Primary Nerve Cells Research Articles

By regulating the IP3R/GRP75/VDAC1 complex to restore mitochondrial dynamic balance, selenomethionine reduces lipopolysaccharide-induced neuronal apoptosis.

Selenium (Se), as one of the essential trace elements, plays an anti-inflammatory, antioxidation, and immune-enhancing effect in the body. In addition, Se can also improve nervous system damage induced by various factors. Earlier studies have described the important role of mitochondrial dynamic imbalance in lipopolysaccharide (LPS)-induced nerve injury. The inositol 1,4,5-triphosphate receptor (IP3R)/glucose-regulated protein 75 (GRP75)/voltage-dependent anion channel 1 (VDAC1) complex is considered to be the key to regulating mitochondrial dynamics. However, it is not clear whether Selenomethionine (SeMet) has any influence on the IP3R/GRP75/VDAC1 complex. Therefore, the aim of this investigation was to determine whether SeMet can alleviate LPS-induced brain damage and to elucidate the function of the IP3R/GRP75/VDAC1 complex in it. We established SeMet and/or LPS exposure models in vivo and in vitro using laying hens and primary chicken nerve cells. We noticed that SeMet reversed endoplasmic reticulum stress (ERS) and the imbalance in mitochondrial dynamics and significantly prevented the occurrence of neuronal apoptosis. We made this finding by morphological observation of the brain tissue of laying hens and the detection of related genes such as ERS, the IP3R/GRP75/VDAC1 complex, calcium signal (Ca2+), mitochondrial dynamics, and apoptosis. Other than that, we also discovered that the IP3R/GRP75/VDAC1 complex was crucial in controlling Ca2+ transport between the endoplasmic reticulumand the mitochondrion when SeMet functions as a neuroprotective agent. In summary, our results revealed the specific mechanism by which SeMet alleviated LPS-induced neuronal apoptosis for the first time. As a consequence, SeMet has great potential in the treatment and prevention of neurological illnesses (like neurodegenerative diseases).

Synthesis, biological activity evaluation and mechanism analysis of new ganglioside GM3 derivatives as potential agents for nervous functional recovery

Neuronal regenerative ability is vital for the treatment of neurodegenerative diseases and neuronal injuries. Recent studies have revealed that Ganglioside GM3 and its derivatives may possess potential neuroprotective and neurite growth-promoting activities. Herein, six GM3 derivatives were synthesized and evaluated their potential neuroprotective effects and neurite outgrowth-promoting activities on a cellular model of Parkinson's disease and primary nerve cells. Amongst these derivatives, derivatives N-14 and 2C-12 demonstrated neuroprotective effects in the MPP + model in SH-SY5Y cells. 2C-12 combined with NGF (nerve growth factor) induced effecially neurite growth in primary nerve cells. Further action mechanism revealed that derivative 2C-12 exerts neuroprotective effects by regulating the Wnt signaling pathway, specifically involving the Wnt7b gene. Overall, this study establishes a foundation for further exploration and development of GM3 derivatives with neurotherapeutic potential.

Demethylnobiletin ameliorates cerebral ischemia-reperfusion injury in rats through Nrf2/HO-1 signaling pathway.

Demethylnobiletin (DN), with a variety of biological activities, is a polymethoxy-flavanone (PMF) found in citrus. In the present study, we explored the biological activities and potential mechanism of DN to improve cerebral ischemia reperfusion injury (CIRI) in rats, and identified DN as a novel neuroprotective agent for patients with ischemic brain injury. Rat CIRI models were established via middle cerebral artery occlusion (MCAO). Primary nerve cells were isolated and cultured in fetal rat cerebral cortex in vitro, and oxygen-glucose deprivation/reperfusion (OGD/R) models of primary nerve cells were induced. After intervention with DN with different concentrations in MCAO rats and OGD/R nerve cells, 2,3,5-triphenyltetrazolium chloride staining was used to quantify cerebral infarction size in CIRI rats. Modified neurological severity score was utilized to assess neurological performance. Histopathologic staining and live/dead cell-viability staining was used to observe apoptosis. Levels of glutathione (GSH), superoxide dismutase (SOD), reactive oxygen species (ROS) and malondialdehyde (MDA) in tissues and cells were detected using commercial kits. DN level in serum and cerebrospinal fluid of MCAO rats were measured by liquid chromatography tandem mass spectrometry. In addition, expression levels of proteins like Kelch like ECH associated protein 1 (Keap1), nuclear factor erythroid 2-related factor 2 (Nfr2) and heme oxygenase 1 (HO-1) in the Nrf2/HO-1 pathway, and apoptosis-related proteins like Cleaved caspase-3, BCL-2-associated X protein (Bax) and B-cell lymphoma-2 (Bcl-2) were determined by Western blot and immunofluorescence. DN can significantly enhance neurological function recovery by reducing cerebral infarction size and weakening neurocytes apoptosis in MCAO rats. It was further found that DN could improve oxidative stress (OS) injury of nerve cells by bringing down MDA and ROS levels and increasing SOD and GSH levels. Notably, DN exerts its pharmacological influences through entering blood-brain barrier. Mechanically, DN can reduce Keap1 expression while activate Nrf2 and HO-1 expression in neurocytes. The protective effect of DN on neurocytes have been demonstrated in both in vitro and in vivo circumstances. It deserves to be developed as a potential neuroprotective agent through regulating the Nrf2/HO-1 signaling pathway to ameliorate neurocytes impairment caused by OS.

