Sodium Channel Voltage-Gated Beta 2 Plays a Vital Role in Brain Aging Associated with Synaptic Plasticity and Expression of COX5A and FGF-2.

The role of sodium channel voltage-gated beta 2 (SCN2B) in brain aging is largely unknown. The present study was therefore designed to determine the role of SCN2B in brain aging by using the senescence-accelerated mice prone 8 (SAMP8), a brain senescence-accelerated animal model, together with the SCN2B transgenic mice. The results showed that SAMP8 exhibited impaired learning and memory functions, assessed by the Morris water maze test, as early as 8 months of age. The messenger RNA (mRNA) and protein expressions of SCN2B were also upregulated in the prefrontal cortex at this age. Treatment with traditional Chinese anti-aging medicine Xueshuangtong (Panax notoginseng saponins, PNS) significantly reversed the SCN2B expressions in the prefrontal cortex, resulting in improved learning and memory. Moreover, SCN2B knockdown transgenic mice were generated and bred to determine the roles of SCN2B in brain senescence. A reduction in the SCN2B level by 60.68% resulted in improvement in the hippocampus-dependent spatial recognition memory and long-term potential (LTP) slope of field excitatory postsynaptic potential (fEPSP), followed by an upregulation of COX5A mRNA levels and downregulation of fibroblast growth factor-2 (FGF-2) mRNA expression. Together, the present findings indicated that SCN2B could play an important role in the aging-related cognitive deterioration, which is associated with the regulations of COX5A and FGF-2. These findings could provide the potential strategy of candidate target to develop antisenescence drugs for the treatment of brain aging.

Co-transplantation of GDNF-overexpressing neural stem cells and fetal dopaminergic neurons mitigates motor symptoms in a rat model of Parkinson's disease.

Striatal transplantation of dopaminergic (DA) neurons or neural stem cells (NSCs) has been reported to improve the symptoms of Parkinson's disease (PD), but the low rate of cell survival, differentiation, and integration in the host brain limits the therapeutic efficacy. We investigated the therapeutic effects of intracranial co-transplantation of mesencephalic NSCs stably overexpressing human glial-derived neurotrophic factor (GDNF-mNSCs) together with fetal DA neurons in the 6-OHDA rat model of PD. Striatal injection of mNSCs labeled by the contrast enhancer superparamagnetic iron oxide (SPIO) resulted in a hypointense signal in the striatum on T2-weighted magnetic resonance images that lasted for at least 8 weeks post-injection, confirming the long-term survival of injected stem cells in vivo. Co-transplantation of GDNF-mNSCs with fetal DA neurons significantly reduced apomorphine-induced rotation, a behavioral endophenotype of PD, compared to sham-treated controls, rats injected with mNSCs expressing empty vector (control mNSCs) plus fetal DA neurons, or rats injected separately with either control mNSCs, GDNF-mNSCs, or fetal DA neurons. In addition, survival and differentiation of mNSCs into DA neurons was significantly greater following co-transplantation of GDNF-mNSCs plus fetal DA neurons compared to the other treatment groups as indicated by the greater number of cell expressing both the mNSCs lineage tracer enhanced green fluorescent protein (eGFP) and the DA neuron marker tyrosine hydroxylase. The success of cell-based therapies for PD may be greatly improved by co-transplantation of fetal DA neurons with mNSCs genetically modified to overexpress trophic factors such as GDNF that support differentiation into DA cells and their survival in vivo.

How does the motor relearning program improve neurological function of brain ischemia monkeys?

The motor relearning program can significantly improve various functional disturbance induced by ischemic cerebrovascular diseases. However, its mechanism of action remains poorly understood. In injured brain tissues, glial fibrillary acidic protein and neurofilament protein changes can reflect the condition of injured neurons and astrocytes, while vascular endothelial growth factor and basic fibroblast growth factor changes can indicate angiogenesis. In the present study, we induced ischemic brain injury in the rhesus macaque by electrocoagulation of the M1 segment of the right middle cerebral artery. The motor relearning program was conducted for 60 days from the third day after model establishment. Immunohistochemistry and single-photon emission CT showed that the numbers of glial fibrillary acidic protein-, neurofilament protein-, vascular endothelial growth factor- and basic fibroblast growth factor-positive cells were significantly increased in the infarcted side compared with the contralateral hemisphere following the motor relearning program. Moreover, cerebral blood flow in the infarcted side was significantly improved. The clinical rating scale for stroke was used to assess neurological function changes in the rhesus macaque following the motor relearning program. Results showed that motor function was improved, and problems with consciousness, self-care ability and balance function were significantly ameliorated. These findings indicate that the motor relearning program significantly promoted neuronal regeneration, repair and angiogenesis in the surroundings of the infarcted hemisphere, and improve neurological function in the rhesus macaque following brain ischemia.

