNF expression in the cerebral cortex was higher in the Tf-p GFAP-BDNF-PEG group than in the Tf-p CMV-BDNF-PEG group.This study demonstrates the successful construction of a non-virus targeted liposome,Tf-p GFAP-BDNF-PEG,which crosses the blood-brain barrier and is distributed in the cerebral cortex.Our work provides an experimental basis for BDNF-related targeted drug delivery in the brain.展开更多
Acupuncture is widely used in the treatment of cerebral hemorrhage,and it improves outcomes in experimental animal models and patients.However,the mechanisms underlying the effectiveness of acupuncture treatment for c...Acupuncture is widely used in the treatment of cerebral hemorrhage,and it improves outcomes in experimental animal models and patients.However,the mechanisms underlying the effectiveness of acupuncture treatment for cerebral hemorrhage are still unclear.In this study,a model of intracerebral hemorrhage was produced by injecting 50μL autologous blood into the caudate nucleus in Wistar rats.Acupuncture at Baihui(DU20)and Qubin(GB7)acupoints was performed at a depth of 1.0 inch,12 hours after blood injection,once every 24 hours.The needle was rotated at 200 r/min for 5 minutes,For each 30-minute session,needling at 200 r/min was performed for three sessions,each lasting 5 minutes.For the positive control group,at 6 hours,and 1,2,3 and 7 days after induction of hemorrhage,the rats were intraperitoneally injected with 1 mL aniracetam(0.75 mg/mL),three times a day.The Bederson behavioral test was used to assess palsy in the contralateral limbs.Western blot assay was used to examine the expression levels of Nestin and basic fibroblast growth factor in the basal ganglia.Immunohistochemistry was performed to count the number of Nestin-and glial cell line-derived neurotrophic factor-positive cells in the basal ganglia.Acupuncture effectively reduced hemorrhage and brain edema,elevated the expression levels of Nestin and basic fibroblast growth factor in the basal ganglia,and increased the number of Nestin-and glial cell line-derived neurotrophic factor-positive cells in the basal ganglia.Together,these findings suggest that acupuncture promotes functional recovery after cerebral hemorrhage by increasing the expression of neurotrophic factors.The study was approved by the Committee for Experimental Animals of Heilongjiang Medical Laboratory Animal Center(approval No.2017061001)on June 10,2017.展开更多
Neuroprotection and neuroregeneration are two of the most promising disease-modifying ther- apies for the incurable and widespread Parkinson＇s disease. In Parkinson＇s disease, progressive degeneration of nigrostriat...Neuroprotection and neuroregeneration are two of the most promising disease-modifying ther- apies for the incurable and widespread Parkinson＇s disease. In Parkinson＇s disease, progressive degeneration of nigrostriatal dopaminergic neurons causes debilitating motor symptoms. Neurotrophic factors play important regulatory roles in the development, survival and maintenance of specific neuronal populations. These factors have the potential to slow down, halt or reverse the loss of nigrostriatal dopaminergic neurons in Parkinsoffs disease. Several neurotrophic fac- tors have been investigated in this regard. This review article discusses the neurodevelopmental roles and therapeutic potential of three dopaminergic neurotrophic factors： glial cell line-derived neurotrophic factor, neurturin and growth/differentiation factor 5.展开更多
Acupuncture for the treatment of Parkinson＇s disease has a precise clinical outcome. This study investigated the effect of electroacupuncture at Fengfu （GV16） and Taichong （LR3） acupoints in rat models of Parkin...Acupuncture for the treatment of Parkinson＇s disease has a precise clinical outcome. This study investigated the effect of electroacupuncture at Fengfu （GV16） and Taichong （LR3） acupoints in rat models of Parkinson＇s disease induced by subcutaneous injection of rotenone into rat neck and back. Reverse transcription-PCR demonstrated that brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor mRNA expression was significantly increased in the substantia nigra of rat models of Parkinson＇s disease, and that abnormal behavior of rats was significantly improved following electroacupuncture treatment. These results indicated that electroacupuncture treatment upregulated brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor mRNA expression in the substantia nigra of rat models of Parkinson＇s disease. Thus, electroacupuncture may be useful in the treatment of Parkinson＇s disease.展开更多
基金the Natural Science Foundation of Jiangsu Department of Education, No. 02KJB310009
