Age-related decrease in brain-derived neurotrophic factor gene expression in the brain of the zitter rat with genetic spongiform encephalopathy

Facilitation of AVP(4–8) on Gene Expression of BDNF and NGF in Rat Brain

Whereas nerve growth factor has been extensively studied in human keratinocytes, little is known on the role of other members of the neurotrophin family. We investigated the expression and function of neurotrophins and neurotrophin receptors in cultured human keratinocytes. We demonstrated by reverse transcription-polymerase chain reaction that keratinocytes synthesize neurotrophin-3, brain-derived neurotrophic factor, and neurotrophin-4/5. These cells also express tyrosinase kinase A and C, the nerve growth factor and neuro-trophin-3 high-affinity receptors, respectively. On the other hand, only the truncated extracellular isoform of tyrosinase kinase B, the high-affinity brain-derived neurotrophic factor and neurotrophin-4/5 receptor, is detected in keratinocytes. Moreover, neurotrophin-3, brain-derived neurotrophic factor, and neurotrophin-4/5 proteins are secreted by human keratinocytes at low levels. Keratinocyte stem cells synthesize the highest amounts of nerve growth factor, while they secrete higher levels of nerve growth factor as compared with transit amplifying cells. Neurotrophin-3 stimulates keratinocyte proliferation, where brain-derived neurotrophic factor or neurotrophin-4/5 does not exert any effect on keratinocyte proliferation. Addition of neurotrophin-3 slightly upregulates the secretion of nerve growth factor, whereas nerve growth factor strongly augments neurotrophin-3 release. Ultraviolet B irradiation downregulates nerve growth factor, whereas it augments neurotrophin-3 and neurotrophin-4/5 protein levels. Ultraviolet A irradiation increases the level of neurotrophin-3, whereas it does not exert any effect on the other neurotrophins. Finally, neurotrophins other than nerve growth factor fail to protect human keratinocytes from ultraviolet B-induced apoptosis. This work delineates a functional neurotrophin network, which may contribute to epidermal homeostasis.

Objective To evaluate the effect of emulsified isoflurane on cognitive function in rats.Methods Seventy-two adult male SD rats,aged 8 weeks,weighing 250-300 g,were randomly divided into 3 groups:normal control group (group C,n =12),intralipid group(group E,n =12),and 8％ emulsified isoflurane group ( group EI,n =48).Morris water maze test was performed at 2 h after administration in group E and at 2 h,1,7,14 d after administration in 12 rats at each time point in group EI.The escape latency,staying time at the original platform quadrant,frequency of crossing the original platform and swimming speed were recorded.Orbital blood samples were taken from 6 rats in each group after water maze test for determination of the plasma corticosterone concentration,and then the animals were sacrificed and their hippocampi were removed for determination of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) contents.Brains were removed from another 6 rats in each group after water maze test for determination of the expression of BDNF and NGF in DG,CA3,CA2,CA1 of hippocampus.Results Compared with group C,in group EI the escape latency at 2 h after administration was prolonged,staying time at the original platform quadrant was shortened,the expression of BDNF in DG and CA3 of hippocampus was down-regulated and the BDNF content in hippocampus was decreased at 2 h and 1 d after administration( P ＜ 0.05 or 0.01).The escape latency was shortened and staying time at the original platform quadrant was prolonged at 7 and 14 d after administration,the content of NGF in hippocampus was increased at 1,7 and 14 d after administration and the expression of BDNF in DG and CA3 of hippocampus was up-regulated at 1d after administration as compared with those at 2 h after administration in group E1( P ＜ 0.05 or 0.01).There was no significant difference in the variables mentioned above between groups E and C( P ＞ 0.05 ).There was no significant difference in plasma corticosterone concentration among the 3 groups ( P ＞ 0.01 ).Conclusion The mechanism by which emulsified isoflurane results in transient cognitive impairment in rats is related to down-regulating the expression of BDNF in hippocampus,but not related to corticosterone and NGF. Key words: Isoflurane ; Fat emulsions, intravenous ; Cognition disorders; Postoperative complications

