神经生长因子和高压氧对新生鼠缺氧缺血性脑损伤后神经再生的影响

新生儿窒息所致缺氧缺血性脑损伤(hypoxic-ischemic brain damage,HIBD),是造成新生儿死亡和导致神经系统后遗症的主要原因,目前临床上以对症支持治疗为主,尚无特效疗法.近年不少文献报道采用神经生长因子(nerve growth factor,NGF)和高压氧(hyperbaric oxygenation,HBO)治疗新生儿HIBD,但疗效不肯定.本研究于2006年4～11月通过建立新生鼠HIBD模型,给予外源性NGF和HBO早期治疗,并结合5-溴脱氧尿嘧啶(BrdU)标记技术,通过动态检测新生鼠脑缺氧缺血后海马齿状回(dentate gyrua,DG)巢蛋白(Nestin)表达、Brdu标记阳性神经细胞数的变化,比较NGF和HBO对新生鼠HIBD后神经增殖再生的影响.

Several studies have demonstrated that L-carnitine exhibits neuroprotective effects on injured sciatic nerve of rats with diabetes mellitus. It is hypothesized that L-carnitine exhibits neuro-protective effects on injured sciatic nerve of rats. Rat sciatic nerve was crush injured by a forceps and exhibited degenerative changes. After intragastric administration of 50 and 100 mg/kg L-carnitine for 30 days, axon area, myelin sheath area, axon diameter, myelin sheath diameter, and numerical density of the myelinated axons of injured sciatic nerve were similar to normal, and the function of injured sciatic nerve also improved signiifcantly. These ifndings suggest that L-carnitine exhibits neuroprotective effects on sciatic nerve crush injury in rats.

Schwann cells, nerve regeneration promoters in peripheral nerve tissue engineering, can be used to repair both the peripheral and central nervous systems. However, isolation and puriifcation of Schwann cells are complicated by contamination with ifbroblasts. Current reported measures are mainly limited by either high cost or complicated procedures with low cell yields or purity. In this study, we collected dorsal root ganglia from neonatal rats from which we obtained highly puriifed Schwann cells using serum-free melanocyte culture medium. The purity of Schwann cells (> 95%) using our method was higher than that using standard medium containing fetal bovine serum. The obtained Schwann cells were implanted into poly(lactic-co-glycolic acid)/chi-tosan conduits to repair 10-mm sciatic nerve defects in rats. Results showed that axonal diameter and area were signiifcantly increased and motor functions were obviously improved in the rat sciatic nerve tissue. Experimental ifndings suggest that serum-free melanocyte culture medium is conducive to purify Schwann cells and poly(lactic-co-glycolic acid)/chitosan nerve conduits combined with Schwann cells contribute to restore sciatic nerve defects.

There are few studies on the neuroprotective effects of syringaldehyde in a rat model of cerebral ischemia. The study aimed to elucidate the mechanisms underlying the neuroprotective effects of syringaldehyde on ischemic brain cells. Rat models of cerebral ischemia were intraperitoneally administered syringaldehyde. At 6 and 24 hours after syringaldehyde administration, cell damage in the brain of cerebral ischemia rats was obviously reduced, superoxide dismutase activity and nuclear respiratory factor 1 expression in the brain tissue were markedly increased, malondi-adehyde level was obviously decreased, apoptosis-related cysteine peptidase caspase-3 and -9 immunoreactivity was obviously decreased, and neurological function was markedly improved. These ifndings suggest that syringaldehyde exerts neuroprotective effects on cerebral ischemia injury through anti-oxidation and anti-apoptosis.