文摘BACKGROUND： Substantia nigra is deep in position and limited in range, the glial cell line-derived neurotrophic factor （GDNF） injection directly into substantia nigra has relatively greater damages with higher difficulty. GDNF injection into striatum, the target area of dopaminergic neuron, may protect the dopaminergic neurons in the compact part of substantia nigra through retrograde transport. OBJECTIVE： To investigate the protective effect of intrastriatal GDNF on dopaminergic neurons in the substantia nigra of mice with Parkinson disease （PD）, and analyze the action pathway. DESIGN： A controlled observation. SETTING： Neurobiological Laboratory of Xuzhou Medical College. MATERIALS： Twenty-four male Kunming mice of 7 - 8 weeks old were used. GDNF, 1-methy1-4-pheny1-1,2,3,6-tetrahydropyridine （MPTP） were purchased from Sigma Company （USA）; LEICAQWin image processing and analytical system. METHODS： The experiments were carded out in the Neurobiological Laboratory of Xuzhou Medical College from September 2005 to October 2006. The PD models were established in adult KunMing mice by intraperitoneal injection of MPTP. The model mice were were randomly divided into four groups with 6 mice in each group： GDNF 4-day group, phosphate buffer solution （PSB） 4-day group, GDNF 6-day group and PSB 6-day group. Mice in the GDNF 4 and 6-day groups were administrated with 1 μ L GDNF solution （20 μ g/L, dispensed with 0.01 mol/L PBS） injected into right striatum at 4 and 6 days after model establishment. Mice in the PSB 4 and 6-day groups were administrated with 0.01 mol/L PBS of the same volume to the same injection at corresponding time points. ② On the 12^th day after model establishment, the midbrain tissue section of each mice was divided into 3 areas from rostral to caudal sides. The positive neurons of tyroxine hydroxylase （TH） and calcium binding protein （CB） with obvious nucleolus and clear outline were randomly selected for the measurement, and the number of positive neurons in unit area was counted. MAIN OUTCOME MEASURES： Number of positive neurons of TH and CB in midbrain substantia nigra of mice in each group. RESULTS： All the 24 mice were involved in the analysis of results. The numbers of TH^＋ and CB^＋ neurons in the GDNF 4-day group （54.33±6.92, 46.33±5.54） were obviously more than those in the PBS 4-day group （27.67±5.01, 21.50±5.96, P 〈 0.01）. The numbers of TH^＋ and CB^＋ neurons in the GDNF 6-day group （75.67±5.39, 69.67±8.69） were obviously more than those in the PBS 6-day group （27.17±4.50, 21.33 ±5.72, P 〈 0.01） and those in the GDNF 4-day group （P 〈 0.01 ）. CONCLUSION： Intrastriatal GDNF can protect dopaminergic neurons in substantia nigra of PD mice, and it may be related to the increase of CB expression.
文摘The efficacy of electroacupuncture in the treatment of peripheral facial paralysis is known, but the specific mechanism has not been clarified. Glial cell-derived neurotrophic factor(GDNF) has been shown to protect neurons by binding to N-cadherin. Our previous results have shown that electroacupuncture could increase the expression of N-cadherin mRNA in facial neurons and promote facial nerve regeneration. In this study, the potential mechanisms by which electroacupuncture promotes nerve regeneration were elucidated through assessing the effects of electroacupuncture on GDNF and N-cadherin expression in facial motoneurons of rabbits with peripheral facial nerve crush injury. New Zealand rabbits were randomly divided into a normal group(normal control, n = 21), injury group(n = 45) and electroacupuncture group(n = 45). Model rabbits underwent facial nerve crush injury only. Rabbits in the electroacupuncture group received facial nerve injury, and then underwent electroacupuncture at Yifeng(TE17), Jiache(ST6), Sibai(ST2), Dicang(ST4), Yangbai(GB14), Quanliao(SI18), and Hegu(LI4; only acupuncture, no electrical stimulation). The results showed that in behavioral assessments, the total scores of blink reflex, vibrissae movement, and position of apex nasi, were markedly lower in the EA group than those in the injury group. Hematoxylin-eosin staining of the right buccinator muscle of each group showed that the cross-sectional area of buccinator was larger in the electroacupuncture group than in the injury group on days 1, 14 and 21 post-surgery. Toluidine blue staining of the right facial nerve tissue of each group revealed that on day 14 post-surgery, there was less axonal demyelination and fewer inflammatory cells in the electroacupuncture group compared with the injury group. Quantitative real time-polymerase chain reaction showed that compared with the injury group, N-cadherin mRNA levels on days 4, 7, 14 and 21 and GDNF mRNA levels on days 4, 7 and 14 were significantly higher in the electroacupuncture group. Western blot assay displayed that compared with the injury group, the expression of GDNF protein levels on days 7, 14 and 21 were significantly upregulated in the electroacupuncture group. The histology with hematoxylin-eosin staining and Nissl staining of brainstem tissues containing facial neurons in the middle and lower part of the pons exhibited that on day 7 post-surgery, there were significantly fewer apoptotic neurons in the electroacupuncture group than in the injury group. By day 21, there was no significantly difference in the number of neurons between the electroacupuncture and normal groups. Taken together, these results have confirmed that electroacupuncture promotes regeneration of peripheral facial nerve injury in rabbits, inhibits neuronal apoptosis, and reduces peripheral inflammatory response, resulting in the recovery of facial muscle function. This is achieved by up-regulating the expression of GDNF and N-cadherin in central facial neurons.