Objective To observe the effect of human β-nerve growth factor (β-NGF) and brain-derived neurotrophic factor (BDNF) gene co-transfection on the differentiation of rat bone marrow stromal stem cells (BMSCs). Methods BMSCs were isolated from the femoral and tibial bone marrow of SD rats by density-gradient centrifugation and identified using immunocytochemistry of Src homology 3 (SH3). The first-passage BMSCs were transfected with pSVCEP-NGF-CAT and pSVCEP-BDNF-CAT plasmids via liposome, and also with pEGFP-C1 as the marker of transfection. After a 4-week cell culture, the expression of EGFP in the transfected cells was detected using fluorescence microscope, and the transfection efficiency was calculated. Laser scanning confocal microscope was employed to observe the expressions of the neural precursor cell markers NGF, BDNF, nestin, neuron specific enolase (NSE), NF and glial fibrillary acidic protein (GFAP) and evaluate the differentiation of the transfected cells. Results The primary and passaged BMSCs with high purity were obtained successfully without any decrease in the proliferation and differentiation potentials. The long spindle-shaped cells in the early passages displayed high proliferative activity, and immunocytochemistry of SH3 identified the purified cells as the bona fide BMSCs. NGF and BDNF genes were stably expressed in the BMSCs after the transfection, and the transfected cells were also positive for nestin, NSE, NF-M and GFAP. Conclusions Effective ectopic expression of human β-NGF and BDNF genes is achieved in rat BMSCs after liposome-mediated gene tmnsfection. The BMSCs are capable of differentiating into neuronal precursor cells that express several neural proteins and resemble the immature neurons or glial cells after induction with neurotrophic factors. Key words: Bone marrow stromal stem cells; Nerve growth factor; Brain-derived neurotrophic factor; Liposome-mediated transfection; Cell differentiation

Previous reports have shown that neuronal and glial cells express functionally active thrombin receptors. The thrombin receptor (PAR-1), a member of a growing family of protease activated receptors (PARs), requires cleavage of the extracellular amino-terminus domain by thrombin to induce signal transduction. Studies from our laboratory have shown that PAR-1 activation following the addition of thrombin or a synthetic thrombin receptor activating peptide (TRAP) induces motoneuron cell death both in vitro and in vivo. In addition to increasing motoneuron cell death, PAR- 1 activation leads to decreases in the mean neurite length and side branching in highly enriched motoneuron cultures. It has been suggested that motoneuron survival depends on access to sufficient target-derived neurotrophic factors through axonal branching and synaptic contacts. However, whether the thrombininduced effects on motoneurons can be prevented by neurotrophic factors is still unknown. Using highly enriched avian motoneuron cultures, we show here that alone, soluble chick skeletal muscle extracts (CMX), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), and glial cell line-derived neurotrophic factor (GDNF) significantly increased motoneuron survival compared to controls, whereas nerve growth factor (NGF) did not have a significant effect on motoneuron survival. Furthermore, cotreatment with muscle-derived agents (i.e., CMX, BDNF, GDNF) significantly prevented the death of motoneurons induced by alpha-thrombin. Yet, non-muscle-derived agents (CNTF and NGF) had little or no significant effect in reversing thrombin-induced motoneuron death. CMX and CNTF significantly increased the mean length of neurites, whereas NGF, BDNF, and GDNF failed to enhance neurite outgrowth compared to controls. Furthermore, CMX and CNTF significantly prevented thrombin-induced inhibition of neurite outgrowth, whereas BDNF and GDNF only partially reversed thrombin-induced inhibition of neurite outgrowth. These findings show differential effects of neurotrophic factors on thrombin-induced motoneuron degeneration and suggest specific overlaps between the trophic and stress pathways activated by some neurotrophic agents and thrombin, respectively.

