Autophagy protects neurons against facial nerve injury in rats / 中国组织工程研究

BACKGROUND:After peripheral facial nerve injury,glial cell-derived neurotrophic factor(GDNF)can play a protective role in facial neurons.It has been found that GDNF can regulate the level of autophagy through mammalian target of rapamycin(mTOR),but it is unclear whether it can regulate facial neurons through the adenylate-activated protein kinase/Unc-51-like kinase 1(AMPK/ULK1)signaling pathway after facial nerve injury. OBJECTIVE:To establish a facial nerve injury model in Sprague-Dawley rats and explore the role of autophagy in facial nerve regeneration and the mechanism by which the GDNF/AMPK/ULK1 signaling pathway promotes facial nerve repair after injury. METHODS:Seventy-two Sprague-Dawley rats were randomly divided into sham group,model group and autophagy inhibitor 3-methyladenine(3-MA)group,with 24 rats in each group.Only the main trunk of the facial nerve was exposed in the sham group,while the remaining two groups were modeled for the compression injury of the facial nerve trunk.After successful modeling,the model group was given intraperitoneal injection of normal saline(15 mg/kg),and the 3-MA group was given intraperitoneal injection of 3-MA(15 mg/kg),both once daily for 7 days.The rats in each group were scored on the Simone 10-point behavioral scale at 1,4,7,14,21 and 28 days after surgery.Nissl staining was performed to observe the morphology and number of facial neuron cells at 7,14,21,and 28 days.The expression levels of p-AMPK,p-ULK1,Beclin1 and GDNF in the facial neuron tissues of rats were detected by western blot assay. RESULTS AND CONCLUSION:Behavioral scoring showed that the improvement of facial paralysis symptoms in the 3-MA group was worse and later than that in the model group(P<0.05).Nissl staining showed that the morphology and number of Nissl bodies in facial neurons in the 3-MA group recovered poorly and the number was less than that in the model group(P<0.05).Western blot detection results showed that the expression of p-AMPK and Beclin1 in the model group was higher than that in the 3-MA group and the sham group(P<0.05).The protein expression of p-ULK1 in the model group was lower than that in the 3-MA group and the sham group(P<0.05).To conclude,autophagy inhibitor delays nerve repair after facial nerve injury,which may be related to down-regulation of GDNF expression,inactivation of AMPK,and phosphorylation of ULK1,thereby inhibiting neuronal autophagy levels.

Correlation between serum GDNF level and neuroimaging changes and cognitive impairment in patients with cerebral small vessel disease / 中华行为医学与脑科学杂志

Objective:To investigate the relationship between serum glial cell line-derived neurotrophic factor (GDNF) levels and neuroimaging changes and cognitive impairment in patients with cerebral small vascular disease (CSVD).Methods:135 patients with CSVD recruited from the Department of Neurology of the First Affiliated Hospital of Xinxiang Medical University from September 2021 to July 2022 were assessed by cranial multimodal magnetic resonance imaging and Montreal cognitive function assessment (MoCA), and the basic data were analyzed at the same time.The serum GDNF concentration of all patients was detected by enzyme-linked immunosorbent assay (ELISA). According to the median GDNF concentration, the patients were divided into low GDNF group and high GDNF group. The baseline data, MoCA score and imaging markers of the two groups were compared by Mann-Whitney U test, chi-square test, logistic regression, Kruskal-Wallis H test and Jonckheere-Terpstra trend test, and the correlation between serum GDNF level and imaging markers and cognitive function of patients with CSVD was analyzed. Results:The median serum GDNF concentration of all CSVD patients was 16.66 pg/mL. Multivariate logistic regression analysis showed that low serum GDNF level was a risk factor for white matter hyperintensity and total image load in patients with CSVD. Serum GDNF level was a protective factor of cognitive impairment in patients with CSVD in multiple logistic regression analysis. The area under the curve of ROC curve analysis of cognitive impairment after CSVD predicted by serum GDNF level was 0.735, the sensitivity was 66.4%, and the specificity was 71.4%. The level of serum GDNF was positively related with visual space and executive function, attention and computational power, delayed recall and orientation( r=0.267, 0.187, 0.219, 0.215, all P<0.05). Conclusion:The serum GDNF level is related to white matter hyperintensities, total imaging load and cognitive impairment in patients with CSVD. Serum GDNF level may play a predictive role in CSVD and cognitive impairment.

