Graded Coexpression of Ion Channel, Neurofilament, and Synaptic Genes in Fast-Spiking Vestibular Nucleus Neurons.

Computations that require speed and temporal precision are implemented throughout the nervous system by neurons capable of firing at very high rates, rapidly encoding and transmitting a rich amount of information, but with substantial metabolic and physical costs. For economical fast spiking and high throughput information processing, neurons need to optimize multiple biophysical properties in parallel, but the mechanisms of this coordination remain unknown. We hypothesized that coordinated gene expression may underlie the coordinated tuning of the biophysical properties required for rapid firing and signal transmission. Taking advantage of the diversity of fast-spiking cell types in the medial vestibular nucleus of mice of both sexes, we examined the relationship between gene expression, ionic currents, and neuronal firing capacity. Across excitatory and inhibitory cell types, genes encoding voltage-gated ion channels responsible for depolarizing and repolarizing the action potential were tightly coexpressed, and their absolute expression levels increased with maximal firing rate. Remarkably, this coordinated gene expression extended to neurofilaments and specific presynaptic molecules, providing a mechanism for coregulating axon caliber and transmitter release to match firing capacity. These findings suggest the presence of a module of genes, which is coexpressed in a graded manner and jointly tunes multiple biophysical properties for economical differentiation of firing capacity. The graded tuning of fast-spiking capacity by the absolute expression levels of specific ion channels provides a counterexample to the widely held assumption that cell-type-specific firing patterns can be achieved via a vast combination of different ion channels.SIGNIFICANCE STATEMENT Although essential roles of fast-spiking neurons in various neural circuits have been widely recognized, it remains unclear how neurons efficiently coordinate the multiple biophysical properties required to maintain high rates of action potential firing and transmitter release. Taking advantage of diverse fast-firing capacities among medial vestibular nucleus neurons of mice, we identify a group of ion channel, synaptic, and structural genes that exhibit mutually correlated expression levels, which covary with firing capacity. Coexpression of this fast-spiking gene module may be a basic strategy for neurons to efficiently and coordinately tune the speed of action potential generation and propagation and transmitter release at presynaptic terminals.

Administration of leuprolide acetate, a GnRH agonist, improves urodynamic parameters in ovariectomized rats.

AIM: To evaluate the effects of a treatment with leuprolide acetate (LA) on bladder overactivity as well as the expression of gonadotropin releasing hormone receptor (GnRH-R), and neurofilaments NF68 and NF200 in female rats with overactive bladder induced by castration. METHODS: Changes in the urodynamic parameters were determined in SHAM, ovariectomized (OVX) and ovariectomized rats treated with LA (OVX-LA). A semi-quantitative analysis for the expression pattern of GnRH-R and neurofilaments NF68 and NF200 were determined. RESULTS: Forty-three days after ovariectomy, rats from the OVX group have significant lower values for intercontractile interval (ICI) and compliance (C); as well as higher values for basal bladder pressure (BP) and frequency of non-voiding contractions (NVC). The systemic application of LA increased voiding volume (Vv) and pressure threshold (ThP) in the OVX-LA animals. The application of LA reduced the high frequency of NVC in the OVX rats. No significant differences were found for Vv and NVCs between the OVX-LA vs SHAM groups. At the mid part of the bladder, the presence of GnRH-R was evidenced in the urothelium of the SHAM group. The OVX animals showed different pattern of immunolabeling for GnRH-R as well as for neurofilaments NF200 and NF68, whereas in the OVX-LA group the immunofluorescence pattern was similar to the one seen in SHAM bladders (P < 0.05 for OVX vs OVX + LA). CONCLUSIONS: the results suggest that systemic application of LA can improve bladder dysfunction in castrated rats, and perhaps considered as a treatment for overactive bladder conditions secondary to menopause.

Bardoxolone methyl induces neuritogenesis in Neuro2a cells.