基金funded by Shefa Neurosciences Research Center at Khatam Al-Anbia Hospital,Tehran,Iran(Grant#86-N-105)
文摘To evaluate the effects of glial cell line-derived neurotrophic factor transplantation combined with adipose-derived stem cells-transdifferentiated motoneuron delivery on spinal cord con-tusion injury, we developed rat models of spinal cord contusion injury, 7 days later, injected adipose-derived stem cells-transdifferentiated motoneurons into the epicenter, rostral and caudal regions of the impact site and simultaneously transplanted glial cell line-derived neuro-trophic factor-gelfoam complex into the myelin sheath. Motoneuron-like cell transplantation combined with glial cell line-derived neurotrophic factor delivery reduced cavity formations and increased cell density in the transplantation site. The combined therapy exhibited superior promoting effects on recovery of motor function to transplantation of glial cell line-derived neurotrophic factor, adipose-derived stem cells or motoneurons alone. These ifndings suggest that motoneuron-like cell transplantation combined with glial cell line-derived neurotrophic factor delivery holds a great promise for repair of spinal cord injury.
基金Shenzhen Science and Technology Project(No.201103061)
文摘Objective:To investigate the interference and expression of human glial cell line-derived neurotrophic factor(hCDNF) and soluble TNF alpha(sTMFRⅠ) receptor genes in neural stem cells and to evaluate the roles of these proteins in the genetic treatment of spinal cord injury.Methods:Full-length of GDNF cDNA(538 bp) and sTMFRⅠcDNA(504 bp) were inserted into the early 1 region of adenovirus genomic DNA respectively and were immediated by the human cytomegalovirus(gene promoter/enhancer). These adenoviruses were propagated in HEK293 cells via homologous recombination for 7-10 days in vivo,then they were used to infect human neural stem ceils.The infection and expression of gene were tested under immunofluorescence.ELISA and Westem-blot after 48 hours.Results:Almost all the cultured cells showed the nestin immunofluorescence positive staining,which was the characteristics of neural stem cell.A great quantity of EGFP and KFP were observed in neural stem cells,which indicated the expression of GDNF and sTMFRⅠ.After transfection of GDNF and sTMFRⅠgenes,many neural stem cells show GFAP and tubulin immunofluorescence positive staining,which meant that most neural stem cells differentiated into neuron at that condition.Conclusions:The infective efficiency of adenovirus is greatly acceptable to neural stem cell,thus adenovirus provide a useful vector for exogenous GDNF and sTMFRⅠgenes expressing in neural stem cells,which is useful for differentiation of neural stem cell.
文摘The neurotrophin and glial cell line-derived neurotrophic factor(GDNF) family of growth factors have been extensively studied because of their proven ability to regulate development of the peripheral nervous system.The neurotrophin family,which includes nerve growth factor(NGF),NT-3,NT4/5 and BDNF,is also known for its ability to regulate the function of adult sensory neurons.Until recently,little was known concerning the role of the GNDF-family(that includes GDNF,artemin,neurturin and persephin) in adult sensory neuron function.Here we describe recent data that indicates that the GDNF family can regulate sensory neuron function,that some of its members are elevated in inflammatory pain models and that application of these growth factors produces pain in vivo.Finally we discuss how these two families of growth factors may converge on a single membrane receptor,TRPV1,to produce long-lasting hyperalgesia.