Unilateral hippocampal lesions in newborn and adult rats: effects on spatial memory and BDNF gene expression

Brain-derived neurotrophic factor (BDNF) plays a key role in energy balance. In population studies, SNPs of the BDNF locus have been linked to obesity, but the mechanism by which these variants cause weight gain is unknown. Here, we examined human hypothalamic BDNF expression in association with 44 BDNF SNPs. We observed that the minor C allele of rs12291063 is associated with lower human ventromedial hypothalamic BDNF expression (p < 0.001) and greater adiposity in both adult and pediatric cohorts (p values < 0.05). We further demonstrated that the major T allele for rs12291063 possesses a binding capacity for the transcriptional regulator, heterogeneous nuclear ribonucleoprotein D0B, knockdown of which disrupts transactivation by the T allele. Binding and transactivation functions are both disrupted by substituting C for T. These findings provide a rationale for BDNF augmentation as a targeted treatment for obesity in individuals who have the rs12291063 CC genotype.

Endothelins (ETs) are suggested to be involved in functional alterations of astrocytes after brain injury, including proliferation, hypertrophy and production of neurotrophic factors. In this study, effects of Ala1,3,11,15-endothelin-1 (Ala1,3,11,15-ET-1), an ETB receptor selective agonist, on neurotrophic factor production were examined in rat brain. A continuous intracerebroventricular administration of Ala1,3,11,15-ET-1 (500 pmol/day for 7 days) increased the numbers of GFAP- and vimentin-positive astrocytes in the hippocampus, caudate putamen and cerebrum. Ala1,3,11,15-ET-1 did not induce neuronal degeneration and activation of microglia/macrophage in these brain regions. The intracerebroventricular administration of Ala1,3,11,15-ET-1 for 7 days caused two- to three-fold increases in glial cell line-derived neurotrophic factors (GDNF) mRNA in the hippocampus and cerebrum. The mRNA levels of brain-derived neurotrophic factors (BDNF) in caudate putamen were increased by Ala1,3,11,15-ET-1. Expressions of nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) mRNA in these regions were not largely affected by Ala1,3,11,15-ET-1, except cerebral NGF mRNA level was increased. The Ala1,3,11,15-ET-1-induced increases in GDNF and BDNF mRNA levels were accompanied by increases in immunoreactive GDNF and BDNF. Immunohistochemical observations showed that GFAP-positive astrocytes expressed GDNF and BDNF in the brain regions of Ala1,3,11,15-ET-1-infused rats. In cultured rat astrocytes, Ala1,3,11,15-ET-1 (100 nm) increased mRNA levels of GDNF and BDNF. These results suggest that activation of brain ETB receptors induced GDNF and BDNF expression in astrocytes.

Alterations of BDNF and NT-3 genes expression in the nucleus paragigantocellularis during morphine dependency and withdrawal

Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are two major members of the neurotrophin family. Using immunohistochemistry and in situ hybridization histochemistry, we examined the effect of L5 spinal nerve ligation (SPNL), a neuropathic pain model, on the expression of BDNF in the uninjured L4 dorsal root ganglion (DRG). After L5 SPNL, both immunoreactivity for BDNF and the hybridization intensity for BDNF mRNA increased mainly in the small- and medium-sized neurons. The percentage of BDNF mRNA-expressing neurons increased in the ipsilateral L4 DRG compared with the contralateral DRG from the third to 28th day after ligation. A significantly greater number of BDNF-immunoreactive neurons were observed in the ipsilateral L4 DRG than contralateral side 14 d after ligation. To test the contribution of BDNF to the thermal hyperalgesia produced in this model, we intrathecally injected anti-BDNF antibody at third day after ligation. This treatment clearly attenuated thermal hyperalgesia for a few hours. Almost all BDNF mRNA-expressing neurons coexpressed trkA, a high-affinity NGF receptor, mRNA. The percentage of BDNF mRNA-expressing cells of trkA cells significantly increased in the ipsilateral L4 DRG 14 d after ligation. Furthermore, we examined the contribution of NGF on this phenotypic change using ELISA, Northern blot analysis, and anti-NGF antibody. NGF content in the ipsilateral L4 DRG linearly increased and reached a statistical significant level 14 d after L5 SPNL. Moreover, at this time point, the increase in NGF mRNA was observed in the ipsilateral L5 DRG and sciatic nerve, but not in the ipsilateral L4 DRG or L4 spinal nerve. Local application of anti-NGF antibody to the L4 spinal nerve beside the L5 spinal nerve-ligation site prevented the development of thermal hyperalgesia for 5 d after ligation. Our data suggest that BDNF, which increased in the uninjured L4 DRG neurons, acts as a sensory neuromodulator in the dorsal horn and contributes to thermal hyperalgesia in this neuropathic pain model. The contribution of locally synthesized NGF to thermal hyperalgesia was also demonstrated. These dynamic alterations in the expression and content of BDNF and NGF in the uninjured DRG neurons might be involved in the pathomechanisms of neuropathic pain.