Molecular Characterization and Function of the Nogo-66 Receptor (NgR1) Gene in the Chinese Tree Shrew

Nogo-66 receptor (NgR1) is a glycosylphosphatidylinositol-linked cell surface receptor with high affinity for Nogo-66. The binding of Nogo-66 to NgR1 plays a key role in inhibiting neurite growth, limiting synaptic plasticity and mediating Mammalian Reovirus (MRV) infection. The Chinese tree shrew (Tupaia belangeri chinensis) is, a new and valuable experimental animal that is widely used in biomedical research. Although susceptible to MRV, little is known about tree shrew NgR1 and its role in MRV infection. In this study, we cloned NgR1 form the Chinese tree shrew by RACE technology and analyzed its characteristics, spatial structure and its tissue expression. We also examined the expression pattern of NgR1 in the response of tree shrew primary nerve cells (tNC) to MRV1/TS/2011 infection. Tree shrew NgR1 was found to have a closer relationship to human NgR1 (90.34%) than to mouse NgR1. Similar to the protein structure of human NgR1, the tree shrew NgR1 has the same leucine-rich repeat (LRR) domain structure that is capped by C-terminal and N-terminal cysteine-rich modules. The tree shrew NgR1 mRNAs were predominantly detected in the central nervous system (CNS), and tree shrew NgR1 can mediate infection by MRV1/TS/2011. Taken together, these results help to elucidate the function of NgR1 and provide a basis for using the tree shrew as an animal model for studies of the nervous system and infectious diseases.

Immunohistochemical characterization of nerve elements in porcine intrinsic laryngeal ganglia.

The present study investigated the chemical coding of neurons and nerve fibres in local laryngeal ganglia in pigs (n=5) using double-labelling immunohistochemistry. Virtually all the neurons were cholinergic in nature (ChAT- or VAChT-positive). Only very solitary, small nerve cells (presumably representing interneurons) stained intensely for adrenergic marker, DβH. Many neurons also contained immunoreactivity for NOS (91%), VIP (62.7%), NPY (24.7%), galanin (10%), SP (1.3%) and CGRP (5.3%). No neurons expressing somatostatin or Leu-enkephalin were observed. Nearly all the neuronal somata were densely supplied with varicose cholinergic nerve terminals, which presumably represented preganglionic axons, and some of them were also closely apposed with CGRP- and/or SP-positive varicose nerve endings, which were putative collaterals of extrinsic primary sensory fibres. In conclusion, this study has revealed that intrinsic neurons in the porcine larynx, like in many other mammalian species studied, should be classified as parasympathetic cholinergic neurons expressing biologically active substances, predominantly NOS and VIP. Furthermore, they are likely to receive inputs from not only preganglionic neurons but also primary sensory nerve cells. Finally, it appears that the information on the occurrence of the local laryngeal ganglia should be regularly included in textbooks dealing with the cranial portion of the parasympathetic nervous system in mammals.

RhEPO Upregulates the PPARγ Pathway in Long-term Cultured Primary Nerve Cells via PI3K/Akt to Delay Cell Senescence.