文摘Glial cell line-derived neurotrophic factor recombinant adenovirus vector-transfected bone marrow mesenchymal stem cells were induced to differentiate into neuron-like cells using inductive medium containing retinoic acid and epidermal growth factor. Cell viability, micro- tubule-associated protein 2-positive cell ratio, and the expression levels of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43 protein in the su- pernatant were significantly higher in glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells compared with empty virus plasmid-transfected bone marrow mes- enchymal stem cells. Furthermore, microtubule-associated protein 2, glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein743 mRNA levels in cell pellets were statistically higher in glial cell line-derived neurotrophic factor/bone marrow mesen- chymal stem cells compared with empty virus plasmid-transfected bone marrow mesenchymal stem cells. These results suggest that glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells have a higher rate of induction into neuron-like cells, and this enhanced differentiation into neuron-like cells may be associated with up-regulated expression of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43.
基金supported by the National Natural Science Foundation of China,No.81772688(to DSG)the Postdoctoral Science Foundation of Jiangsu Province of China,No.1202119C(to HL)
文摘Glial cell line-derived neurotrophic factor(GDNF) plays a critical role in neuronal survival and function. GDNF has two major splice variants in the brain,α-pro-GDNF and β-pro-GDNF, and both isoforms have strong neuroprotective effects on dopamine neurons. However, the expression of the GDNF splice variants in dopaminergic neurons in the brain remains unclear. Therefore, in this study, we investigated the mRNA and protein expression of α-and β-pro-GDNF in the mouse brain by real-time quantitative polymerase chain reaction, using splice variant-specific primers, and western blot analysis. At the mRNA level,β-pro-GDNF expression was significantly greater than that of α-pro-GDNF in the mouse brain. In contrast, at the protein level,α-pro-GDNF expression was markedly greater than that of β-pro-GDNF. To clarify the mechanism underlying this inverse relationship in mRNA and protein expression levels of the GDNF splice variants, we analyzed the expression of sorting protein-related receptor with A-type repeats(SorLA) by real-time quantitative polymerase chain reaction. At the mRNA level, SorLA was positively associated with β-pro-GDNF expression, but not with α-pro-GDNF expression. This suggests that the differential expression of α-and β-pro-GDNF in the mouse brain is related to SorLA expression. As a sorting protein, SorLA could contribute to the inverse relationship among the mRNA and protein levels of the GDNF isoforms. This study was approved by the Animal Ethics Committee of Xuzhou Medical University, China on July 14, 2016.
基金supported by the Science andTechnology Development Program of Guangdong Province, No.2009b030801329
文摘The present study co-cultured human embryonic olfactory ensheathJng 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.
基金Specialized Research Fund for the Doc-toral Program of Higher Education, No. 20060183053
文摘Several studies have suggested that exogenous glial cell line-derived neurotrophic factor may pro-tect neurons from cerebral ischemic injury. However, the mechanisms underlying the neuroprotec-tive effects of endogenous glial cell line-derived neurotrophic factor remain unclear. The present experiments sought to elucidate the influence of various conditioned media on neuronal apoptosis, using a normal culture medium for astrocytes, an astrocyte medium highly expressing glial cell line-derived neurotrophic factor, and an astrocyte medium in which glial cell line-derived neurotro-phic factor expression was silenced using RNAi technology. The results confirmed that the use of RNAi silencing to target pretreated glial cell line-derived neurotrophic factor expression promoted neuronal apoptosis. In addition, oxygen and glucose deprivation preconditioning was found to upregulate glial cell line-derived neurotrophic factor expression, and significantly reduce neuronal apoptosis.