Serum NOS3 and GDNF levels in patients with arteriosclerotic cerebral infarction and their clinical significance / 中风与神经疾病杂志

@#Objective　To investigate the expression differences of serum endothelial nitric oxide synthase (NOS3) and glial cell-derived neurotrophic factor (GDNF) in patients with arteriosclerotic cerebral infarction and their role in the prognosis of patients.Methods　A total of 126 patients with atherosclerotic cerebral infarction admitted to our hospital from January 2021 to January 2022 and 130 healthy people in the same period were selected.Patients were divided into groups according to infarct volume,dysfunction and prognosis,and serum NOS3 and GDNF levels were compared between different groups.ROC curve was used to analyze the predictive value of NOS3 and GDNF for poor prognosis.Results　The proportion of diabetes and hypertension in patients with atherosclerotic cerebral infarction increased,and the serum levels of NOS3 and GDNF decreased (P<0.05).There was no significant difference in the levels of NOS3 and GDNF among patients with different infarct volumes (P>0.05).The levels of NOS3 and GDNF in mild dysfunction group,moderate dysfunction group and severe dysfunction group decreased successively (P<0.05).The AUC of NOS3 and GDNF levels in predicting poor prognosis were 0.858 and 0.779,respectively.Conclusion　The serum levels of NOS3 and GDNF in patients with atherosclerotic cerebral infarction are decreased,and their levels are related to the degree of neurological impairment and prognosis of patients,which is expected to be a biological indicator to evaluate the prognosis of patients.

Involvement of GDNF/PI3K/AKT pathway in promoting facial nerve regeneration using electro acupuncture in a rabbit model of facial nerve crush injury / 中国组织工程研究

BACKGROUND: The mechanism of electroacupuncture on multi-acupoints in the treatment of peripheral facial paralysis is still unknown. Glial cell-derived neurotrophic factor (GDNF) is currently the most effective factor in promoting the survival of motoneurons in vitro, and the PI3K/AKT pathway plays an important role in protecting damaged motoneurons. There is yet no research on GDNF/PI3K/AKT pathway involved in electroacupuncture promoting facial nerve regeneration in rabbits. OBJECTIVE: To observe the effect of electroacupuncture on regeneration after peripheral facial nerve crush injury, and to explore the protective mechanism of electroacupuncture on facial motoneurons through the GDNF/PI3K/AKT signaling pathway. METHODS: Sixty-six adult healthy New Zealand white rabbits provided by the Animal Experimental Center of Southwest Medical University were randomly divided into a normal group and a model group. The facial nerves on the right side in the model group were subjected to a crush injury. Then the animal models were randomly divided into a model control group and an electroacupuncture group. Animals in the model control group recovered naturally, while those in the electroacupuncture group underwent electroacupuncture at Yifeng, Jiache, Sibai, Dicang, Yangbai, and Quanliao acupoints daily for 30 minutes. The improvement of facial paralysis symptoms in experimental animals were observed and scored. Tissue samples were directly taken form the normal group, and pons tissues with facial neurons were taken in the model group at 1, 4, 7, 14, and 28 days postoperatively. The morphologies of facial motoneurons and Nissl bodies were observed by hematoxylin-eosin staining and Nissl staining, respectively. Immunohistochemical techniques and western blot assay were used to detect the protein expression of GDNF, PI3K, AKT, and p-AKT in the facial motoneurons. The study protocol was approved by the Animal Ethics Committee of Southwest Medical University with approval No. 20170120001. RESULTS AND CONCLUSION: The symptoms of facial paralysis were that the animal’s mouth was drooped at the affected side, with lodging tentacles and the movement being weakened, and the eyelids that could not be lifted, which recovered faster and more completely in the electroacupuncture group than the model control group. The morphological changes of facial neurons and changes of Nissl bodies in the electroacupuncture group were lighter than those in the model control group. At each time point postoperatively, the stronger GDNF immune response could be seen in the electroacupuncture group, and the number of GDNF-positive cells was higher than that of the model control group except 1 day postoperatively (P < 0.001). The expressions of GDNF, PI3K, p-AKT proteins in the facial motoneurons were significantly increased in the electroacupuncture group compared with the model control group (P < 0.05; P < 0.01; P < 0.001). To conclude, electroacupuncture can effectively treat the peripheral facial paralysis caused by the crushed injury of facial nerve and promote the recovery of facial neurons. The up-regulation of GDNF expression in the facial motoneurons and the activation of PI3K/AKT signaling pathway may be the underlying protective mechanism of electroacupuncture.