Directional induction of dopaminergic neurons from neural stem cells using substantia nigra homogenates and basic fibroblast growth factor.

To date, complex components of available reagents have been used for directional induction of neural stem cells into dopaminergic neurons, resulting in a poor ability to repeat experiments. This study sought to investigate whether a homogenate of the substantia nigra of adult rats and/or basic fibroblast growth factor could directionally induce neural stem cells derived from the subventricular zone of embryonic rats to differentiate into dopaminergic neurons. Tyrosine hydroxylase-positive cells were observed exclusively after induction with the homogenate supernatant of the substantia nigra from adult rats and basic fibroblast growth factor for 48 hours in vitro. However, in the groups treated with homogenate supernatant or basic fibroblast growth factor alone, tyrosine hydroxylase expression was not observed. Moreover, the content of dopamine in the culture medium of subventricular zone neurons was significantly increased at 48 hours after induction with the homogenate supernatant of the substantia nigra from adult rats and basic fibroblast growth factor. Experimental findings indicate that the homogenate supernatant of the substantia nigra from adult rats and basic fibroblast growth factor could directionally induce neural stem cells derived from the subventricular zone of embryonic rats to differentiate into dopaminergic neurons in the substantia nigra with the ability to secrete dopamine.

[Kallikrein-binding protein promotes axonal regeneration and protect rat retinal ganglion cells following optic nerve injury].

OBJECTIVE: To explore the effect of kallikrein-binding protein (KPB) in protecting retinal ganglion cells (RGCs) and promoting axonal regeneration following optical nerve injury in rats. METHODS: Crush injury of the optic nerve at 0.5-1.0 mm from the eyeball was induced in rats, which received subsequent KBP injection into the vitreous cavity (experimental group) and PBS injection (control group). At 7, 14 and 21 days after the injury, the rats were sacrificed and frozen sections of the eyeball were prepared to observe the structure and thickness of the retina and count the number of survival RGCs with HE staining. The optic nerves were collected for Western blotting to assess the effect of KBP on the RGCs and axonal regeneration. RESULTS: RGC counts and retinal thickness showed significant differences between the two groups. Western blotting also demonstrated a significant difference in the expression of the nerve regeneration marker protein GAP-43 between the two groups. CONCLUSION: KBP offers protection on RGCs and promotes regeneration of the optic nerve axons after optic nerve injury in rats.

[Changes in glial fibrillary acidic protein and growth-associated protein-43 expressions in retinal ganglial cells during axonal regeneration].

OBJECTIVE: To explore the changes in the expressions of glial fibrillary acidic protein (GFAP) and growth- associated protein-43 (GAP-43) in retinal ganglial cells after neural transplantation. METHODS: Thirty-nine rats were randomized into normal control group, nerve amputation group and nerve amputation with peripheral nerve transplantation group. Immunohistochemistry was used to detect the changes in the expressions of GFAP and GAP-43 at different time points after the operations, and real-time PCR was employed to detect the mRNA expressions of 13 genes in the retinal ganglial cells of the rats. RESULTS: Immunohistochemistry showed obviously increased GFAP expressions in the retina following the nerve amputation. GFAP expression was down-regulated while GAP-43 expression upregulated in the retinal ganglial cells after peripheral nerve transplantation. Real-time PCR results showed that 5 days after the operations, retinal GFAP and GAP-43 expressions increased significantly in the nerve amputation group and peripheral nerve transplantation groups as compared with those in the control group, but GAP-43 expression decreased significantly in the former two groups afterwards. CONCLUSION: The regenerated retina may adjust the production of GFAP. The retinal ganglial cells express GAP-43 during retinal regeneration. Up-regulation of the expression of GAP-43 provides the evidence for nerve regeneration following the nerve transplantation.

