碱性成纤维细胞生长因子、表皮生长因子对脑缺血大鼠内源性神经干细胞增殖的影响

目的探讨碱性成纤维细胞生长因子(bFGF)、表皮生长因子(EGF)对大鼠局灶性脑缺血模型内源性神经干细胞增殖的影响。方法通过大脑中动脉阻塞法建立大鼠的局灶性脑缺血模型,随机分组后分别皮下注射生理盐水、bFGF、EGF以及bFGF EGF;每日1次,共3d,此后每3d1次。采用免疫组化法,以5溴脱氧嘧啶尿苷(Brdu)标记神经干细胞,观察并比较各组大鼠制模后第7d、14d、21d侧脑室室管膜下区(SVZ)和海马齿状回Brdu阳性细胞的表达。结果制模后,各组大鼠双侧SVZ和海马齿状回均出现Brdu阳性细胞,且阳性细胞数随时间递减;与对照组相比,药物干预组Brdu阳性细胞数显著增加(P<0.05～0.01);与单药组相比,bFGF EGF组(联合组)Brdu阳性细胞数增加更明显(P<0.05～0.01);各药物干预组在制模第7dBrdu阳性细胞数最多(P<0.05～0.01)。结论皮下注射bFGF、EGF可促进脑缺血大鼠模型内源性神经干细胞的增殖;bFGF和EGF联合应用对脑缺血大鼠神经干细胞的增殖效应有协同作用。     

Bridging peripheral nerves using a deacetyl chitin conduit combined with short-term electrical stimulation

Previous studies have demonstrated that deacetyl chitin conduit nerve bridging or electrical stimulation can effectively promote the regeneration of the injured peripheral nerve. We hypoth-esized that the combination of these two approaches could result in enhanced regeneration. Rats with right sciatic nerve injury were subjected to deacetyl chitin conduit bridging combined with electrical stimulation （0.1 ms, 3 V, 20 Hz, for 1 hour）. At 6 and 12 weeks after treatment, nerve conduction velocity, myelinated axon number, ifber diameter, axon diameter and the thickness of the myelin sheath in the stimulation group were better than in the non-stimulation group. The results indicate that deacetyl chitin conduit bridging combined with temporary electrical stimu-lation can promote peripheral nerve repair.     

Response of the sensorimotor cortex of cerebral palsy rats receiving transplantation of vascular endothelial growth factor 165-transfected neural stem cells

Neural stem cells are characterized by the ability to differentiate and stably express exogenous ge- nes. Vascular endothelial growth factor plays a role in protecting local blood vessels and neurons of newborn rats with hypoxic-ischemic encephalopathy. Transplantation of vascular endothelial growth factor-transfected neural stem cells may be neuroprotective in rats with cerebral palsy. In this study, 7-day-old Sprague-Dawley rats were divided into five groups： （1） sham operation （control）, （2） cerebral palsy model alone or with （3） phosphate-buffered saline, （4） vascular en- dothelial growth factor 165 ＋ neural stem cells, or （5） neural stem cells alone. The cerebral palsy model was established by ligating the left common carotid artery followed by exposure to hypox- ia. Phosphate-buffered saline, vascular endothelial growth factor ＋ neural stem cells, and neural stem cells alone were administered into the sensorimotor cortex using the stereotaxic instrument and microsyringe. After transplantation, the radial-arm water maze test and holding test were performed. Immunohistochemistry for vascular endothelial growth factor and histology using hematoxylin-eosin were performed on cerebral cortex. Results revealed that the number of vas- cular endothelial growth factor-positive cells in cerebral palsy rats transplanted with vascular endothelial growth factor-transfected neural stem cells was increased, the time for finding water and the finding repetitions were reduced, the holding time was prolonged, and the degree of cell degeneration or necrosis was reduced. These findings indicate that the transplantation of vascu- lar endothelial growth factor-transfected neural stem cells alleviates brain damage and cognitive deficits, and is neuroprotective in neonatal rats with hypoxia ischemic-mediated cerebral palsy.     

Intrathecal injection of epidermal growth factor and fibroblast growth factor 2 promotes proliferation of neural precursor cells in the spinal cords of mice with mutant human SOD1 gene

We investigated three steps of neural precursor cell activation--proliferation, migration, and differentiation--in amyotrophic lateral sclerosis spinal cord treated with intrathecal infusion of epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2) into the lumbar spinal cord region of normal and symptomatic transgenic (Tg) mice with a mutant human Cu/Zn superoxide dismutase (SOD1) gene. We observed that 5-bromodeoxyuridine (BrdU) + nestin double-labeled neural precursor cells increased in the spinal cords of Tg mice compared with non-Tg mice, with a much greater increase produced by EGF and FGF2 treatment. The number of BrdU + nestin double-labeled cells was larger than that of BrdU + ionized calcium-binding adapter molecule-1 (Iba1), BrdU + glial fibrillary acidic protein (GFAP), or BrdU + highly polysialylated neural cell adhesion molecule (PSA-NCAM) double-labeled cells, but none expressed neuronal nuclear antigen (NeuN). On further analysis of the gray matter of Tg mice, the number of BrdU + nestin and BrdU + PSA-NCAM double-labeled cells increased more in the ventral horns than the dorsal horns, which was again greatly enhanced by EGF and FGF2 treatment. Because neural precursor cells reside close to the ependyma of central canal, the present study suggests that proliferation and migration of neural precursor cells to the ventral horns is greatly activated in symptomatic Tg mice and is further enhanced by EGF and FGF2 treatment and, furthermore, that the neural precursor cells preferentially differentiate into neuronal precursor cells instead of astrocytes in Tg mice with EGF and FGF2 treatment.     