Retinoic acid affects apoptosis of neurons injured by hypoxic-ischemic brain damage via retinoic acid receptor alpha / 中华物理医学与康复杂志

Objective To explore the effect of the retinoic acid (RA) on the apoptosis of neurons caused by hypoxic ischemic brain damage (HIBD).Methods Seventy-two newborn Sprague-Dawley rats were randomly divided into an RA deficiency (RAD) group,an RA normal (RAN) group and a control group,each of 24.The HIBD model was established in the RAD and RAN groups using Rice's method.The left common carotid artery was exposed,ligated and cut,inducing hypoxia.In the control group the left common carotid artery was exposed without any other treatment.Three and 7 days after the operation,neuron apoptosis in the brain tissue was evaluated using TUNEL staining.The degree of HIBD was quantified using modified neurological severity scores (mNSS) 7,14,21 and 28 days after the operation.Primary neurons were cultured in vitro,and oxygen glucose deprivation (OGD) was induced,then control,OGD and RA+ OGD groups were formed.The gene transcription and the protein activity of retinoic acid receptor alpha (RARcα),GDNF (glial cell line-derived neurotrophic factor) and Caspase-8 were examined with polymerase chain reactions (PCR) and Western blotting.The RA+OGD group was exposed to RA and SiRNA adenovirus,and divided into a silenced group and a negative transfection group according to the infection.Results The average mNSS of the RAD group was significantly higher than that of the RAN group.TUNEL staining showed that the apoptotic cells in the cortex increased from day 3 to 7 after the operation,but significantly more in the RAD group than in the RAN group.The gene transcription and protein activity of RARα and GDNF in the RA+OGD group were significantly higher than in the OGD group,and those of Caspase-8 were significantly lower.The gene transcription and protein activity of RARα and GDNF in the silenced group were significantly lower than in the negative transfection group,while those of Caspase-8 were just the opposite.Conclusion RA can inhibit the apoptosis of primary neurons after HIBD by up-regulating the expression of GDNF and down-regulating that of Caspase-8 via RARα.

Acupuncture alters pro-inflammatory cytokines in the plasma of maternally separated rat pups / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the potential alleviating effects of acupuncture on maternal separation (MS)-induced changes in plasma pro-inflammatory cytokine levels of rat pups.</p><p><b>METHODS</b>On postnatal day 15, rat pups were randomly assigned to 4 groups (n=6 per group) using a random number table: normal, MS, MS with acupuncture stimulation at Shenmen (HT 7) acupoint (MS+HT 7), and MS with acupuncture stimulation at Chengshan (BL 57) acupoint (MS+BL 57) groups. Rat pups in the normal group were housed with their mothers under standard conditions; those in the MS, MS+HT 7 and MS+BL 57 groups were maternally separated and individually maintained. Acupuncture stimulation was performed at HT 7 or BL 57 acupoints once a day for 7 consecutive days. A tail suspension test was performed to measure immobility time of rats and the plasma was collected on postnatal day 21, then levels of corticosterone (CORT), interleukin (IL)-1β, IL-6 and glial cell-derived neurotrophic factor (GDNF) in plasma were measured.</p><p><b>RESULTS</b>Compared with the normal group, the immobility time and the plasma levels of CORT, IL-1β, IL-6 and GDNF in the MS group were significantly increased (P<0.05 or P<0.01). Compared with the MS group, the immobility time and the plasma levels of CORT, IL-1β, IL-6 and GDNF were significantly reduced in the MS+HT 7 group (P<0.05 or P<0.01). Moreover, the immobility time and plasma levels of IL-1β and IL-6 in the MS+HT 7 group were significantly lower than those in the MS+BL 57 group (P<0.05).</p><p><b>CONCLUSION</b>Acupuncture stimulation at HT 7 can alleviate the behavioral impairment and changes of the cytokines by MS, indicating that acupuncture can help to relieve MS-induced depression.</p>