Microarray profile of brain aging-related genes in the frontal cortex of SAMP8.

This study examined the protein expression profile changes in the brain of senescence-accelerated mice/prone 8 (SAMP8) model. Two approaches, namely microarray and RT-PCR, were used in the study. Four genes, which are orthologous to human, were found to differentially express in the aging brain of mice. In this study, we examined the differentially expressed genes in the frontal cortex of the SAMP8 mice of two different ages (4 and 12 month old). Four orthologous genes (i.e., guanine nucleotide binding protein-alpha q polypeptide, kinesin family member 1B, sortilin 1, and somatostatin) showed significant changes in expression with aging. This study may provide important information on the mechanism of aging or aging-related diseases such as Alzheimer's diseases.

Effects of pcDNA3-beta-NGF gene-modified BMSC on the rat model of Parkinson's disease.

This study determined the effects of pcDNA3-beta-nerve growth factor (NGF) gene-modified bone marrow stromal cells (BMSC) on the rat model of Parkinson's disease (PD). The recombinant plasmid pcDNA3-beta-NGF was transfected into BMSC, and NGF expression and its biological activity in vitro were detected. BMSC modified by the NGF gene were then grafted into the corpus striatum of PD rats, and the rotation behavior was evaluated at 1, 2, 4, and 6 weeks post-transplantation. A significant improvement in rotation behavior was observed in PD rats subjected to cell transplantation, especially in PD rats receiving NGF-modified BMSC. The genetically modified BMSC survived and expressed beta-NGF but did not differentiate into tyrosine hydroxylase-positive cells in vivo. The present findings suggested that genetically modified BMSC could be effective for PD treatment, and the mechanisms might involve the neuroprotective effects of beta-NGF.

Transplantation of NGF-gene-modified bone marrow stromal cells into a rat model of Alzheimer' disease.

It is well known that bone marrow stromal cells (BMSC) grafted into the hippocampus of the rat model of Alzheimer's disease (AD) could survive and differentiate into cholinergic neurons as well as contribute towards functional restoration. The present study evaluated the effects of BMSC as a seed cell modified by nerve growth factor (NGF) gene into the hippocampus of AD rats. The beta-amyloid protein was injected bilaterally into the rat hippocampus to reproduce the AD model. After the human total RNA was extracted, the NGF gene was amplified by reverse transcription-polymerase chain reaction, then cloned into the pcDNA3. BMSC derived from a green fluorescence protein transgenic mouse were isolated, cultured, identified, and transfected by the NGF recombinant. The NGF-gene-modified BMSC were then transplanted into the hippocampus of AD rats. The results showed that implanted BMSC survived, migrated and expressed NGF as well as differentiated into ChAT-positive neurons. A significant improvement in learning and memory in AD rats was also seen in NGF-gene-modified BMSC group, when compared with the BMSC group. The present findings suggested that BMSC provided an effective carrier for delivery of NGF into AD rats, and the administration of NGF-gene-modified BMSC may be considered as a potential strategy for the development of effective therapies for the treatment of AD.

Bone marrow stromal cells of transgenic mice can improve the cognitive ability of an Alzheimer's disease rat model.

This study investigated the effects of bone marrow stromal cells transplantation on Alzheimer's disease (AD). Bone marrow stromal cells (BMSC) were obtained from the bone marrow of transgenic mice expressing green fluorescent protein and transplanted into the hippocampus of rats, which had received an injection of beta amyloid protein into the hippocampus 8 days earlier. Morris Water Maze test was used to observe behavior 2 weeks after transplantation. The survival and differentiation of the grafts were studied immunohistochemically. Behavior improved significantly in the transplanted group. The transplanted BMSC survived and presented ChAT-like neurons, indicating that these transplanted cells might differentiate into cholinergic neurons and the procedure could be a promising therapy for Alzheimer's disease.

[Changes in number of EGF positive neurons in ventral horn and contralateral cortex motor area of rhesus after hemisection spinal cord injury].