Previous studies have confirmed that both recombinant human erythropoietin (rhEPO) and peroxisome proliferator-activated receptors γ (PPARγ) activator pioglitazone can protect senescent nerve cells, and their mechanisms involve enhancing cell antioxidant capacity and reducing cell apoptosis. However, whether the PPARγ pathway is involved in the rhEPO anti-aging process in neuronal cells is still unclear. In this study, to explore the relationship between rhEPO and the PPARγ pathway at the cellular level, primary nerve cells cultured for 22days were used to simulate the natural aging process of nerve cells. Starting on the 11th day of culture, rhEPO, LY294002, and GW9662 were added for treatment. Immunochemical methods and SA-β-gal staining were used to observe the changes in cellular antioxidant capacity and the fraction of senescent cells. The results showed that PPARγ blockade retarded the effect of rhEPO on the cellular antioxidant capacity and altered the fraction of senescent cells. It was confirmed that PPARγ was involved in rhEPO's anti-aging process in neuronal cells. Real-time fluorescent quantitative RT-PCR, Western blotting, and immunofluorescence staining were used to observe the changes in PPARγ pathway-related factors in nerve cells after rhEPO treatment. The results showed that rhEPO significantly upregulated the expression of PPARγ coactivator-1α (PGC-1α), PPARγ, and nuclear PPARγ in cells but did not affect the level of phosphorylated PPARγ protein, confirming that rhEPO has the ability to upregulate the PPARγ pathway. PI3K/Akt and PPARγ pathway blockade experiments were used to explore the relationships among rhEPO, PI3K/Akt, and PPARγ. The results showed that after PPARγ blockade, rhEPO had no significant effect on the PI3K/Akt pathway-related factor p-Akt, while after PI3K/Akt blockade, rhEPO's effects on PPARγ-related factors (PGC-1α, PPARγ, and nuclear PPARγ) were significantly decreased. It is suggested that rhEPO delays the PI3K/Akt pathway in the process of neuronal senescence, which is located upstream of PPARγ regulation. In conclusion, this study confirmed that rhEPO can upregulate the expression of PGC-1α and PPARγ in cells and the level of PPARγ protein in the nucleus to enhance the antioxidant capacity of cells and delay the senescence of nerve cells through the PI3K/Akt pathway. These findings will provide ideas for finding new targets for neuroprotection research and will also provide a theoretical basis and experimental evidence for rhEPO anti-aging research in neural cells.

Recombinant human erythropoietin protects long-term cultured ageing primary nerve cells by upregulating the PI3K/Akt pathway.

ObjectivePrevious studies have found that recombinant human erythropoietin (rhEPO) protects long-term cultured ageing primary nerve cells by enhancing the endogenous antioxidant capacity of cells; however, its signalling pathways are not clear. This study aimed to explore the relationship between the rhEPO and PI3K/Akt pathways in the protection of senescent nerve cells at the cellular level.MethodsPrimary nerve cells were cultured for 22 days to mimic the natural ageing process of nerve cells. rhEPO and LY294002 were administered as an intervention on the 11th day of culture. Western blot, immunochemistry, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide, immunofluorescence double-labelling staining, Annexin V-FITC/PI double-labelling flow cytometry, and SA-β-gal staining experiments were used to observe the expression levels of erythropoietin receptor (EPOR) and phosphorylated Akt (p-Akt) protein and the related indices of nerve cell senescence.ResultsWestern blot experiments showed that in ageing long-term cultured primary neurons, the EPOR and p-Akt decreased and rhEPO upregulated the expression levels of EPOR and p-Akt protein. The rest showed that the PI3K/Akt pathway blockade reduced the antioxidation capacity, cell viability, cell morphology, and ratio of apoptotic cells and senescent cells of rhEPO on ageing long-term cultured primary nerve cells.ConclusionsThis study explored the relationship between the rhEPO and PI3K/Akt pathways in the protection of ageing nerve cells at the cellular level and found that rhEPO protects long-term cultured ageing primary nerve cells by upregulating the PI3K/Akt pathway. These findings provide a theoretical basis and experimental evidence for the antiaeging mechanism of EPO in the nervous system.

H2S protects hippocampal neurons against hypoxia-reoxygenation injury by promoting RhoA phosphorylation at Ser188

Inhibition of RhoA-ROCK pathway is involved in the H2S-induced cerebral vasodilatation and H2S-mediated protection on endothelial cells against oxygen-glucose deprivation/reoxygenation injury. However, the inhibitory mechanism of H2S on RhoA-ROCK pathway is still unclear. The aim of this study was to investigate the target and mechanism of H2S in inhibition of RhoA/ROCK. GST-RhoAwild and GST-RhoAS188A proteins were constructed and expressed, and were used for phosphorylation assay in vitro. Recombinant RhoAwild-pEGFP-N1 and RhoAS188A-pEGFP-N1 plasmids were constructed and transfected into primary hippocampal nerve cells (HNCs) to evaluate the neuroprotective mechanism of endothelial H2S by using transwell co-culture system with endothelial cells from cystathionine-γ-lyase knockout (CSE−/−) mice and 3-mercaptopyruvate sulfurtransferase knockout (3-MST−/−) rats, respectively. We found that NaHS, exogenous H2S donor, promoted RhoA phosphorylation at Ser188 in the presence of cGMP-dependent protein kinase 1 (PKG1) in vitro. Besides, both exogenous and endothelial H2S facilitated the RhoA phosphorylation at Ser188 in HNCs, which induced the reduction of RhoA activity and membrane transposition, as well as ROCK2 activity and expression. To further investigate the role of endothelial H2S on RhoA phosphorylation, we detected H2S release from ECs of CSE+/+ and CSE−/− mice, and 3-MST+/+ and 3-MST−/− rats, respectively, and found that H2S produced by ECs in the culture medium is mainly catalyzed by CSE synthase. Moreover, we revealed that both endothelial H2S, mainly catalyzed by CSE, and exogenous H2S protected the HNCs against hypoxia-reoxygenation injury via phosphorylating RhoA at Ser188.