Cynomorium songaricum improves sperm count and motility and serum testosterone level and promotes proliferation of undifferentiated spermatogonia in oligoasthenospermia rats / 中华男科学杂志

<p><b>Objective</b>To investigate the effects of cynomorium songaricum (CS) decoction on the testis weight, serum testosterone level, and sperm parameters of rats with oligoasthenospermia (OAS), explore its action mechanism of improving the proliferation of undifferentiated spermatogonial cells, and provide some experimental and theoretical evidence for the development of new Chinese drugs for OAS.</p><p><b>METHODS</b>Thirty 8-week-old male SD rats were randomly divided into five groups of equal number: blank control, model control, high-dose CS, medium-dose CS, and low-dose CS. OAS models were established by intraperitoneal injection of cyclophosphamide and, a month later, treated intragastrically with normal saline or CS at 2, 1, and 0.5 g per kg of the body weight per day, all for 4 weeks. Then, the testes of the animals were harvested to obtain the testicular weight, sperm concentration and motility, and the level of serum testosterone (T), detect the expressions of the transcription factor 1 (Oct4), Thy-1 cell surface antigen (Thy1), promyelocytic leukemia zinc finger (PLZF), KIT proto-oncogene receptor tyrosine kinase (C-kit) and glial cell-derived neurotrophic factor (GDNF) in the testis tissue of the rats in the low-dose CS group by real-time PCR.</p><p><b>RESULTS</b>The testis weights in the blank control, model control, high-dose CS, medium-dose CS, and low-dose CS groups were (1.52±0.06), (1.55±0.06), (1.43±0.30), (1.35±0.40) and (1.34±0.04) g, respectively, not significantly different in the blank and model controls from those in the CS groups (P>0.05). The visual field sperm count per 10 HP was significantly increased in the high-, medium-, and low-dose CS groups (202±20, 196±5 and 216±25) as compared with the blank and model controls (200±15 and 134±30) (P<0.05). The mRNA expressions of the Oct4, Thy1, PLZF and GDNF genes were remarkably higher in the low-dose CS group than in the controls (P<0.05), but that of the C-kit gene showed no significant difference from the latter (P>0.05). The visual field sperm motility per 10 HP was markedly increased in the blank control (［52.1±5.5］%), model control (［38.1±2.5］%), high-dose CS (［59.1±9.5］%), medium-dose CS (［58.7±9.5］%), and low-dose CS (［49.6±1.0］%) groups, and so was the level of serum testosterone (［190±87.5］, ［82.5±25.8］, ［229±75.6］, ［331±86.7］ and ［185±82.4］ mmol/L), both remarkably higher in the CS groups than in the model controls (P<0.05) but with no statistically significant difference between the CS groups and the blank controls (P>0.05).</p><p><b>CONCLUSIONS</b>CS can significantly improve sperm concentration, sperm motility and serum T level in OAS rats, probably by inducing the expression of GDNF in the rat Sertoli cells, promoting the proliferation of undifferentiated spermatogonial cells, and enhancing spermatogenesis.</p>

Effect on transplantation of bone marrow mononuclear cells on neuron-like cell differentiation and glial cell-derived neurotrophic factor expression in rats with intracerebral hemorrhage / 中华神经医学杂志