OBJECTIVE: To observe the changes in the amount of epidermal growth factor (EGF) immunopositive neurons in ventral horn and contralateral cortex motor area of rhesus following hemisection spinal cord injury (hSCI). METHODS: Eighteen adult healthy rhesus were randomly divided into six groups: Sham-operation group; Day 7, Day 14, Month 1. Month 2 and Month 3 hemisection spinal cord injury groups. In the hSCI groups, the monkeys were subjected to left hemisection of T11 spinal cord, and then were put to death at the corresponding time after operation. The rostral part 5 mm proximal to the lesioned point of spinal cord and the caudal part 5 mm distal to the lesioned point were taken from each monkey. The contralateral cortex motor area was taken out, too. Frozen sections were incubated in specific polyclonal anti-EGF antibody; the immunohistochemical SP method was adopted in the study. RESULTS: In 3 months after hSCI, the number of EGF immunopositive neurons in the ventral horn of spinal cord near the lesion and in the contralateral cortex motor area of brain decreased as compared with those of the sham-operation group (P<0.05). The number of positive neurons decreased first, then came back, and later after hSCI, decreased again (P<0.05). Besides this, the number of positive neurons varied in different parts at the same time point. CONCLUSION: The EGF immunopositive neurons decreased apparently in the ventral horn of spinal cord near the lesion and in the contralateral cortex motor area in 3 months after hSCI. Hemisection spinal cord injury affected the expression of EGF for motor neurons in ventral horn on the lesioned side as well as on the intact side. Early after hSCI the number of positive neurons decreased sharply and then came back spontaneously in the ventral horn of spinal cord near the lesion and in the contralateral cortex of brain.

Immunohistochemical distribution of NGF, BDNF, NT-3, and NT-4 in adult rhesus monkey brains.

Immunohistochemical distribution and cellular localization of neurotrophins was investigated in adult monkey brains using antisera against nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). Western blot analysis showed that each antibody specifically recognized appropriate bands of approximately 14.7 kDa, 14.2 kDa, 13.6 kDa, and 14.5 kDa, for NGF, BDNF, NT-3, and NT-4, respectively. These positions coincided with the molecular masses of the neurotrophins studied. Furthermore, sections exposed to primary antiserum preadsorbed with full-length NGF, BDNF, NT-3, and NT-4 exhibited no detectable immunoreactivity, demonstrating specificities of the antibodies against the tissues prepared from rhesus monkeys. The study provided a systematic report on the distribution of NGF, BDNF, NT-3, and NT-4 in the monkey brain. Varying intensity of immunostaining was observed in the somata and processes of a wide variety of neurons and glial cells in the cerebrum, cerebellum, hippocampus, and other regions of the brain. Neurons in some regions such as the cerebral cortex and the hippocampus, which stained for neurotrophins, also expressed neurotrophic factor mRNA. In some other brain regions, there was discrepancy of protein distribution and mRNA expression reported previously, indicating a retrograde or anterograde action mode of neurotrophins. Results of this study provide a morphological basis for the elucidation of the roles of NGF, BDNF, NT-3, and NT-4 in adult primate brains.

[Comparison of retrograde amnesia changes within different injury levels of cerebral concussion in rats].

OBJECTIVE: To investigate the retrograde amnesia changes within different injury levels of cerebral concussion in rats. METHODS: A metallic pendulum striker device of brain injury was deployed to duplicate CC models of different injury levels within Sprague-Dawley (S-D) rats. The investigated animals were divided into two groups according to classification standard, that is, Pure Cerebral Concussion (PCC) group and Complicated Cerebral Concussion (CCC) group. One control group was used, and each group included 8 animals. The retrograde amnesia of each group was assessed by Morris Water Maze (MWM) Test from 3 days preinjury to 7 days postconcussion. RESULTS: Compared with the control group, the retrograde amnesia was detected within 3 days in PCC group, and 5 days in CCC group after injury. At the same time, the two groups both manifested space recognition deficit. CONCLUSION: The retrograde amnesia existed in both pure cerebral concussion group and complicated cerebral concussion. Furthermore, the lasting time of retrograde amnesia in animals correlates to the injury level of brain concussion.

[Distribution and change of NGF, BDNF and NT3 in the hippocampus of rat with AD].