Various retinal injuries induced by ocular hypertension have been shown to induce plastic chang-es in retinal synapses, but the potential regulatory mechanism of synaptic plasticity after retinal injury was still unclear. A rat model of acute ocular hypertension was established by injecting saline intravitreally for an hour, and elevating the intraocular pressure to 14.63 kPa (110 mmHg). Western blot assay and immunolfuorescence results showed that synaptophysin expression had a distinct spatiotemporal change that increased in the inner plexiform layer within 1 day and spread across the outer plexiform layer after 3 days. Glial ifbrillary acidic protein expression in retinae was greatly increased after 3 days, and reached a peak at 7 days, which was also consistent with the peak time of synaptophysin expression in the outer plexiform layer following the in-creased intraocular pressure. Fluorocitrate, a glial metabolic inhibitor, was intravitreally injected to inhibit glial cell activation following high intraocular pressure. This signiifcantly inhibited the enhanced glial ifbrillary acidic protein expression induced by high intraocular pressure injury. Synaptophysin expression also decreased in the inner plexiform layer within a day and the wid-ened distribution in the outer plexiform layer had disappeared by 3 days. The results suggested that retinal glial cell activation might play an important role in the process of retinal synaptic plasticity induced by acute high intraocular pressure through affecting the expression and distri-bution of synaptic functional proteins, such as synaptophysin.

The spinal cord has the ability to regenerate but the microenvironment generated after trauma reduces that capacity. An increase in Src family kinase (SFK) activity has been implicated in neuropathological conditions associated with central nervous system trauma. Therefore, we hypothesized that a decrease in SFK activation by a long-term treatment with 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyramidine (PP2), a selective SFK inhibitor, after spinal cord contusion with the New York University (NYU) impactor device would generate a permissive environment that improves axonal sprouting and/or behavioral activity. Results demonstrated that long-term blockade of SFK activation with PP2 increases locomotor activity at 7, 14, 21 and 28 days post-injury in the Basso, Beattie, and Bresnahan open ifeld test, round and square beam crossing tests. In addition, an increase in white matter spared tissue and sero-tonin ifber density was observed in animals treated with PP2. However, blockade of SFK activity did not change the astrocytic response or inifltration of cells from the immune system at 28 days post-injury. Moreover, a reduced SFK activity with PP2 diminished Ephexin (a guanine nucle-otide exchange factor) phosphorylation in the acute phase (4 days post-injury) after trauma. Together, these ifndings suggest a potential role of SFK in the regulation of spared tissue and/or axonal outgrowth that may result in functional locomotor recovery during the pathophysiolo-gy generated after spinal cord injury. Our study also points out that ephexin1 phosphorylation (activation) by SFK action may be involved in the repulsive microenvironment generated after spinal cord injury.

In addition to its lipid-lowering effect, atorvastatin exerts anti-inflammatory and antioxidant effects as well. In this study, we hypothesized that atorvastatin could protect against cerebral isch-emia/reperfusion injury. The middle cerebral artery ischemia/reperfusion model was established, and atorvastatin, 6.5 mg/kg, was administered by gavage. We found that, after cerebral ischemia/reperfusion injury, levels of the inflammation-related factors E-selectin and myeloperoxidase were upregulated, the oxidative stress-related marker malondialdehyde was increased, and super-oxide dismutase activity was decreased in the ischemic cerebral cortex. Atorvastatin pretreatment signiifcantly inhibited these changes. Our ifndings indicate that atorvastatin protects against ce-rebral ischemia/reperfusion injury through anti-inlfammatory and antioxidant effects.

Our previous study showed that when glutamate receptor (GluR)6 C terminus-containing pep-tide conjugated with the human immunodeficiency virus Tat protein (GluR6)-9c is delivered into hippocampal neurons in a brain ischemic model, the activation of mixed lineage kinase 3 (MLK3) and c-Jun NH2-terminal kinase (JNK) is inhibited via GluR6-postsynaptic density pro-tein 95 (PSD95). In the present study, we investigated whether the recombinant adenovirus (Ad) carrying GluR6c could suppress the assembly of the GluR6-PSD95-MLK3 signaling module and decrease neuronal cell death induced by kainate in hippocampal CA1 subregion. A seizure model in Sprague-Dawley rats was induced by intraperitoneal injections of kainate. The effect of Ad-Glur6-9c on the phosphorylation of JNK, MLK3 and mitogen-activated kinase kinase 7 (MKK7) was observed with western immunoblots and immunohistochemistry. Our findings revealed that overexpression of GluR6c inhibited the interaction of GluR6 with PSD95 and prevented the kainate-induced activation of JNK, MLK3 and MKK7. Furthermore, kainate-mediated neuronal cell death was signiifcantly suppressed by GluR6c. Taken together, GluR6 may play a pivotal role in neuronal cell death.