Presenilin-Deficient Neurons and Astrocytes Display Normal Mitochondrial Phenotypes.

Presenilin 1 (PS1) and Presenilin 2 (PS2) are predominantly known as the catalytic subunits of the γ-secretase complex that generates the amyloid-β (Aβ) peptide, the major constituent of the senile plaques found in the brain of Alzheimer's disease (AD) patients. Apart from their role in γ-secretase activity, a growing number of cellular functions have been recently attributed to PSs. Notably, PSs were found to be enriched in mitochondria-associated membranes (MAMs) where mitochondria and endoplasmic reticulum (ER) interact. PS2 was more specifically reported to regulate calcium shuttling between these two organelles by controlling the formation of functional MAMs. We have previously demonstrated in mouse embryonic fibroblasts (MEF) an altered mitochondrial morphology along with reduced mitochondrial respiration and increased glycolysis in PS2-deficient cells (PS2KO). This phenotype was restored by the stable re-expression of human PS2. Still, all these results were obtained in immortalized cells, and one bottom-line question is to know whether these observations hold true in central nervous system (CNS) cells. To that end, we carried out primary cultures of PS1 knockdown (KD), PS2KO, and PS1KD/PS2KO (PSdKO) neurons and astrocytes. They were obtained from the same litter by crossing PS2 heterozygous; PS1 floxed (PS2+/−; PS1flox/flox) animals. Genetic downregulation of PS1 was achieved by lentiviral expression of the Cre recombinase in primary cultures. Strikingly, we did not observe any mitochondrial phenotype in PS1KD, PS2KO, or PSdKO primary cultures in basal conditions. Mitochondrial respiration and membrane potential were similar in all models, as were the glycolytic flux and NAD+/NADH ratio. Likewise, mitochondrial morphology and content was unaltered by PS expression. We further investigated the differences between results we obtained here in primary nerve cells and those previously reported in MEF cell lines by analyzing PS2KO primary fibroblasts. We found no mitochondrial dysfunction in this model, in line with observations in PS2KO primary neurons and astrocytes. Together, our results indicate that the mitochondrial phenotype observed in immortalized PS2-deficient cell lines cannot be extrapolated to primary neurons, astrocytes, and even to primary fibroblasts. The PS-dependent mitochondrial phenotype reported so far might therefore be the consequence of a cell immortalization process and should be critically reconsidered regarding its relevance to AD.

Bicyclic RGD peptides enhance nerve growth in synthetic PEG-based Anisogels.

Nerve regeneration scaffolds often consist of soft hydrogels modified with extracellular matrix (ECM) proteins or fragments, as well as linear and cyclic peptides. One of the commonly used integrin-mediated cell adhesive peptide sequences is Arg-Gly-Asp (RGD). Despite its straightforward coupling mechanisms to artificial extracellular matrix (aECM) constructs, linear RGD peptides suffer from low stability towards degradation and lack integrin selectivity. Cyclization of RGD improves the affinity towards integrin subtypes but lacks selectivity. In this study, a new class of short bicyclic peptides with RGD in a cyclic loop and ‘random screened’ tri-amino acid peptide sequences in the second loop is investigated as a biochemical cue for cell growth inside three-dimensional (3D) synthetic poly(ethylene glycol) (PEG)-based Anisogels. These peptides impart high integrin affinity and selectivity towards either αvβ3 or α5β1 integrin subunits. Enzymatic conjugation of such bicyclic peptides to the PEG backbone enables the formulation of an aECM hydrogel that supports nerve growth. Furthermore, different proteolytic cleavable moieties are incorporated and compared to promote cell migration and proliferation, resulting in enhanced cell growth with different degradable peptide crosslinkers. Mouse fibroblasts and primary nerve cells from embryonic chick dorsal root ganglions (DRGs) show superior growth in bicyclic RGD peptide conjugated gels selective towards αvβ3 or α5β1, compared to monocyclic or linear RGD peptides, with a slight preference to αvβ3 selective bicyclic peptides in the case of nerve growth. Synthetic Anisogels, modified with bicyclic RGD peptides and containing short aligned, magneto-responsive fibers, show oriented DRG outgrowth parallel to the fibers. This report shows the potential of PEG hydrogels coupled with bicyclic RGD peptides as an aECM model and paves the way for a new class of integrin selective biomolecules for cell growth and nerve regeneration.