BACKGROUND: Bardoxolone methyl (RTA 402, CDDOMe) has been long known for its anti-inflammatory and exceptional cytotoxic activity. The biological responses to CDDOMe are truly dose dependent. And owing to the structural modifications introduced in its parent molecule oleanolic acid, CDDOMe is able to form reversible adducts with cellular proteins containing redox sensitive cysteine residues. This nature of CDDOMe makes it a multifunctional molecule targeting multiple signaling pathways. This study was initiated to study the response of Neuro2a, a mouse neuroblastoma cell line to CDDOMe. METHODS: Neuro2a cells were treated with CDDOMe and all trans retinoic acid (ATRA) for 4days and observed for neurite outgrowth. The neurite length was estimated using ImageJ software (Neuron growth plugin). Cell viability was investigated using MTT dye reduction and trypan blue dye exclusion method. Gene expression of differentiation markers was analyzed using quantitative PCR. Cellular localization of Tuj1 and synaptophysin in differentiated Neuro2a cells was observed using immunofluorescence. RESULTS: CDDOMe ceased proliferation and induced dramatic neurite outgrowth in Neuro2a cells. These morphological changes were accompanied by time dependent increase in the mRNA levels of tyrosine hydroxylase, neurofilament 200 and synaptophysin. Besides, cytoskeleton protein Tuj1 and the synaptic vesicle protein synaptophysin were also observed to be localized in the neurites induced by CDDOMe. CONCLUSIONS: These early shreds of evidence suggest that CDDOMe induces differentiation in Neuro2a cells at concentrations ranging from 0.2 to 0.4µM and indeed contributes the existing knowledge on CDDOMe induced activities in cells.

Human vs. Mouse Nociceptors - Similarities and Differences.

The somatosensory system allows us to detect a diverse range of physical and chemical stimuli including noxious ones, which can initiate protective reflexes to prevent tissue damage. However, the sensation of pain can - under pathological circumstances - outlive its usefulness and perpetrate ongoing suffering. Rodent model systems have been tremendously useful to help understand basic mechanisms of pain perception. Unfortunately, the translation of this knowledge into novel therapies has been challenging. We have investigated similarities and differences of human and mouse peptidergic (TRKA expressing) nociceptors using dual-color fluorescence in situ hybridization of dorsal root ganglia. By comparing the transcripts of a selected group of well-established nociceptive markers, we observed significant differences for some of them. We found co-expression of Trpv1, a key player for sensitization and inflammatory pain, with TrkA in a larger population in humans compared to mice. Similar results could be obtained for Nav1.8 and Nav1.9, two voltage gated sodium channels implicated in pathological forms of pain. Additionally, co-expression of Ret and TrkA was also found to be more abundant in human neurons. Moreover, the neurofilament heavy polypeptide was detected in all human sensory DRG neurons compared to a more selective expression pattern observed in rodents. To our knowledge, this is the first time that such detailed comparative analysis has been performed and we believe that our findings will direct future experimentation geared to understand the difficulties we face in translating findings from rodent models to humans.

Changes in neurofilament 200 and tyrosine hydroxylase expression in the cardiac innervation of diabetic rats during aging.

Changes in sensory and sympathetic innervation during diabetes mellitus (DM) can be a predictor of arrhythmias, silent myocardial ischemia, and chronic heart failure, but knowledge about these changes is still unsatisfactory. We analyzed whether prolonged DM induces changes in density of sensory and sympathetic nerve terminals of rat's heart and whether it contributes to cardiomyopathy during aging. DM was induced by i/p injecting 55 mg/kg streptozotocin to male Sprague-Dawley rats, while a control group received a citrate buffer. DM in the rats was validated by measuring blood glucose level. Animals were sacrificed after 2 weeks, 2 months, 6 months, and 12 months. Five areas of cardiac sections were analyzed. Antibodies raised against tyrosine hydroxylase (TH) and neurofilament 200 kDa (NF 200) were used to detect sympathetic and sensory fibers. TH immunoreactive fiber density increased in DM groups 2 weeks after induction, reaching a peek after 2 months, while in the later stages of DM (6 and 12 months), there was no significant difference compared to control. NF 200 immunoreactive fiber density increased 2 weeks after induction compared to control. There was no consistent pattern of change during the given period in both the DM or control groups. In the DM group, we found thickening of the left ventricle wall (P<.05) as the sign of cardiomyopathy. Our findings suggest that hyperglycemia as a hallmark of DM in early stages can lead to proliferation of sympathetic and sensory nerve terminals. This finding can contribute to a better understanding of the occurrence of arrhythmias and silent myocardial ischemia in DM.