Previous studies have shown that neuroiflament protein M expression is upregulated in the early stage of spinal cord ischemia/reperfusion injury, indicating that this protein may play a role in the injury process. In the present study, we compared protein expression in spinal cord tissue of rabbits after 25 minutes of ischemia followed by 0, 12, 24, or 48 hours of reperfusion with that of sham operated rabbits, using proteomic two-dimensional gel electrophoresis and mass spec-trometry. In addition, the nerve repair-related neurofilament protein M with the unregulated expression was detected with immunohistochemistry and western blot analysis. Two-dimen-sional gel electrophoresis and mass spectrometry showed that, compared with the sham group, upregulation of protein expression was most signiifcant in the spinal cords of rabbits that had undergone ischemia and 24 hours of reperfusion. Immunohistochemical analysis revealed that neuroiflament protein M was located in the membrane and cytoplasm of neuronal soma and axons at each time point after injury. Western blot analysis showed that neuroiflament protein M expression increased with reperfusion time until it peaked at 24 hours and returned to baseline level after 48 hours. Furthermore, neuroiflament protein M is phosphorylated under oxidative stress, and expression changes were parallel for the phosphorylated and non-phosphorylated forms. Neurofilament protein M plays an important role in spinal cord ischemia/reperfusion injury, and its functions are achieved through oxidative phosphorylation.

Previous studies have conifrmed that the beclin 1 complex plays a key role in the initial stage of autophagy and deregulated autophagy might involve in amyotrophic lateral sclerosis. However, the mechanism underlying altered autophagy associated with the beclin 1 complex remains un-clear. In this study, we transfected the Cu/Zn superoxide dismutase 1 G93A mutant protein into the motor neuron-like cell line NSC34 cultured in vitro. Western blotting and co-immunopre-cipitation showed that the Cu/Zn superoxide dismutase 1 G93A mutant enhanced the turnover of autophagic marker microtubule-associated protein light chain 3II (LC3II) and stimulated the conversion of EGFP-LC3I to EGFP-LC3II, but had little inlfuence on the binding capacity of the autophagy modulators ATG14L, rubicon, UVRAG, and hVps34 to beclin 1 during auto-phagosome formation. These results suggest that the amyotrophic lateral sclerosis-linked Cu/Zn superoxide dismutase 1 G93A mutant can upregulate autophagic activity in NSC34 cells, but that this does not markedly affect beclin 1 complex components.

In the present study, we transplanted adipose-derived mesenchymal stem cells into the hippo-campi of APP/PS1 transgenic Alzheimer’s disease model mice. Immunofluorescence staining revealed that the number of newly generated (BrdU+) cells in the subgranular zone of the dentate gyrus in the hippocampus was signiifcantly higher in Alzheimer’s disease mice after adipose-de-rived mesenchymal stem cell transplantation, and there was also a significant increase in the number of BrdU+/DCX+neuroblasts in these animals. Adipose-derived mesenchymal stem cell transplantation enhanced neurogenic activity in the subventricular zone as well. Furthermore, adipose-derived mesenchymal stem cell transplantation reduced oxidative stress and alleviated cognitive impairment in the mice. Based on these ifndings, we propose that adipose-derived mes-enchymal stem cell transplantation enhances endogenous neurogenesis in both the subgranular and subventricular zones in APP/PS1 transgenic Alzheimer’s disease mice, thereby facilitating functional recovery.

Platelet-rich plasma containing various growth factors can promote nerve regeneration. An in-side-out vein graft can substitute nerve autograft to repair short nerve defects. It is hypothesized that an inside-out vein graft iflled with platelet-rich plasma shows better effects in the repair of short sciatic nerve defects. In this study, an inside-out vein autograft iflled with platelet-rich plasma was used to bridge a 10 mm-long sciatic nerve defect in rats. The sciatic nerve function of rats with an inside-out vein autograft iflled with platelet-rich plasma was better improved than that of rats with a simple inside-out vein autograft. At 6 and 8 weeks, the sciatic nerve function of rats with an inside-out vein autograft filled with platelet-rich plasma was better than that of rats undergoing nerve autografting. Compared with the sciatic nerve repaired with a simple inside-out vein autograft, the number of myelinated axons was higher, axon diameter and myelin sheath were greater in the sciatic nerve repaired with an inside-out vein autograft iflled with platelet-rich plasma and they were similar to those in the sciatic nerve repaired with nerve autograft. These findings suggest that an inside-out vein graft filled with platelet-rich plasma can substitute nerve autograft to repair short sciatic nerve defects.