The microbiota is essential in the host's physiology, development, reproduction, immune system, nutrient metabolism, in brain chemistry and behavior. The gut microbiota plays a crucial role in the bidirectional gut–brain axis, a communication that integrates the gut and central nervous system (CNS) activities, and thus, the concept of microbiota–gut–brain axis is emerging where the microbes have considered as signaling components in the gut-brain axis. Animal studies reveals, in particular, that gut bacteria influence the brain-derived neurotrophic factor (BDNF) levels, and behavior specially after probiotic administration. How this alterations in brain chemistry are related to specific behavioral changes is unclear but it will likely be a focus of future research efforts. Among these animal studies, to our knowledge, no studies on the microbiota–gut–brain axis in zebrafish (Danio rerio) have been carried out. We hypothesized that a continuous administration of an exogenous probiotic might also influence the host's behavior and neurochemical gene expression. The purpose of this study was to determine whether probiotic strain can modulate gut commensal bacteria influencing brain neurochemistry and behavior in zebrafish. Thus, we treated adult zebrafish for 28 days with Lactobacillus rhamnosus, a probiotic strain which is one of the main components of the commensal microflora of human intestinal tract and it is widely used as a probiotic in mammals to adult male and female AB wild tipe zebrafish. We established differences between treated with probiotic strain and control group in shoaling behavior pattern, using a Video Tracker software; we quantified brain-derived neurotrophic factor (BDNF) gene expression by using RT-qPCR; we at last analyzed the microbiota profiles within two experimental groups by sing the culture-independent methods such as Denaturing Gradient Gel Electrophoresis (DGGE) and Next-Generation Sequencing (NGS). The probiotic treated group, compared to the control group, showed a statistically significant near two-fold increase in BDNF gene expression, different shoaling behavioural pattern and a shift in microbiota composition with a significant increase of Firmicutes and a reduction of Proteobacteria. The results of each approach may support the existence of a microbiota–gut–brain axis, in adult zebrafish and in line with numerous animal studies we can speculate that microbiota manipulation could influence behavior and brain expression of BDNF.

Differential effects of long-term treatment with typical and atypical antipsychotics on NGF and BDNF levels in rat striatum and hippocampus