基金supported by the National Natural Science Foundation of China,No.81372698
文摘Pitx3 is strongly associated with the phenotype, differentiation, and survival of dopaminergic neurons. The relationship between Pitx3 and glial cell line-derived neurotrophic factor（GDNF） in dopaminergic neurons remains poorly understood. The present investigation sought to construct and screen a lentivirus expression plasmid carrying a rat Pitx3 short hairpin（sh）RNA and to assess the impact of Pitx3 gene knockdown on GDNF transcriptional activity in MES23.5 dopaminergic neurons. Three pairs of interference sequences were designed and separately ligated into GV102 expression vectors. These recombinant plasmids were transfected into MES23.5 cells and western blot assays were performed to detect Pitx3 protein expression. Finally, the most effective Pitx3 sh RNA and a dual-luciferase reporter gene plasmid carrying the GDNF promoter region（GDNF-luciferase） were cotransfected into MES23.5 cells. Sequencing showed that the synthesized sequences were identical to the three Pitx3 interference sequences. Inverted fluorescence microscopy revealed that the lentivirus expression plasmids carrying Pitx3-sh RNA had 40-50% transfection efficiency. Western blot assay confirmed that the corresponding Pitx3 of the third knockdown sequence had the lowest expression level. Dual-luciferase reporter gene results showed that the GDNF transcriptional activity in dopaminergic cells cotransfected with both plasmids was decreased compared with those transfected with GDNF-luciferase alone. Together, the results showed that the designed Pitx3-sh RNA interference sequence decreased Pitx3 protein expression, which decreased GDNF transcriptional activity.
基金the National Natural Science Foundation of China, No. 30471759
文摘The purpose of this study was to evaluate the effect of poly（lactide-co-glycolic acid） delayed-release microspheres,which were prepared using glial cell line-derived neurotrophic factor（GDNF）,on the delayed-release,controllability,and protection of GDNF activity.The present study is the first to combine chondroitinase ABC,GDNF,and Nogo A antibody delayed-release microspheres for the treatment of spinal cord injury.Results show that the combined therapy of chondroitinase ABC,GDNF,and Nogo A antibody microspheres can increase the immunoreaction of neurofilament 200 in the injured spinal cord,and this therapeutic effect was better than chondroitinase ABC,GDNF,or Nogo A antibody microspheres administered singularly.
文摘We have been previously reported that disuse muscle atrophy exacerbates both motor neuron (MN) degeneration in caudal regions remote from a spinal cord injury, and decrease in glial cell line-derived neurotrophic factor (GDNF) protein level in paralyzed muscle. In this study we found that disuse muscle atrophy exacerbated the decrease in GDNF protein level in the L4/5 spinal cord, which was not immunopositive for GDNF. Our results were consistent with the fact that in the lumbar spinal cord of rats with mid-thoracic contusion, GDNF expression was not detected, while expression of GDNF receptors (GFRα1 and RET) was. Our study showed that administration of exogenous recombinant GDNF into the atrophic muscle partially rescued α-MN degeneration in the L4/5 spinal cord. These results suggest that the depletion of GDNF protein by muscle atrophy exacerbates α-MN degeneration in caudal regions remote from the injury.
基金funded by a grant from Jilin Province Development and Reform Commission of China,No.JF2012C008-3Jilin Province Industrial Innovation Special Fund Project of China,No.JF2016C050-2the Joint Project between Jilin University and Jilin You-bang Pharmaceutical Co.Ltd.,No.2015YX323
文摘Brain-derived neurotrophic factor（BDNF） plays an important role in the repair of central nervous system injury,but cannot directly traverse the blood-brain barrier.Liposomes are a new type of non-viral vector,able to carry macromolecules across the blood-brain barrier and into the brain.Here,we investigate whether BDNF could be transported across the blood-brain barrier by tail-vein injection of liposomes conjugated to transferrin（Tf） and polyethylene glycol（PEG）,and carrying BDNF modified with cytomegalovirus promoter（pC MV） or glial fibrillary acidic protein promoter（p GFAP）（Tf-p CMV-BDNF-PEG and Tf-p GFAP-BDNF-PEG,respectively）.Both liposomes were able to traverse the blood-brain barrier,and BDNF was mainly expressed in the cerebral cortex.BDNF expression in the cerebral cortex was higher in the Tf-p GFAP-BDNF-PEG group than in the Tf-p CMV-BDNF-PEG group.This study demonstrates the successful construction of a non-virus targeted liposome,Tf-p GFAP-BDNF-PEG,which crosses the blood-brain barrier and is distributed in the cerebral cortex.Our work provides an experimental basis for BDNF-related targeted drug delivery in the brain.