Melatonin has been shown to diminish edema in rats. Melatonin can be used to treat spinal cord injury. This study presumed that melatonin could relieve spinal cord edema and examined how it might act. Our experiments found that melatonin (100 mg/kg, i.p.) could reduce the water content of the spinal cord, and suppress the expression of aquaporin-4 and glial ifbrillary acidic protein after spinal cord injury. This suggests that the mechanism by which melatonin alleviates the damage to the spinal cord by edema might be related to the expression of aquaporin-4 and glial ifbrillary acidic protein.

The Chinese compound Kaixin Jieyu Fang can be used to treat vascular depression;however, the underlying mechanism remains unclear. This study established a rat model of chronic cerebral ischemia-caused white matter damage by ligation of the bilateral common carotid arteries. Rats received daily intragastric administration of a suspension of Kaixin Jieyu Fang powder. After 3, 7 and 21 days of treatment, the degree of white matter damage in the cerebral ischemia rat model was alleviated, Bcl-2 protein and mRNA expression in brain tissue increased, and Bax protein and mRNA expression decreased. These results indicate that Kaixin Jieyu Fang can alleviate cere-bral white matter damage, and the underlying mechanism is associated with regulation of Bcl-2/Bax protein and mRNA expression, which is one of possible mechanism behind the protective effect of Kaixin Jieyu Fang against vascular depression.

The purpose of this work was to investigate whether, by intranasal administration, the nerve growth factor bypasses the blood-brain barrier and turns over the spinal cord neurons and if such therapeutic approach could be of value in the treatment of spinal cord injury. Adult Sprague-Dawley rats with intact and injured spinal cord received daily intranasal nerve growth factor administration in both nostrils for 1 day or for 3 consecutive weeks. We found an in-creased content of nerve growth factor and enhanced expression of nerve growth factor receptor in the spinal cord 24 hours after a single intranasal administration of nerve growth factor in healthy rats, while daily treatment for 3 weeks in a model of spinal cord injury improved the deifcits in locomotor behaviour and increased spinal content of both nerve growth factor and nerve growth factor receptors. These outcomes suggest that the intranasal nerve growth factor bypasses blood-brain barrier and affects spinal cord neurons in spinal cord injury. They also suggest exploiting the possible therapeutic role of intranasally delivered nerve growth factor for the neuroprotection of damaged spinal nerve cells.

Danhong injection (DHI), a Chinese Materia Medica standardized product extracted from Radix Salviae miltiorrhizae and Flos Carthami tinctorii, is widely used in China for treating acute isch-emic stroke. In the present study, we explored the neuroprotective efifcacy of DHI in a rat model of temporary middle cerebral artery occlusion, and evaluated the potential mechanisms under-lying its effects. Pretreatment with DHI (0.9 and 1.8 mL/kg) resulted in a signiifcantly smaller infarct volume and better neurological scores than pretreatment with saline. Furthermore, DHI significantly reduced the permeability of the blood-brain barrier, increased occludin protein expression and decreased neutrophil inifltration, as well as profoundly suppressing the upreg-ulation of matrix metallopeptidase-9 expression seen in rats that had received vehicle. Matrix metallopeptidase-2 expression was not affected by ischemia or DHI. Moreover, DHI (1.8 mL/kg) administered 3 hours after the onset of ischemia also improved neurological scores and reduced infarct size. Our results indicate that the neuroprotective efifcacy of DHI in a rat model of ce-rebral ischemia-reperfusion injury is mediated by a protective effect on the blood-brain barrier and the reversal of neutrophil inifltration.