OBJECTIVE: To investigate the distribution and change of NGF, BDNF and NT3 in hippocampus of rat with Alzheimer disease(AD) by immunohistochemistry. METHODS: AD model was established by injecting beta amyloid protein into the hippocampus of rat. The rats were killed ten days after injection. The hippocampus sections were made coronally on a freezing microtome. Brain sections were processed by immunohistochemical procedure with molocloned antibodies against NGF, BDNF and NT3. The number of positive neurons of NGF, BDNF and NT3 was counted and analyzed statistically. RESULTS: It was found that the number of NGF positive neurons increased and the immunostaining intensity became stronger as compared with the control (P<0.01). The number of BDNF positive neurons decreased and the immunostaining intensity weakened (P<0.01). There Our was no change in the number and immunostaining intensity of NT3 positive neurons (P>0.05). CONCLUSION: results show that NGF, BDNF, NT3 have experienced different changes in hippocampus of rat with AD, suggesting that they play different roles in the course of AD and bear relation to the physiological function of cholinergic neurons in hippocampus of rat with AD. In particular, BDNF exerts crucial effect on the degeneration of neuronal function in the hippocampus of rat with AD.

[Distribution of epidermal growth factor in central nervous system of adult rhesus monkey].

OBJECTIVE: To investigate the distribution of epidermal growth factor (EGF) in the CNS of adult rhesus monkey. METHODS: Frozen sections were incubated in specific polyclonal anti-EGF antibody by the immunohistochemical SP method. RESULTS: The EGF immunopositive reaction was observed in the plasma of neurons. The neurons with strong positive immuno-reaction signals were detected in cerebral cortex, cerebellar Purkinje cells, cerebellar nuclei, pyramidal neurons of hippocampus, caudate nucleus, lentiform nucleus, claustrum, nuclei in diencephalons, substantia nigra, cranial nerve nuclei, reticular formation in brain stem, pontine nuclei, red nucleus, superior and inferior olivary nucleus, gracile nucleus, cuneate nucleus, also the ventral horn, lateral horn, dorsal horn and the central gray matter in spinal cord. Furthermore, a few EGF immunopositive glias and fibres were observed in some white matter of central nervous system. CONCLUSION: EGF immunoreactive material was extensively distributed in the CNS of the adult rhesus monkey. The results suggest that EGF may be concerned with various types of neurons and other cells.

Postmortem changes in the central nervous system and adrenal medulla of the heroin addicts.

This study describes the pathological changes in 20 heroin addicts (12 male and 8 female) autopsied 24 h after sudden death. The central nervous system (including the pituitary body) and the adrenal medulla were studied, along with those from age-matched controls who died from traffic accidents. Immunohistochemistry and histological (Hematoxylin and eosin) observation were performed. Some neuronal cells in every region of the CNS were positive for opioid receptors but these cells were most numerous in the hippocampus. Positive opioid fibers were most abundant in the basal ganglia region. Histopathology indicated coagulative changes of cytoplasm and dissolution of Nissl bodies of neuron. Edema of nerve fibers was frequently demonstrated. Pituitary body showed an evident decrease or even absence of basophils in the pars anterior. The adrenal medulla featured a down regulation of chromaffin granules. Degeneration of CNS neurons and fibers, alterations in hormonal and blood pressure regulation therefore would be the prime targets of heroin addiction in human subjects.

bcl2, bax, and nestin in the brains of patients with neurodegeneration and those of normal aging.

This study was conducted by employing specimens from the frontal cortices of Alzheimer, multiple-infarct dementia patients, and those of normal aging (age matched to patients). The objective was to evaluate and compare the bcl2, bax, and nestin patterns in these three groups. Using immunocytochemistry, it was observed that bcl2 and bax active sites were colocalized in 45% of cells in Alzheimer, 52% of cells in multiple infarct, and 30% of cells in normal aging. bcl2 and bax could also be separately located in cells of all three groups. bax cells were most prominent in number in Alzheimer patients and least prominent in normal aging. nestin was found in all three groups but was most prominent in the multiple-infarct patients. Both astrocytes and neurons demonstrated positive nestin sites. The difference in pattern between groups will lead to further understanding of cellular changes in neurodegenerative patients and those of normal aging.