Mice lacking the intracellular glucocorticoid‐regenerating enzyme 11β‐hydroxysteroid dehydrogenase type 1 (11β‐HSD1) are protected from age‐related spatial memory deficits. 11β‐HSD1 is expressed predominantly in the brain, liver and adipose tissue. Reduced glucocorticoid levels in the brain in the absence of 11β‐HSD1 may underlie the improved memory in aged 11β‐HSD1 deficient mice. However, the improved glucose tolerance, insulin sensitisation and cardioprotective lipid profile associated with reduced peripheral glucocorticoid regeneration may potentially contribute to the cognitive phenotype of aged 11β‐HSD1 deficient mice. In the present study, transgenic mice with forebrain‐specific overexpression of 11β‐HSD1 (Tg) were intercrossed with global 11β‐HSD1 knockout mice (HSD1KO) to examine the influence of forebrain and peripheral 11β‐HSD1 activity on spatial memory in aged mice. Transgene‐mediated delivery of 11β‐HSD1 to the hippocampus and cortex of aged HSD1KO mice reversed the improved spatial memory retention in the Y‐maze but not spatial learning in the watermaze. Brain‐derived neurotrophic factor (BDNF) mRNA levels in the hippocampus of aged HSD1KO mice were increased compared to aged wild‐type mice. Rescue of forebrain 11β‐HSD1 reduced BDNF mRNA in aged HSD1KO mice to levels comparable to aged wild‐type mice. These findings indicate that 11β‐HSD1 regenerated glucocorticoids in the forebrain and decreased levels of BDNF mRNA in the hippocampus play a role in spatial memory deficits in aged wild‐type mice, although 11β‐HSD1 activity in peripheral tissues may also contribute to spatial learning impairments in aged mice.

Objective To investigate the applied value of brain-derived neurotrophic factor (BDNF)-transfected adipose-derived stem cells (ADSCs) hy lentivirus in spinal transplantation.Methods Rat ADSCs were isolated and cultured in vitro under sterile conditions.Then they were transfected with GDNF by high titer lentivirus.Thirty-six SD rats were randomly divided into 3 groups,respectively treated with ADSCs transfected with GDNF by lentivirus (A),ADSCs (B),normal saline (C).Basso-Beattie-Bresnahan (BBB) score,immunofluorescence stain,and the expression levels of BDNF,nerve growth factor (NGF) and glial fibrillary acidic protein (GFAP) were observed.Results On the postoperative week (POW) 3 and 4,the BBB scores in group A were (15.02 ± 1.25) and (17.43 ± 1.05) points,obviously higher than in group B (10.35 ±2.25) and (13.44 ± 1.83) points and group C (5.88 ±2.46) and (6.94 ± 3.22) points.The immunofluorescence stain revealed that the transplanted ADSCs migrated to the injury site.On POW 3,the BDNF,NGF and GFAP expression levels in group A were (38.70 ± 2.75),(35.62 ± 2.74) and (13.17 ± 3.09) cells/HP,significantly different from those in group B (28.80 ±2.05),(24.99 ± 3.58) and (21.83 ± 5.99) cells/HP and group C (24.60 ± 2.44),(20.33 ± 2.25)and (27.10 ±6.47) cells/HP.Conclusion After transplantation of ADSCs in rats with spinal cord injury,the expression of BDNF and NGF was increased,and that of GFAP was suppressed,which was promoted by the modification of BDNF.Maybe it is one of the mechanisms of promoting restoration of injured spinal cord and improving motor function. Key words: Brain derived neural growth factor; Adipose derived mesenchymal stem cell; Acute spinal cord injury

To establish the co-culture model of cancer cells and nerve, and to study the influence of esophageal cancer on nerve fibers. Mouse dorsal root ganglion (DRG) was cultured in sterile conditions by primary isolation. Co-culture model was established using matrigel matrix-embedded DRG and EC109 (esophageal cancer cell line) transfected with green fluorescent protein. Morphological changes of DRG, number and area of neurites were quantified with microscopy and image analysis. Furthermore, the mRNA expression of nerve growth factor(NGF) and brain derived neurotrophic factor(BDNF) was detected by real-time PCR. In mixture cultivation model of EC109 and DRG cells, directional outgrowth of neurite projecting to EC109 was observed, and the length of neurite was markedly longer in proximal field compared to distal field. The number and area of neurite were 87 and 346 μm(2) in proximal field respectively, and 23 and 141 μm(2) in distal field on the 7th day. The expressions of NGF and BDNF were up-regulated in esophageal cancer cells. The esophageal cancer may play an important role in nerve fiber growth and guidance, which may be associated with the up-regulation of NGF and BDNF expressions.