Chiro-Optical Modulation for NURR1 Production from Stem Cells.

Nuclear receptor related 1 (NURR1) is an essential protein for maintenance of dopaminergic neurons in adult midbrain of which deficiency leads to Parkinson's disease. To enhance the NURR1 production of neural cells, various approaches are under investigation. Here we report that NURR1 is highly expressed in stem cells by exposure to an L-polarized blue light emitting diode (LED). Compared to stem cells cultured in the absence of a LED, under polarized green and red LEDs, the stem cells exposed to a polarized blue LED significantly enhanced neuronal biomarkers such as neurofilament M (NFM) and neuron specific enolase (NSE) at both mRNA and protein levels. In particular, NURR1 was selectively enhanced by the stem cells exposed to the L-polarized blue LED. Stem cells exposed to the L-polarized blue LED increased mitochondrial ATP and intracellular calcium ions, which support neuronal differentiation of the stem cells. This study suggests that chiro-optical treatments by using polarized light with a specific wavelength can be used for engineering of stem cells with enhanced specific biochemicals, which may open a new method for a specific disease.

Squid Giant Axon Contains Neurofilament Protein mRNA but does not Synthesize Neurofilament Proteins.

When isolated squid giant axons are incubated in radioactive amino acids, abundant newly synthesized proteins are found in the axoplasm. These proteins are translated in the adaxonal Schwann cells and subsequently transferred into the giant axon. The question as to whether any de novo protein synthesis occurs in the giant axon itself is difficult to resolve because the small contribution of the proteins possibly synthesized intra-axonally is not easily distinguished from the large amounts of the proteins being supplied from the Schwann cells. In this paper, we reexamine this issue by studying the synthesis of endogenous neurofilament (NF) proteins in the axon. Our laboratory previously showed that NF mRNA and protein are present in the squid giant axon, but not in the surrounding adaxonal glia. Therefore, if the isolated squid axon could be shown to contain newly synthesized NF protein de novo, it could not arise from the adaxonal glia. The results of experiments in this paper show that abundant 3H-labeled NF protein is synthesized in the squid giant fiber lobe containing the giant axon's neuronal cell bodies, but despite the presence of NF mRNA in the giant axon no labeled NF protein is detected in the giant axon. This lends support to the glia-axon protein transfer hypothesis which posits that the squid giant axon obtains newly synthesized protein by Schwann cell transfer and not through intra-axonal protein synthesis, and further suggests that the NF mRNA in the axon is in a translationally repressed state.

Direct conversion of mouse fibroblasts to GABAergic neurons with combined medium without the introduction of transcription factors or miRNAs.