Objective To investigate the effect of transplantation of bone marrow mononuclear cells (BMMNCs) on neuron-like cell differentiation and glial cell-derived neurotrophie factor (GDNF) expression in rats with intracerebral hemorrhage (ICH).Methods ICH was induced by stereotactic injection of type Ⅳ collagenase into the striatum of rats,and the successful models were randomly divided into model group (ICH group) and BMMNCs transplantation group (BMMNCs group); BMMNCs group was injected with 1 mL cell suspension containing 3 ×107 cells marked with 5-Bromo-2-deoxyuridine (Brdu).The neurological functions of the rats were observed by modified neurological severity scale (mNSS) test 1,7,14 and 28 days after the injection; the GDNF expression in cerebral tissue was observed by Western blotting 1,7,14 days after the injection; double immunofluorescent labeling was utilized to trace the differentiation of BMMNCs in the rat brains 28 days after the injection.Results As compared with those in the ICH group,the mNSS scores in the BMMNCs group were significantly lower (P＜0.05) and the GDNF expression was significantly increased at all time points (P＜0.05); Brdu and neuron specific nuclear protein (NeuN) double-labeled positive cells were found in the hemorrhage focus in the BMMNCs group 28 d after injection.Conclusion BMMNCs transplantation can improve the neural behavioral function,increase the GDNF expression,and induce BMMNCs differentiating into neuron-like cells.

Differentiation of GDNF and NT-3 Dual Gene-modified Rat Bone Marrow Mesenchymal Stem Cells into Enteric Neuron-like Cells / 华中科技大学学报（医学）（英德文版）

Bone marrow mesenchymal stem cells (BMSCs) have been shown to be multipotent cells that possess high self-replicating capacity.The purpose of our study was to investigate the feasibility of using enteric neuron-like cells obtained by in vitro induction and differentiated from rat BMSCs for the treatment of Hirschsprung's disease (HD).Glial cell-derived neurotrophic factor (GDNF) and neurotrophin-3 (NT-3) are neurotrophic factors that play important roles in neuronal development,differentiation,survival and function.Meanwhile,GDNF mutations are a major cause of HD.In this study,BMSCs were transfected with eukaryotic expression plasmids co-expressing GDNF and NT-3,and the transfected cells displayed neuron-like changes after differentiation induced by fetal gut culture medium (FGCM).Immunofluorescence assay showed positive expression of the neuronal marker NSE and the enteric neuronal markers PGP9.5,VIP and nNOS.Reverse transcription-polymerase chain reaction (RT-PCR) revealed the expression of GDNF and NT-3 in transfected BMSCs.The present study indicates that genetically modified BMSCs co-expressing GDNF and NT-3 are able to differentiate into enteric neuronal cells and express enteric nerve markers when induced by FGCM.This study provides an experimental basis for gene therapy to treat enteric nervous system-related disorders,such as HD.

The effects of constraint-induced movement therapy on expression of tyrosine hydroxylase and glial cell derived neurotrophic factor in Parkinson's disease model rats / 中华物理医学与康复杂志

Objective To explore the effects of constraint-induced movement therapy (CIMT) on the expression of tyrosine hydroxylase (TH) and glial cell derived neurotrophic factor (GDNF) in Parkinson's disease (PD) model rats. MethodsPD models were established by microinjection of 6-hydroxydopamine (6-OHDA) solution into substantia nigra of rats' right cerebral hemisphere.Forty-two model rats were divided randomly into an exercise group and a control group 1 week after microinjection.The exercise group rats were forced to use their impaired limbs by placing their nonimpaired fore-limbs in casts.The control group rats were housed in the same environment without any special treatment.Two weeks after 6-OHDA infusion and exercise training,the behavioral changes of rats were examined after intraperitoneal injection apomorphine ( APO).The content of dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) was measured by high performance liquid chromatography with electrochemistry ( HPLAEC) ; the expressions of TH and GDNF in striatum were detected by immunohistochemical methods and TH,GDNF mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR).Results After 2 weeks of training,the rotating laps of the rats in exercise group within 30 min after APO induction,reduced to a significantly greater extent when compared to the control group (P ＜ 0.05).The content of DA and it's metabolites DOPAC in striatum homogenate was significantly higher in exercise group than that in the control group ( P ＜ 0.05 ),and the expression levels,of TH and GDNF protein/ mRNA were also significantly higher in the exercise group than those in control group ( P ＜ 0.05 ).Conclusions CIMT can improve the behavioral performance of PD rats,probably through promoting the expressions of TH and GDNF protein/mRNA in striatum,and increasing DA and it's metabolites DOPAC level.