脑梗死大鼠神经前体细胞增殖水平的研究

目的研究脑梗死病灶周围及海马处神经前体细胞增殖水平的动态变化。方法采用易卒中型肾性高血压大鼠(RHRSP)，电凝大脑中动脉(MCA)主干制成脑梗死(MCAO)模型。行大鼠神经功能评定，免疫组化观察并计数梗死灶边缘、对侧镜区及双侧海马5-溴脱氧尿核苷(Bromodeoxyuridine，BrdU)标记的细胞。结果MCAO后大鼠神经功能评分减低，5d时恢复正常。MCAO后梗死灶边缘、对侧镜区及双侧海马均有BrdU阳性细胞分布，且病灶侧多于病灶对侧，集中分布于病灶周围。结论脑缺血可诱导神经前体细胞增殖并移向病灶，可能成为脑梗死恢复的重要物质基础。     

Neuronal apoptosis in cerebral ischemia/reperfusion area following electrical stimulation of fastigial nucleus

Previous studies have indicated that electrical stimulation of the cerebellar fastigial nucleus in rats may reduce brain infarct size, increase the expression of Ku70 in cerebral ischemia/ reperfusion area, and decrease the number of apoptotic neurons. However, the anti-apoptotic mechanism of Ku70 remains unclear. In this study, fastigial nucleus stimulation was given to rats 24, 48, and 72 hours before cerebral ischemia/reperfusion injury. Results from the electrical stim- ulation group revealed that rats exhibited a reduction in brain infarct size, a significant increase in the expression of KuT0 in cerebral ischemia/reperfusion regions, and a decreased number of terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL）-positive cells. Double immunofluorescence staining revealed no co-localization of Ku70 with TUNEL-positive cells. However, Ku70 partly co-localized with Bax protein in the cytoplasm of rats with cerebral ischemia/reperfusion injury. These findings suggest an involvement of Ku70 with Bax in the cy- toplasm of rats exposed to electrical stimulation of the cerebellar fastigial nucleus, and may thus provide an understanding into the anti-apoptotic activity of KuT0 in cerebral ischemia/reperfu- sion injury.     

Acute hepatocyte growth factor treatment induces long-term neuroprotection and stroke recovery via mechanisms involving neural precursor cell proliferation and differentiation

Hepatocyte growth factor (HGF) is an interesting candidate for acute stroke treatment as shown by continuous infusion or gene delivery protocols. However, little is known about HGF-mediated long-term effects. The present study therefore analyzed long-term effects of an acute intrastriatal HGF treatment (5 μg) after a 45-minute stroke, with regard to brain injury and neurologic recovery. Hepatocyte growth factor induced long-term neuroprotection as assessed by infarct volume and neuronal cell death analysis for as long as 4 weeks after stroke, which was associated with sustained neurologic recovery as evidenced by corner-turn and tight-rope tests. Analyzing underlying mechanisms of HGF-induced sustained neuroprotection, enhanced cell proliferation followed by increased neuronal differentiation of neural precursor cells (NPCs) was observed in the ischemic striatum of HGF-treated mice, which persisted for up to 4 weeks. In line with this, HGF promoted neurosphere formation as well as proliferation of NPC and decreased caspase-3-dependent hypoxic injury in vitro. Preservation of blood–brain barrier integrity 24 hours after stroke was furthermore noticed in animals receiving HGF, which was associated with the inhibition of matrix metalloproteases (MMP)-2 and MMP-9 at 4 and 24 hours, respectively. We suggest that sustained recruitment of proliferating cells together with improved neurovascular remodeling provides an explanation for HGF-induced long-term neuroprotection.     