Degeneration or loss of GABAergic neurons frequently may lead to many neuropsychiatric disorders such as epilepsy and autism spectrum disorders. So far no clinically effective therapies can slow and halt the progression of these diseases. Cell-replacement therapy is a promising strategy for treatment of these neuropsychiatric diseases. Although increasing evidence showed that mammalian somatic cells can be directly converted into functional neurons using specific transcription factors or miRNAs via virus delivery, the application of these induced neurons is potentially problematic, due to integration of vectors into the host genome, which results in the disruption or dysfunction of nearby genes. Here, we show that mouse fibroblasts could be efficiently reprogrammed into GABAergic neurons in a combined medium composed of conditioned medium from neurotrophin-3 modified Olfactory Ensheathing Cells (NT3-OECs) plus SB431542, GDNF and RA. Following 3 weeks of induction, these cells derived from fibroblasts acquired the morphological and phenotypical GABAerigic neuronal properties, as demonstrated by the expression of neuronal markers including Tuj1, NeuN, Neurofilament-L, GABA, GABA receptors and GABA transporter 1. More importantly, these converted cells acquired neuronal functional properties such as synapse formation and increasing intracellular free calcium influx when treated with BayK, a specific activator of L-type calcium channel. Therefore, our findings demonstrate for the first time that fibroblasts can be directly converted into GABAergic neurons without ectopic expression of specific transcription factors or miRNA. This study may provide a promising cell source for the application of cell replacement therapy in neuropsychiatric disorders.

Effect of stab injury in the rat cerebral cortex on temporal pattern of expression of neuronal cytoskeletal proteins: an immunohistochemical study.

Compelling evidence now points to the critical role of the cytoskeleton in neurodegeneration. In the present study, using an immunohistochemical approach, we have shown that cortical stab injury (CSI) in adult Wistar rats significantly affects temporal pattern of expression of neurofilament proteins (NFs), a major cytoskeleton components of neurons, and microtubule-associated proteins (MAP2). At 3 days post-injury (dpi) most of the NFs immunoreactivity was found in pyknotic neurons and in fragmentized axonal processes in the perilesioned cortex. These cytoskeletal alterations became more pronounced by 10dpi. At the subcellular level CSI also showed significant impact on NFs and MAP-2 expression. Thus, at 3dpi most of the dendrites disappeared, while large neuronal somata appeared like open circles pointing to membrane disintegration. Conversely, at 10dpi neuronal perikarya and a few new apical dendrites were strongly labeled. Since aberrant NF phosphorylation is a pathological hallmark of many human neurodegenerative disorders, as well as is found after stressor stimuli, the present results shed light into the expression of neurofilaments after the stab brain injury.

Common genetic variants in NEFL influence gene expression and neuroblastoma risk.

The genetic etiology of sporadic neuroblastoma is still largely obscure. In a genome-wide association study, we identified single-nucleotide polymorphisms (SNP) associated with neuroblastoma at the CASC15, BARD1, LMO1, DUSP12, HSD17B12, HACE1, and LIN28B gene loci, but these explain only a small fraction of neuroblastoma heritability. Other neuroblastoma susceptibility genes are likely hidden among signals discarded by the multiple testing corrections. In this study, we evaluated eight additional genes selected as candidates for further study based on proven involvement in neuroblastoma differentiation. SNPs at these candidate genes were tested for association with disease susceptibility in 2,101 cases and 4,202 controls, with the associations found replicated in an independent cohort of 459 cases and 809 controls. Replicated associations were further studied for cis-effect using gene expression, transient overexpression, silencing, and cellular differentiation assays. The neurofilament gene NEFL harbored three SNPs associated with neuroblastoma (rs11994014: Pcombined = 0.0050; OR, 0.88; rs2979704: Pcombined = 0.0072; OR, 0.87; rs1059111: Pcombined = 0.0049; OR, 0.86). The protective allele of rs1059111 correlated with increased NEFL expression. Biologic investigations showed that ectopic overexpression of NEFL inhibited cell growth specifically in neuroblastoma cells carrying the protective allele. NEFL overexpression also enhanced differentiation and impaired the proliferation and anchorage-independent growth of cells with protective allele and basal NEFL expression, while impairing invasiveness and proliferation of cells homozygous for the risk genotype. Clinically, high levels of NEFL expression in primary neuroblastoma specimens were associated with better overall survival (P = 0.03; HR, 0.68). Our results show that common variants of NEFL influence neuroblastoma susceptibility and they establish that NEFL expression influences disease initiation and progression.

Elevated neurofilament light chain (NFL) mRNA levels in prediabetic peripheral neuropathy.