CTRP6(C1q/Tumor Necrosis Factor (TNF)-related protein-6) alleviated the sevoflurane induced injury of mice central nervous system by promoting the expression of p-Akt (phosphorylated Akt)

ABSTRACT Postoperative cognitive impairment and nervous system damage caused by anesthetics seriously affect patient’s postoperative recovery. Recent study has revealed that CTRP6 could alleviate apoptosis, inflammation and oxidative stress of nerve cells, thereby relieving nervous system damage induced by cerebral ischemia reperfusion. However, whether CTRP6 could relieve sevoflurane induced central nervous system injury is unclear. We stimulated C57BL/6 mice with sevoflurane and injected CTRP6 overexpression adenovirus vector. Next, H&E staining and TUNEL assays were performed to examine the effect of CTRP6 on sevoflurane induced injury of central nervous system. Finally, we isolated primary nerve cells of hippocampus. Flow cytometry and commercial kits were used for the detection of apoptosis and ROS levels of these cells. Western blotting was used for the detection of the expression level of p-Akt in central nervous tissues and primary cells. Results showed that sevoflurane induced injury and apoptosis of central nervous tissues. Overexpression of CTRP6 relieved apoptosis and injury of these tissues. CTRP6 inhibited the expression of cleaved caspase-3 and cleaved PARP in these tissues. Sevoflurane promoted apoptosis of primary cells and enhanced the expression of ROS and MDA in these cells. Overexpression of CTRP6 alleviated apoptosis and suppressed production of ROS and MDA in these cells. In addition, CTRP6 also enhanced the expression of p-Akt in primary cells. Taken together, our results suggested that CTRP6 relieved sevoflurane induced injury of central nervous tissues by promoting the expression of p-Akt. Therefore, the targeted drug of CTRP6 should be explored for the remission of these symptoms.

Relationship between Erk1/2 signal pathway and nerve cell apoptosis rats with ischemic stroke

To investigate the relationship between the Erk1/2 signal pathway and neuronal apoptosis in ischemic stroke rats. Male SD(Sprague Dawley) rats (n = 24) were randomly divided into three groups, each containing 8 rats: sham-operated group, MCAO(Midle cerebral artery oclusion) group, and MCAO + U0126 intervention group (U0126 group). In in vitro trial, primary cortical nerve cells were divided into three groups: control group, OGD(Oxygen and glucose deprivation) group, and U0126 intervention group (U0126 group). In vivo protein expression levels of Erk1/2, p-Erk1/2 and Bcl-2 were determined using western blot. The expressions of Bcl-2, Bcl-xl and Bax were assayed using immunohistochemical staining. Nerve cell mortality in cerebral tissue was detected using TUNEL staining. In in vitro trials, cell apoptosis was assayed with flow cytometry and LDH release. The activity of caspase-3 was determined. Nerve cell apoptosis was determined using Hoechst33258 staining method. In in vivo trial, it was found that the protein expression level of p-ERK1/2 in cerebral tissue in the MCAO group was significantly increased, when compared with that of the sham-operated group, while the protein expression level of p-Erk1/2 in the U0126 group was significantly lower than that in the MCAO group. The expression levels of Bcl-2 and Bcl-xl in the MCAO group were significantly lower than the corresponding expression levels in the sham-operated group, while the expressions of Bcl-2 and Bcl-xl in the U0126 group were significantly lower than those in MCAO group. In MCAO group, the expression of Bax was significantly higher than that in the sham-operated group, while Bax expression was higher in U0126 than in MCAO group. There were significantly higher number of dead nerve cells in MCAO group than in the sham-operated group, while nerve cell mortality in U0126 group was significantly lower than in MCAO group. In in vitro trials, flow cytometry revealed significantly higher apoptosis of OGD-treated nerve cells, relative to the control group. Nerve cells exposed to U0126 and treated with ODR (Oxygen-dependent repressor) were significantly decreased in population, when compared with single OGD treatment group. The LDH release level of nerve cells treated OGD was significantly increased, when compared with that of the control group. However, LDH release level of nerve cells treated with OGD after U0126 intervention was significantly decreased, relative to the single OGD treatment group. The dilution of nerve cell nucleus after OGD treatment was significantly increased, when compared with that of the control group. For nerve cells treated with ODR after U0126 intervention, the nuclear dilution was significantly decreased, relative to that of nerve cell nucleus in the single OGD treatment group. The OGD treatment led to significant increase in nerve cell caspase-3 activity, relative the control group. However, the caspase-3 activity of nerve cells treated with ODR after U0126 intervention was significantly decreased, when compared with single OGD treatment group. The activation of Erk1/2 signal pathway during ischemic stroke promotes apoptosis of nerve cells. Based on these findings, it can be reasonably inferred that the ERK1/2 signal pathway may be an important target for treating ischemic stroke.