[Effect of different FSB concentration and first-time exchange of total volume medium on proliferation of bone marrow stromal cells from GFP transgenic mouse in vitro].

OBJECTIVE: To evaluate the effect of different fetal bovine serum (FBS) concentration and first-time exchange of total volume medium on the proliferation of bone marrow stromal cells (BMSCs) from green fluorescence protein (GFP) transgenic mouse in vitro. METHODS: Bone marrow cells isolated from GFP transgenic mice were cultured in DMEM/F12 containing 10%, 20%, 30% FBS respectively; the first exchange of the total volume medium was made at different times (4 h, 6 h, 8 h, 10 h, 12 h, 24 h, 48 h and 72 h) after 3 d primary culture; then the total volume medicum was exchanged every three days. The amplification of BMSCs was determined. The passage 5 BMSCs cultured in DMEM/F12 containing 10% and 20% FBS were examined with the antibodies CD44, CD45 and CD54 at the time of first exchange of the total volume medium. RESULTS: The cultured cells proliferated well in DMEM/F12 containing 10% FBS and 20% FBS. With the extension of the time for first exchange of total volume medium, the density of the adhered cells increased, but the purity of BMSCs decreased. CONCLUSION: The method of making cells adhere to culture plastic in different time for cultivating and purifying BMSCs from GFP transgenic mice is effective, the appropriate concentration of FBS is 10%-20% and the best time for the the first exchange of total volume medium is 8 hour.

[Establishment of maca mulatta model of resuscitation after selective cerebral ultra-deep hypothermic blood flow occlusion].

OBJECTIVE: To study the feasibility of resuscitation after selective cerebral ultra-deep hypothermia and blood flow occlusion. METHODS: Ten 4-10 year-old maca mulattas were divided into 3 groups: four-vessel occlusion group, two-vessel occlusion group and identical temperature perfusion group. MRI were examinated before and after operation, the vital signs and the hemodynamical parameters were observed during the experiment, neurological deficient evaluation was performed after operation. RESULTS: In all of the ten monkeys, the hemodynamical parameters of two-vessel occulation were steady during the operation, and all of them lived after filling 60 minutes. MRI were normal after operation, and the function of neurological deficient scale was normal. The others of identical temperature perfusion group and four-vessel occlusion group were not resuscitation after filling 60 minutes and died. CONCLUSION: Monkey could resuscitate from selective cerebral ultra-deep hypothermia and blood flow occlusion of bilateral common carotid artery in 60 minutes.

Effects of acupuncture on the expression of glial cell line-derived neurotrophic factor (GDNF) and basic fibroblast growth factor (FGF-2/bFGF) in the left sixth lumbar dorsal root ganglion following removal of adjacent dorsal root ganglia.

This investigation studied the temporal changes in the expression of GDNF and FGF-2 in the left sixth lumbar (L6) dorsal root ganglion (DRG) after acupuncture in adult cats subjected to unilateral removal of adjacent DRG. The cats were divided into three groups. Group I were normal control animals. Group II cats were subjected to removal of DRG associated with the left L1-L5 and L7-S2 spinal nerves, sparing the L6 DRG. Group III cats received similar treatment as Group II ones, but in addition were subjected to acupuncture on the left side at acupuncture points (xuewei) the day after the operation. Both Groups II and III animals were perfused under anesthesia at 7 and 14 days post-operation (dpo) and their left DRG were processed for the immunohistochemical demonstration of GDNF and FGF-2. Following removal of adjacent DRG, the average number of all GDNF stained neurons in L6 DRG was decreased at 7 dpo and more so at 14 dpo. Acupuncture reversed this trend, as demonstrated in the increased average number of immunopositive small-to-medium sized neurons. FGF-2 expression was also less marked at 7 dpo but returned to normal at 14 dpo. Acupuncture significantly increased the average number of FGF-2 positive neurons compared with that in operated animals. This increase was observed in both large and small-to-medium sized neurons. In conclusion, our results demonstrated that the average number of GDNF and FGF-2 neurons in L6 DRG was decreased after unilateral removal of adjacent DRG but acupuncture could reverse some of the changes.