基金supported by the National Natural Science Foundation of China,Nos.81473764,81273824,30772840(to WZ)the Doctoral Fund of Ministry of Education of China,No.20102327110003(to WZ)+4 种基金the Natural Science Foundation of Heilongjiang Province of China,No.ZD201204(to WZ)the Special Fund for Technological Innovation Research of Harbin of China,No.2012RFXXS062(to WZ)the Doctoral Innovation Fund of Heilongjiang University of Chinese Medicine of China,No.2015bs03(to QXC)the Chunhui Plans Research Cooperation Project of China,No.Z2007-1-15010(to WZ)the University Nursing Program for Young Scholars with Creative Talents in Heilongjiang Province of China,No.UNPYSCT-2018234(to QXC)
文摘Acupuncture is widely used in the treatment of cerebral hemorrhage,and it improves outcomes in experimental animal models and patients.However,the mechanisms underlying the effectiveness of acupuncture treatment for cerebral hemorrhage are still unclear.In this study,a model of intracerebral hemorrhage was produced by injecting 50μL autologous blood into the caudate nucleus in Wistar rats.Acupuncture at Baihui(DU20)and Qubin(GB7)acupoints was performed at a depth of 1.0 inch,12 hours after blood injection,once every 24 hours.The needle was rotated at 200 r/min for 5 minutes,For each 30-minute session,needling at 200 r/min was performed for three sessions,each lasting 5 minutes.For the positive control group,at 6 hours,and 1,2,3 and 7 days after induction of hemorrhage,the rats were intraperitoneally injected with 1 mL aniracetam(0.75 mg/mL),three times a day.The Bederson behavioral test was used to assess palsy in the contralateral limbs.Western blot assay was used to examine the expression levels of Nestin and basic fibroblast growth factor in the basal ganglia.Immunohistochemistry was performed to count the number of Nestin-and glial cell line-derived neurotrophic factor-positive cells in the basal ganglia.Acupuncture effectively reduced hemorrhage and brain edema,elevated the expression levels of Nestin and basic fibroblast growth factor in the basal ganglia,and increased the number of Nestin-and glial cell line-derived neurotrophic factor-positive cells in the basal ganglia.Together,these findings suggest that acupuncture promotes functional recovery after cerebral hemorrhage by increasing the expression of neurotrophic factors.The study was approved by the Committee for Experimental Animals of Heilongjiang Medical Laboratory Animal Center(approval No.2017061001)on June 10,2017.
基金supported by grants from the Irish Research Council(R13702 and R15897SVH/AS/G’OK)+3 种基金the Health Research Board of Ireland(HRA/2009/127GO’K/AS)Science Foundation Ireland(10/RFP/NES2786GO’K)
文摘Neuroprotection and neuroregeneration are two of the most promising disease-modifying ther- apies for the incurable and widespread Parkinson＇s disease. In Parkinson＇s disease, progressive degeneration of nigrostriatal dopaminergic neurons causes debilitating motor symptoms. Neurotrophic factors play important regulatory roles in the development, survival and maintenance of specific neuronal populations. These factors have the potential to slow down, halt or reverse the loss of nigrostriatal dopaminergic neurons in Parkinsoffs disease. Several neurotrophic fac- tors have been investigated in this regard. This review article discusses the neurodevelopmental roles and therapeutic potential of three dopaminergic neurotrophic factors： glial cell line-derived neurotrophic factor, neurturin and growth/differentiation factor 5.
基金supported by the National Natural Science Foundation of China,No.30973787,30973809the Open Research Fund of Zhejiang First-foremost Key Subject-Acupuncture & Moxibustion,No.ZTK2010A10+1 种基金the Natural Science Foundation of Hubei Province,No.2009CDA068the Integrated Traditional and Western Medicine project by the Health Department of Hubei Province,No.2010Z-Z01
文摘Acupuncture for the treatment of Parkinson＇s disease has a precise clinical outcome. This study investigated the effect of electroacupuncture at Fengfu （GV16） and Taichong （LR3） acupoints in rat models of Parkinson＇s disease induced by subcutaneous injection of rotenone into rat neck and back. Reverse transcription-PCR demonstrated that brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor mRNA expression was significantly increased in the substantia nigra of rat models of Parkinson＇s disease, and that abnormal behavior of rats was significantly improved following electroacupuncture treatment. These results indicated that electroacupuncture treatment upregulated brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor mRNA expression in the substantia nigra of rat models of Parkinson＇s disease. Thus, electroacupuncture may be useful in the treatment of Parkinson＇s disease.