MiR-463-3p inhibits tibial nerve regeneration via post-transcriptional suppression of SPRR1A

Background miRNAs have been involved in neural development, degeneration, and regeneration. MiR-463-3p is expressed in reproductive and nervous systems. In this study, the role of miR-463-3p in tibial nerve injury and regeneration was explored. Materials and methods A model of tibial nerve injury was established with the crush method, and the levels of miR-463-3p were detected at days 0, 3, 7, 12, 18 and 24 post-injury. Then, primary tibial nerve cells were isolated from newborn mice, and miR-463-3p was respectively overexpressed and knocked down in cultured cells. Behaviors of tibial nerve cells were detected. Furthermore, bioinformatics technology was used to investigate the underlying mechanism. Results The expression miR-463-3p was robustly increased in the injured tibial nerve in vivo and in tibial nerve cells treated with oxygen-glucose deprivation. The data on gain- and loss-of-function demonstrated that miR-463-3p negatively regulated including neurite length, percentage of cells with neurites, and cell branching in tibial nerve cells. Small proline-rich repeat protein 1 A (SPRR1A), an identified nerve regeneration associated genes, was identified as a target gene of miR-463-3p. Conclusion Inhibition of miR-463-3p could increase SPRR1A expression in the tibial nerve tissue and improve regeneration of the tibial nerve post-injury in vivo.

Activation of PPARγ pathway enhances cellular anti-oxidant capacity to protect long-term cultured primary rat neural cells from apoptosis

To study the protective effect of enhanced peroxisome proliferator activated receptor γ (PPARγ) pathway against apoptosis of long-term cultured primary nerve cells. A natural aging model was established in primary rat nerve cells by long-term culture for 22 days. The cells were divided into control group, 0.1, 1.0, 5.0, and 10 μmol/L GW9662 intervention groups, and 0.1, 1.0, 5.0, and 10 μmol/L pioglitazone intervention groups. The cell viability was assessed using MTT assay and the cell morphological changes were observed after the treatments to determine the optimal concentrations of GW9662 and pioglitazone. Double immunofluorescence labeling and flow cytometry were used to observe the changes in the number of viable cells and cell apoptosis following the treatments; immunocytochemical staining was used to assess the changes in the anti-oxidation ability of the treated cells. The optimal concentrations of GW9662 and pioglitazone determined based on the cell viability and morphological changes were both 1 μmol/L. Compared with the control group, GW9662 treatment significantly lowered while pioglitazone significantly increased the total cell number and nerve cell counts (P < 0.05), and nerve cells in the cell cultures maintained a constant ratio at about 80% in all the groups (P > 0.05). GW9662 significantly enhanced while pioglitazone significantly lowered the cell apoptosis rates compared with the control group (P < 0.05). GW9662 obviously lowered SOD activity and GSH content in G group (P < 0.05) and increased MDA content in the cells (P < 0.05), and pioglitazone resulted in reverse changes in SOD, GSH and MDA contents in the cells (P < 0.05). Activation of PPARγ pathway protects long-term cultured primary nerve cells by enhancing cellular anti-oxidant capacity and reducing cell apoptosis, suggesting a potential strategy for anti-aging treatment of the nervous system through intervention of the PPARγ pathway.

Irisin regulates the expression of BDNF and glycometabolism in diabetic rats.

Irisin is a proteolytic product of the fibronectin type III domain-containing protein 5. The aim of the present study was to verify whether irisin is involved in the pathogenesis of diabetic mild cognitive impairment and elucidate the associated mechanisms. Diabetic rats were divided into four groups: Control, Model, Irisin (overexpression of irisin) and Irisin-short hairpin (sh)-RNA (irisin interference). The levels of irisin, brain-derived neurotrophic factor (BDNF), glycosylated hemoglobin A1c (GHbA1c) and advanced glycated end products (AGEs) in the serum were determined using ELISA. The expression of BDNF in the hippocampal tissue was evaluated by immunohistochemical analysis. Compared with the Control group, the levels of irisin and BDNF were markedly decreased in the Model and Irisin-shRNA groups, whereas those of GHbA1c and AGEs were markedly increased. However, the levels of irisin and BDNF in the Irisin group were significantly higher than those in the Model group, whereas the levels of GHbA1c and AGEs in the Irisin group were significantly lower. Irisin-shRNA significantly downregulated the expression of irisin and BDNF, and upregulated the levels of GHbA1c and AGEs, compared with those in the Model group. Rat primary hippocampal nerve cells were isolated and identified by microtubule-associated protein-2 labeling. The vitality of primary cells from diabetic rats, evaluated using a methyl thiazolyl tetrazolium assay, was markedly decreased and further reduced following the injection of irisin-shRNA, however, it was markedly improved following irisin treatment. The mRNA and protein levels of BDNF in the primary cells were evaluated by fluorogenic reverse transcription-quantitative polymerase chain reaction and western blot analyses, respectively, following the exposure of cells to different concentrations of glucose: 0 (control), 5.5, 15 and 25 mmol/l for 12, 24 and 48 h. The mRNA and protein expression levels of BDNF in the primary cells following exposure to glucose were significantly lower than those observed in the control. Further exposure to glucose led to a significant decrease in the expression of BDNF. In conclusion, irisin may regulate the expression of BDNF and glycometabolism in diabetic rats.