Evidence suggests that peripheral nerve injury occurs during the early stages of disease with mild glycemic dysregulation. Two proteins, neuron-specific enolase (NSE) and neurofilament light chain (NFL), have been examined previously as possible markers of neuronal damage in the pathophysiology of neuropathies. Herein, we aimed to determine the potential value of circulatory NSE and NFL mRNA levels in prediabetic patients and in those with peripheral neuropathy. This prospective clinical study included 45 prediabetic patients and 30 age- and sex-matched controls. All prediabetic patients were assessed with respect to diabetes-related microvascular complications, such as peripheral neuropathy, retinopathy and nephropathy. mRNA levels of NSE and NFL were determined in the blood by real-time polymerase chain reaction. NSE mRNA levels were similar between prediabetic and control groups (p > 0.05), whereas NFL mRNA levels were significantly higher in prediabetics than in controls (p < 0.001). NSE mRNA levels did not significantly differ between prediabetic patients with and without peripheral neuropathy (p > 0.05), while NFL mRNA levels were significantly higher in prediabetics with peripheral neuropathy than in those without (p = 0.038). According to correlation analysis, NFL mRNA levels were positively correlated with the Douleur Neuropathique 4 questionnaire score in prediabetic patients (r = 0.302, p = 0.044). This is the first study to suggest blood NFL mRNA as a surrogate marker for early prediction of prediabetic peripheral neuropathy, while NSE mRNA levels may be of no diagnostic value in prediabetic patients.

[Morphological changes in the lumbar dorsal root ganglion of the domestic porcine after pulsed radiofrequency stimulation].

Pulsed radiofrequency (PRF) is a percutaneous minimal invasive procedure that can be used when conservative pain therapy methods have been ineffective. The effectiveness of PRF was demonstrated in various good quality randomized control studies, but mechanisms of action are still unclear. The aim of our study is to analyse the histological effects of PRF on the domestic porcine dorsal root ganglion (DRG), and evaluate the expression of biomarkers in gangliocytes. 3 domestic porcines were investigated. Under general anaesthesia and X-ray control, DRG PRF was performed. Four lumbar DRGs (L1, L2, L3, L4) were randomly treated. The opposite side DRGs was used as control. One month after the procedure the animal was euthanized. The lumbar region of the spine was placed in 10% formaldehyde for a month. After this fixation DRG samples were prepared for slide analysis. They were embedded in paraffin in order to obtain 3 microm thick sections, which were then cut by microtome and collected on slide glasses. Using standard immunohistochemical reactions, the materials were tinted to define biomarkers NF, GFAP, Hsp-70 expression and apoptosis by TUNEL kit. The number of cells with NF (26.0 +/- 3.0 vs 16.1 +/- 3.3; p < 0.05), GFAP (12.0 +/- 1.3 vs 3.2 +/- 0.9; p < 0.05) and Hsp-70 (10.0 +/- 1.6 vs 4.2 +/- 1.0; p < 0.05) expression, were larger in the PRF side comparing with the control side. Additionally, glial cells in spinal ganglia of both sides demonstrated immunoreactivity. The instances of apoptosis were not significantly different, in statistical terms, between the control and experimental sides (18.0 +/- 4.0 vs 20.0 +/- 4.0; p = 0.35). PRF in spinal gangliocytes of lumbar region increases neural tissue cytoskeleton factors like NF and GFAP suggesting about active regeneration processes into the cells 1 month after the procedure. Spinal gangliocytes one month after PRF treatment notably increases Hsp-70 expression suggesting about activation of cellular activity and inhibitory role reducing of oxidative stress. Similar number of apoptotic cells in spinal ganglia of lumbar region after PRF and control side suggests about inhibitory role of PRF on programmed cell death and stimulation of cell survival.

Nucleoside diphosphate kinase Nm23-M1 involves in oligodendroglial versus neuronal cell fate decision in vitro.

