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.

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.

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.

Molecular evidence of the neuroprotective effect of Ginkgo biloba (EGb761) using bax/bcl-2 ratio after brain ischemia in senescence-accelerated mice, strain prone-8.

In the present studies, we investigated the molecular mechanism of one of the active ingredients of Ginkgo biloba, EGb761, to affect the levels of several apoptotic markers in six brain regions following global ischemia in senescence-accelerated mice. A 4-day treatment with EGb761 significantly decreased bax/bcl-2 ratios in all brain regions in both young and aged mice. Our findings indicate that the bax/bcl-2 ratio provides a suitable index of apoptosis and modulation of these markers may explain the neuroprotective action of EGb761.

[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.

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.

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.

[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.

[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.

[Evaluation of neurological function following establishment of spinal cord hemisection model in rhesus].

OBJECTIVE: To establish spinal cord half-transection model in rhesus and investigate the Four neurological function and morphologic changes following spinal cord hemisection in rhesus. METHODS: Four cynomolgus monkeys were subjected to T-11 laminectomy and resection of a 1-mm length of hemispinal cord, while the controls underwent the identical laminectomy procedure but not the half-transection of the spinal cord. Neurological function of hindlimb was evaluated using modified Tarlov' grading, and cortical somatosensary evoked potentials (CSEP) were recorded in the 3rd postoperative month after spinal cord injury (SCI). The animals were sacrificed for histological examination. All the slices were processed with H-E staining and the number of neurons in the anterior horn of grey matter was counted. RESULTS: Irregular cavity was observed at the lesion site in the 3rd postoperative month. Distinct handicap of locomotor function in hindlimbs was observed in the half-transaction group immediately after SCI. As time went on, the locomotor function improved partially. Partial recovery of hindlimb function of adult monkey was noted in half-transection group from 14 days to 3 months after SCI, compared with that seen 24 hours postoperatively. The total number of neurons in the anterior horn of grey matter identical with hemitransection side was significantly smaller than that of the other side in the same segment (P < 0.05). CONCLUSION: A reproducible model of SCI in the nonhuman primate was established. Spontaneous partial recovery of the ipsilateral hindlimb function occurred in the monkey with spinal cord hemisected during different periods, which indicated the functional plasticity in the spinal cord after hemisection injury.

[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.

[Cloning the gene of rat premature nerve growth factor and constructing its eukaryotic expression vector pEGFP-NGF].

OBJECTIVE: To clone the entire coding sequence of rat premature nerve growth factor (NGF) beta subunit and construct its eukaryotic expression vector. METHODS: The gene of premature nerve growth factor (NGF) beta subunit was amplified by RT-PCR from SD rat brain. RT-PCR product was ligated into pMD 18-T Vector, the recombinant plasmid was identified by the restriction enzymes, PCR and DNA sequence analysis. Then the gene of premature nerve growth factor (NGF) beta subunit was cloned into the eukaryotic expression vector pEGFP-N1. The recombinant plasmid pEGFP-NGF was identified by the restriction enzymes analysis. RESULTS: The DNA sequence was identical to the published sequence encoding NGF gene, the restriction enzymes mapping product of the recombinant plasmid pEGFP-NGF was nearly 750 bp which matched the expected size. CONCLUSION: The entire coding sequence of premature nerve growth factor (NGF) beta subunit was successfully cloned and its eukaryotic expression vector pEGFP-NGF was constructed, which will provide the basis for studying the gene of NGF.

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.

[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.