Structural and Functional Substitution of Deleted Primary Sensory Neurons by New Growth from Intrinsic Spinal Cord Nerve Cells: An Alternative Concept in Reconstruction of Spinal Cord Circuits.

In a recent clinical report, return of the tendon stretch reflex was demonstrated after spinal cord surgery in a case of total traumatic brachial plexus avulsion injury. Peripheral nerve grafts had been implanted into the spinal cord to reconnect to the peripheral nerves for motor and sensory function. The dorsal root ganglia (DRG) containing the primary sensory nerve cells had been surgically removed in order for secondary or spinal cord sensory neurons to extend into the periphery and replace the deleted DRG neurons. The present experimental study uses a rat injury model first to corroborate the clinical finding of a re-established spinal reflex arch, and second, to elucidate some of the potential mechanisms underlying these findings by means of morphological, immunohistochemical, and electrophysiological assessments. Our findings indicate that, after spinal cord surgery, the central nervous system sensory system could replace the traumatically detached original peripheral sensory connections through new neurite growth from dendrites.

RhEPO Enhances Cellular Anti-oxidant Capacity to Protect Long-Term Cultured Aging Primary Nerve Cells

Erythropoietin (EPO) may protect the nervous system of animals against aging damage, making it a potential anti-aging drug for the nervous system. However, experimental evidence from natural aging nerve cell models is lacking, and the efficacy of EPO and underlying mechanism of this effect warrant further study. Thus, the present study used long-term cultured primary nerve cells to successfully mimic the natural aging process of nerve cells. Starting on the 11th day of culture, cells were treated with different concentrations of recombinant human erythropoietin (rhEPO). Using double immunofluorescence labeling, we found that rhEPO significantly improved the morphology of long-term cultured primary nerve cells and increased the total number of long-term cultured primary cells. However, rhEPO did not improve the ratio of nerve cells. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to measure nerve cell activity and showed that rhEPO significantly improved the activity of long-term cultured primary nerve cells. Moreover, Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double immunofluorescence labeling flow cytometry revealed that rhEPO reduced the apoptotic rate of long-term cultured primary nerve cells. Senescence-associated β-galactosidase (SA-β-gal) immunohistochemistry staining showed that rhEPO significantly reduced the aging rate of long-term cultured primary nerve cells. Immunochemistry revealed that rhEPO enhanced intracellular superoxide dismutase (SOD) activity and glutathione (GSH) abundance and reduced the intracellular malondialdehyde (MDA) level. In addition, this effect depended on the dose, was maximized at a dose of 100U/ml and was more pronounced than that of vitamin E. In summary, this study finds that rhEPO protects long-term cultured primary nerve cells from aging in a dose-dependent manner. The mechanism of this effect may be associated with the enhancement of the intracellular anti-oxidant capacity. These findings provide a theoretical basis to further the anti-aging mechanism of EPO in the nervous system, and they provide experimental evidence at the cellular level for the clinical application of EPO to protect the nervous system from aging.

Which form of collagen is suitable for nerve cell culture?

In this study, we investigated the effects of hydrolyzed and non-hydrolyzed collagen and two-dimensional and three-dimensional collagen matrices on cell survival, attachment and neurite outgrowth of primary cultured nerve cells using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay and inverted microscopy. Hydrolyzed collagen facilitated nerve cell survival and neurite outgrowth, but it had no obvious influences on cell attachment. In contrast, non-hydrolyzed two-dimensional collagen matrix had no obvious effects on neurite outgrowth. These findings suggest that hydrolyzed collagen is an ideal nerve cell culture media.