The adult glial progenitor cells were recently shown to be able to produce neurons in central nervous system (CNS) and to become multipotent in vitro. Although the fate decision of glial progenitors was studied extensively, the signals and factors which regulate the timing of neuronal differentiation still remain unknown. To elucidate the mechanisms underlying the neuronal differentiation from glial progenitors, we modified the gene expression profile in NG2(+) glial progenitor cells using enhanced retroviral mutagen (ERM) technique followed by phenotype screening to identify possible gene(s) responsible for glial-neuronal cell fate determination. Among the identified molecules, we found the gene named non-metastatic cell 1 which encodes a nucleoside diphosphate kinase protein A (Nm23-M1 or NME1). So far, the Nm23 members have been shown to be involved in various molecular processes including tumor metastasis, cell proliferation, differentiation and cell fate determination. In the present study, we provide evidence suggesting the role of NME1 in glial-neuronal cell fate determination in vitro. We showed that NME1 is widely expressed in neuronal structures throughout adult mouse CNS. Our immunohistochemical results revealed that NME1 is strongly colocalized with NF200 through white matter of spinal cord and brain. Interestingly, NME1 overexpression in oligodendrocyte progenitor OLN-93 cells potently induced the acquisition of neuronal fate, while its silencing was shown to promote oligodendrocyte differentiation. Furthermore, we demonstrated that dual-functional role of NME1 is achieved through cAMP-dependent protein kinase (PKA). Our data therefore suggested that NME1 acts as a switcher or reprogramming factor which involves in oligodentrocyte versus neuron cell fate specification in vitro.

Involvement of endoplasmic reticulum stress in optic nerve degeneration following N-methyl-D-aspartate-induced retinal damage in mice.

We evaluated time-dependent optic nerve degeneration and the role of endoplasmic reticulum (ER) stress in this process following retinal ganglion cell death in mice. Retinal damage was induced by intravitreal injection of N-methyl-D-aspartate (NMDA). Neurofilament heavy (NFH)- and phosphorylated NFH (pNFH)-positive axons were time-dependently decreased in optic nerves at 1, 3, 7, 14, and 28 days after NMDA injection. Expression of glial fibrillary acidic protein (GFAP)-positive astroglial cells and ionized calcium-binding adaptor molecule 1 (Iba1)-positive microglial cells showed a significant increase in the optic nerve at 7, 14, and 28 days after NMDA injection. In contrast, expression of myelin basic protein (MBP)-positive oligodendrocytes showed a significant decrease in the optic nerve at 7, 14, and 28 days after NMDA injection. In quantitative RT-PCR analysis, expressions of glucose-regulated protein 78 (Grp78)/BiP, Grp94, Calreticulin, C/EBP homologous protein (Chop), and the ER degradation enhancer mannosidase alpha-like 1 (Edem1) genes were increased in the optic nerve at 14 days after NMDA injection. In addition, the Grp94 gene was increased at 7 days after NMDA injection, and the Edem1 gene was increased at 3, 7, and 28 days after NMDA injection. GRP78 and CHOP proteins were colocalized with MBP in the optic nerve after NMDA injection. These findings suggest that the axonal degeneration is dramatic until 7 days after NMDA injection and that glial cells may play some role in the degeneration of the optic nerve. Furthermore, ER stress may play a pivotal role in the decrease of MBP-positive oligodendrocytes after NMDA-induced retinal damage.

The spinal precerebellar nuclei: calcium binding proteins and gene expression profile in the mouse.