The purpose of this study was to assess fetal bovine acellular dermal matrix as a scaffold for supporting the differentiation of bone marrow mesenchymal stem cells into neural cells fol-lowing induction with neural differentiation medium. We performed long-term, continuous observation of cell morphology, growth, differentiation, and neuronal development using several microscopy techniques in conjunction with immunohistochemistry. We examined speciifc neu-ronal proteins and Nissl bodies involved in the differentiation process in order to determine the neuronal differentiation of bone marrow mesenchymal stem cells. The results show that bone marrow mesenchymal stem cells that differentiate on fetal bovine acellular dermal matrix display neuronal morphology with unipolar and bi/multipolar neurite elongations that express neuro-nal-speciifc proteins, includingβIII tubulin. The bone marrow mesenchymal stem cells grown on fetal bovine acellular dermal matrix and induced for long periods of time with neural differen-tiation medium differentiated into a multilayered neural network-like structure with long nerve ifbers that was composed of several parallel microifbers and neuronal cells, forming a complete neural circuit with dendrite-dendrite to axon-dendrite to dendrite-axon synapses. In addition, growth cones with filopodia were observed using scanning electron microscopy. Paraffin sec-tioning showed differentiated bone marrow mesenchymal stem cells with the typical features of neuronal phenotype, such as a large, round nucleus and a cytoplasm full of Nissl bodies. The data suggest that the biological scaffold fetal bovine acellular dermal matrix is capable of supporting human bone marrow mesenchymal stem cell differentiation into functional neurons and the subsequent formation of tissue engineered nerve.

The role of type-2 astrocytes in the repair of central nervous system injury remains poorly un-derstood. In this study, using a relatively simple culture condition in vitro, type-2 astrocytes, differentiated from oligodendrocyte precursor cells by induction with bone morphogenetic pro-tein-4, were co-cultured with dorsal root ganglion neurons. We examined the effects of type-2 astrocytes differentiated from oligodendrocyte precursor cells on the survival and growth of dorsal root ganglion neurons. Results demonstrated that the number of dorsal root ganglion neurons was higher following co-culture of oligodendrocyte precursor cells and type-2 astrocytes than when cultured alone, but lower than that of neurons co-cultured with type-1 astrocytes. The length of the longest process and the length of all processes of a single neuron were shortest in neurons cultured alone, followed by neurons co-cultured with type-2 astrocytes, then neurons co-cultured with oligodendrocyte precursor cells, and longest in neurons co-cultured with type-1 astrocytes. These results indicate that co-culture with type-2 astrocytes can increase neuronal survival rate and process length. However, compared with type-1 astrocytes and oligodendrocyte precursor cells, the promotion effects of type-2 astrocytes on the growth of dorsal root ganglion neurons were weaker.

Angelica sinensis has antioxidative and neuroprotective effects. In the present study, we aimed to determine the neuroprotective effect of polysaccharides isolated from Angelica sinensis. In a pre-liminary experiment, Angelica sinensis polysaccharides not only protected PC12 neuronal cells from H2O2-induced cytotoxicity, but also reduced apoptosis and intracellular reactive oxygen species levels, and increased the mitochondrial membrane potential induced by H2O2 treatment. In a rat model of local cerebral ischemia, we further demonstrated that Angelica sinensis poly-saccharides enhanced the antioxidant activity in cerebral cortical neurons, increased the number of microvessels, and improved blood lfow after ischemia. Our ifndings highlight the protective role of polysaccharides isolated from Angelica sinensis against nerve cell injury and impairment caused by oxidative stress.

Ganoderma lucidum is a traditional Chinese medicine, which has been shown to have both an-ti-oxidative and anti-inlfammatory effects, and noticeably decreases both the infarct area and neuronal apoptosis of the ischemic cortex. This study aimed to investigate the protective effects and mechanisms of pretreatment with ganoderma lucidum (by intragastric administration) in cerebral ischemia/reperfusion injury in rats. Our results showed that pretreatment with ganoder-ma lucidum for 3 and 7 days reduced neuronal loss in the hippocampus, diminished the content of malondialdehyde in the hippocampus and serum, decreased the levels of tumor necrosis fac-tor-αand interleukin-8 in the hippocampus, and increased the activity of superoxide dismutase in the hippocampus and serum. hTese results suggest that pretreatment with ganoderma lucidum was protective against cerebral ischemia/reperfusion injury through its anti-oxidative and an-ti-inlfammatory actions.