Investigation of cell–substrate interactions by focused ion beam preparation and scanning electron microscopy

Cell–substrate interactions, which are an important issue in tissue engineering, have been studied using focused ion beam (FIB) milling and scanning electron microscopy (SEM). Sample cross-sections were generated at predefined positions (target preparation) to investigate the interdependency of growing cells and the substrate material. The experiments focus on two cell culturing systems, hepatocytes (HepG2) on nanoporous aluminum oxide (alumina) membranes and mouse fibroblasts (L929) and primary nerve cells on silicon chips comprised of microneedles. Cross-sections of these soft/hard hybrid systems cannot be prepared by conventional techniques like microtomy. Morphological investigations of hepatocytes growing on nanoporous alumina membranes demonstrate that there is in-growth of microvilli from the cell surface into porous membranes having pore diameters larger than 200 nm. Furthermore, for various cell cultures on microneedle arrays contact between the cells and the microneedles can be observed at high resolution. Based on FIB milled cross-sections and SEM micrographs cells which are only in contact with microneedles and cells which are penetrated by microneedles can be clearly distinguished. Target preparation of biological samples by the FIB technique especially offers the possibility of preparing not only soft materials but also hybrid samples (soft/hard materials). Followed by high resolution imaging by SEM, new insights into cell surface interactions can be obtained.

Cell toxicity assessment methodologies applied in the study of the toxicity of nano-alumina to nerve cells

To observe the effect of nano-alumina on nerve cell viability through different detection kits of cell viability, using micro-alumina and nano-carbon as controls. Primary culturing nerve cells of mouse in vitro, which were exposed to 7 doses of 0 µmol/L, 62.5 µmol/L, 125.0 µmol/L, 250.0 µmol/L, 500.0 µmol/L, 1.0 mmol/L, 2.0 mmol/L concentrations of nano-alumina (nano-Al), micro alumina (micro-Al) and nano-carbon (nano-C), detecting cell viability (A(570) values) with CCK-8, MTT and LDH methods. (1) The results of CCK-8 kit showed that, in doses of 250.0 µmol/L - 2.0 mmol/L, the cell viability values of nano-alumina (the values of A(570) were 0.878 ± 0.009, 0.823 ± 0.016, 0.647 ± 0.008, 0.594 ± 0.013, respectively) were significantly lower than that of micro-Al (the values of A(570) were 0.960 ± 0.008, 0.951 ± 0.036, 0.833 ± 0.008, 0.708 ± 0.012, respectively) and nano-C (the values of A(570) were 0.977 ± 0.003, 0.973 ± 0.002, 0.924 ± 0.006, 0.891 ± 0.023, respectively). While, comparing nano-Al with the same dose of micro-Al, there was significant difference (the t values were -0.082, -0.128, -0.186, -0.114, respectively, P < 0.01), and so as to the comparison of nano-Al with the same dose of nano-C (the t values were -0.099, -0.150, -0.277, -0.297, respectively, P < 0.01). (2) MTT results showed that in the doses of 500.0 µmol/L and 1.0 mmol/L, the cell viability of nano-Al (the values of A(570) were 0.648 ± 0.095 and 0.575 ± 0.061) were lower than that of micro-Al (the values of A(570) were 0.830 ± 0.044 and 0.816 ± 0.014) and nano-C (the values of A(570) were 0.889 ± 0.009 and 0.765 ± 0.049), and the differences were significant (nano-Al compared with the same dose of micro-Al, the t values were -0.183 and -0.242, P < 0.01; nano-Al compared with the same dose of nano-C, the t values were -0.241 and -0.190, P < 0.01). (3) LDH results showed that in the dose from 125.0 µmol/L to 2.0 mmol/L, the LDH release of nano-Al group (the values of A(570) were 1.862 ± 0.102, 1.905 ± 0.066, 1.930 ± 0.037, 1.946 ± 0.033, 1.967 ± 0.068, respectively) were higher than that of nano-C (the values of A(570) were 1.484 ± 0.110, 1.559 ± 0.039, 1.663 ± 0.014, 1.732 ± 0.076, 1.765 ± 0.073, respectively), and the differences were significant (the t values were -0.377, 0.346, 0.266, 0.213, 0.202, respectively, P < 0.01). In the dose from 125.0 µmol/L to 1.0 mmol/L, the LDH release of nano-Al group were higher than that of micro-Al (the values of A(570) were 1.578 ± 0.011, 1.639 ± 0.025, 1.727 ± 0.024, 1.808 ± 0.020, respectively), and the differences were significant (the t values were 0.284, 0.266, 0.202, 0.172, respectively, P < 0.01). The toxicity of nano-Al is greater than nano-C and micro-Al on the viability of nerve cells; LDH is more suitable for detecting changes of cell viability after the effect of nano-materials than CCK-8 and MTT.