We have localized the spinocerebellar neuron groups in C57BL/6J mice by injecting the retrograde neuronal tracer Fluoro-Gold into the cerebellum and examined the distribution of SMI 32 and the calcium-binding proteins (CBPs), calbindin-D-28K (Cb), calretinin (Cr), and parvalbumin (Pv) in the spinal precerebellar nuclei. The spinal precerebellar neuron clusters identified were the dorsal nucleus, central cervical nucleus, lumbar border precerebellar nucleus, lumbar precerebellar nucleus, and sacral precerebellar nucleus. Some dispersed neurons in the deep dorsal horn and spinal laminae 6-8 also projected to the cerebellum. Cb, Cr, Pv, and SMI 32 were present in all major spinal precerebellar nuclei and Pv was the most commonly observed CBP. A number of genes expressed in hindbrain precerebellar nuclei are also expressed in spinal precerebellar groups, but there were some differences in gene expression profile between the different spinal precerebellar nuclei, pointing to functional diversity amongst them.

Transduction of E13 murine neural precursor cells by non-immunogenic recombinant adeno-associated viruses induces major changes in neuronal phenotype.

Neural precursor cells (NPCs) provide a cellular model to compare transduction efficiency and toxicity for a series of recombinant adeno-associated viruses (rAAVs). Results led to the choice of rAAV9 as a preferred candidate to transduce NPCs for in vivo transplantation. Importantly, transduction promoted a neuronal phenotype characterized by neurofilament M (NFM) with a concomitant decrease in the embryonic marker, nestin, without significant change in glial fibrillary acidic protein (GFAP). In marked contrast to recent studies for induced pluripotent stem cells (iPSCs), exposure to rAAVs is non-immunogenic and these do not result in genetic abnormalities, thus bolstering the earlier use of NPCs such as those isolated from E13 murine cells for clinical applications. Mechanisms of cellular interactions were explored by treatment with genistein, a pan-specific inhibitor of protein receptor tyrosine kinases (PRTKs) that blocked the transduction and differentiation, thus implying a central role for this pathway for inducing infectivity along with observed phenotypic changes and as a method for drug design. Implantation of transduced NPCs into adult mouse hippocampus survived up to 28 days producing a time line for targeting or migration to dentate gyrus and CA3-1 compatible with future clinical applications. Furthermore, a majority showed commitment to highly differentiated neuronal phenotypes. Lack of toxicity and immune response of rAAVs plus ability for expansion of NPCs in vitro auger well for their isolation and suggest potential therapeutic applications in repair or replacement of diseased neurons in neurodegeneration.

Bone marrow-derived mesenchymal stem cells expressing the bFGF transgene promote axon regeneration and functional recovery after spinal cord injury in rats.

OBJECTIVE: To investigate neurological effects of transplanting bone marrow-derived mesenchymal stem cells (BMSCs) transfected with the basic fibroblast growth factor (bFGF) gene in spinal cord-injured rats. METHODS: Ninety-six male adult Sprague-Dawley rats were randomized into four groups: (1) pcDNA3.1-bFGF group; (2) pcDNA3.1 group; (3) BMSCs group; and (4) vehicle control (DMEM) group. After the rat model of acute spinal cord injury (SCI) was established, 1×10(6) BMSCs or cells transfected with pcDNA3.1-bFGF or pcDNA3.1 were injected into rats of groups 1-3. At days 1, 7, 14, and 21 after injection, the Basso-Beattie-Bresnahan (BBB) locomotor rating scale was used to evaluate recovery of motor function. Expression changes of bFGF, myelin basic protein (MBP), and NF200 were examined by immunohistochemistry. RESULTS: The BBB score of DMEM group was significantly lower than those of groups 1-3 (P<0.05), but the score of pcDNA3.1-bFGF group was significantly higher than that of BMSCs group or pcDNA3.1 group at day 14 or 21 after injection (P<0.01). The number of bFGF-positive neurons in rats of pcDNA3.1-bFGF group was significantly higher than those of groups 1-3 at any time point (P<0.05). The optical density values of NF200-positive neurons and MBP-positive MBP axons in rats of pcDNA3.1-bFGF group were significantly higher than those of groups 1-3 at day 7 or 14 after injection (P<0.05). CONCLUSIONS: bFGF gene-modified BMSCs not only effectively promoted axonal outgrowth but also enhanced recovery of neurological function after SCI in rats, and may be a good candidate to evaluate gene therapy of SCI in man.

Combined pre- and postnatal ethanol exposure in rats disturbs the myelination of optic axons.

AIMS: To analyse myelination and outgrowth of the optic axons in relation to the neuro-ophthalmological manifestations of ethanol (EtOH) abuse during pregnancy. METHODS: An experimental model of chronic EtOH exposure was developed in rats and their offspring by subjecting the dams to a liquid diet (35% of the daily total calories as either EtOH or maltose-dextrose nutritional controls (Con). Eyeballs and optic nerves were obtained at key developmental stages and processed for morphologic, immunocytochemical and immunoblotting procedures, using alternatively antibodies against myelin basic protein (MBP) or neurofilament (NF) protein, and image analysing. RESULTS: A significant delay in onset of optic axons myelination, as well as a significant reduction in optic nerve size (P < 0.001), optic axons number (P < 0.001), myelinated axons density (P < 0.001), number of myelin lamellae linked to axon diameter (P < 0.001) and optic axon cross-sectional area (P < 0.001) were detected in the global morphometric assessment of the EtOH nerves with respect to the Con. Expression of MBP and NF was noticeably reduced in the EtOH optic nerves when compared with the Con. CONCLUSION: Disturbed myelination of optic axons, caused by EtOH abuse, strongly disrupts the optic nerve development and the establishment of definitive retinal and optic nerve targets, and subsequently the visual patterns.

Effect of diode laser on proliferation and differentiation of PC12 cells.

This study investigated the effects of diode (GaAlAs) laser irradiation at an effective energy density of 5 or 20 J/cm(2) on cell growth factor-induced differentiation and proliferation in pheochromocytoma cells (PC12 cells), and whether those effects were related to activation of the p38 pathway. Laser irradiation at 20 J/cm(2) significantly decreased the number of PC12 cells, while no difference was seen between the 5 J/cm(2) group and the control group (p<0.05). Western blotting revealed marked expression of neurofilament and ß-tubulin, indicating greater neurite differentiation in the irradiation groups than in the control group at 48 hr. Irradiation also enhanced expression of phospho-p38. The decrease in number of cells after laser irradiation was accelerated by p38 inhibitor, while neurite differentiation was up-regulated by laser irradiation, even when the p38 pathway was blocked. This suggests that laser irradiation up-regulated neurite differentiation in PC12 cells involving p38 and another pathway.

Neuregulin-1ß regulates outgrowth of neurites and migration of neurofilament 200 neurons from dorsal root ganglial explants in vitro.

Neuregulin-1ß (NRG-1ß) signaling has multiple functions in neurons. To assess NRG-1ß on neurite outgrowth and neuronal migration in vitro, organotypic dorsal root ganglion (DRG) neuronal culture model was established. Neurite outgrowth and neuronal migration were evaluated using this culture model in the presence (5nmol/L, 10nmol/L, 20nmol/L) or absence of NRG-1ß. Neurofilament 200 (NF-200)-immunoreactive (IR) neurons were determined as the migrating neurons. The number of nerve fiber bundles extended from DRG explant increased significantly in the presence of NRG-1ß (5nmol/L, 23.0±2.2, P<0.05; 10nmol/L, 27.0±2.7, P<0.001; 20nmol/L, 30.8±3.7, P<0.001) as compared with that in the absence of NRG-1ß (19.0±2.2). The number of neurons migrating from DRG explants increased significantly in the presence of NRG-1ß (5nmol/L, 39.6±5.0, P<0.05; 10nmol/L, 54.6±6.7, P<0.001; 20nmol/L, 62.2±5.7, P<0.001) as compared with that in the absence of NRG-1ß (31.6±4.0). Moreover, the increase of the number of nerve fiber bundles and the number of migrating NF-200-IR neurons was dose-dependent for NRG-1ß addition. The data in this study imply that NRG-1ß promotes neurite outgrowth and neuronal migration from DRG explants in vitro.