碱性成纤维细胞因子在局灶性脑缺血后内源性神经干细胞增殖过程中对β-catenin的影响

BACKGROUND： The Wnt/β-catenin signaling pathway plays an important role in neural development. ,β-catenin is an important component of the Wnt/β-catenin signaling pathway. The Wnt signaling pathway has been shown to regulate the interaction of neural stem cells with the extracellular matrix.
OBJECTIVE： To investigate the effects of basic fibroblast growth factor （bFGF） on β-catenin protein and mRNA expression, and on hippocampal neural stem cell proliferation in a rat model of cerebral ischemia/reperfusion. DESIGN, TIME AND SETTING： A randomized, controlled, neurobiology experiment was performed in Shenyang Medical College between August 2006 and August 2008. MATERIALS： A total of 72 healthy male Wistar rats, aged 3 months, were used in this study. bFGF was provided by Beijing SL Pharmaceutical Co.,Ltd., China. METHODS： Rats were randomly divided into 3 groups： sham-operated, ischemia/reperfusion, and bFGF-treated （n = 24 per group）. Focal cerebral ischemia/reperfusion was induced in rats from the ischemia/reperfusion group and the bFGF-treated group by 2 hour right middle cerebral artery occlusion and 2 hour restoration of blood flow using the suture method. The ischemia/reperfusion and bFGF-treated groups were intraperitoneally administered 500 IU/mL of bFGF, or the same volume of physiological saline, once a day at postoperative days 1 3, and once every 3 days thereafter. Simultaneously, the sham-operated group underwent experimental procedures identical to the ischemia/reperfusion and bFGF-treated groups, with the exception of ischemia/reperfusion induction and drug administration. At 2 hours, 2, 6, 13, and 20 days after ischemiaJreperfusion induction, 50 mg/kg bromodeoxyuridine （BrdU） was administered to each group, twice daily, to label proliferating neural stem cells. MAIN OUTCOME MEASURES： The effects of bFGF on BrdU labeling, and ,8 -catenin mRNA and protein expression, in neural stem cells were examined by immunohistochemistry, Western blot, RT-PCR, and in situ hybridization techniques. RESULTS： In the sham-operated group, only a few BrdU-immunoreactive neural stem cells were found. In the ischemia/reperfusion group, BrdU-immunoreactive cells began to increase from 3 days after ischemia/reperfusion induction, reached a peak level at 7 days, and gradually reduced from 21 days. At 3, 7, 14, and 21 days after ischemia/reperfusion induction, the numbers of BrdU-immunoreactive cells were significantly greater in the bFGF-treated group than in the ischemia/reperfusion group. The sham-operated group exhibited slight expression of β-catenin and β-catenin mRNA. In the ischemia/reperfusion group, the expression of β-catenin and β-catenin mRNA gradually increased with reperfusion time, peaked at 14 days after reperfusion, and gradually decreased thereafter; by 21 days, the expression was markedly lower. Following bFGF injection, the expression of hippocampal BrdU, β-catenin, and β-catenin mRNA had apparently increased in each group. CONCLUSION： bFGF promotes neural stem cell proliferation, and the expression of β-catenin and β-catenin mRNA in the ischemic brain tissue. These findings indicate that bFGF promotion of neural stem cell proliferation may be mediated by Wnt/β-catenin signaling pathway.     

大鼠脑梗死后神经前体细胞的增殖及电针作用的实验研究

目的　研究脑梗死病灶周围及海马处神经前体细胞增殖水平的动态变化及电针治疗对其的影响。方法　采用易卒中型肾性高血压大鼠 (RHRSP) ,电凝法凝闭大脑中动脉 (MCAO)。用Garcia等的综合评分法评定大鼠的神经行为学功能 ,免疫组化观察梗死灶边缘、对侧镜区及双侧海马 5 溴脱氧尿核苷 (Bromodeoxyuridine,BrdU)标记细胞的变化。结果　MCAO后大鼠轻偏瘫 ,5天时神经行为学功能恢复正常。MCAO后梗死灶边缘、双侧镜区及双侧海马均有BrdU阳性细胞分布 ,且病灶侧多于病灶对侧 ,病灶周围分布密集。电针治疗促使梗死灶边缘BrdU阳性细胞增多 ,随着治疗时间增加细胞增多更明显。结论　脑梗死可诱导病灶周边及海马神经前体细胞增殖水平上调 ,2周内神经前体细胞随着电针治疗时间的增加而增多。神经前体细胞可能是脑梗死康复的重要物质基础。     

Electrical stimulation accelerates nerve regeneration and functional recovery in delayed peripheral nerve injury in rats

The present study aims to investigate the potential of brief electrical stimulation (ES; 3 V, 20 Hz, 20 min) in improving functional recovery in delayed nerve injury repair (DNIR). The sciatic nerve of Sprague Dawley rats was transected, and the repair of nerve injury was delayed for different time durations (2, 4, 12 and 24 weeks). Brief depolarizing ES was applied to the proximal nerve stump when the transected nerve stumps were bridged with a hollow nerve conduit (5 mm in length) after delayed periods. We found that the diameter and number of regenerated axons, the thickness of myelin sheath, as well as the number of Fluoro‐Gold retrograde‐labeled motoneurons and sensory neurons were significantly increased by ES, suggesting that brief ES to proximal nerve stumps is capable of promoting nerve regeneration in DNIR with different delayed durations, with the longest duration of 24 weeks. In addition, the amplitude of compound muscle action potential (gastrocnemius muscle) and nerve conduction velocity were also enhanced, and gastrocnemius muscle atrophy was partially reversed by brief ES, indicating that brief ES to proximal nerve stump was able to improve functional recovery in DNIR. Furthermore, brief ES was capable of increasing brain‐derived neurotrophic factor (BDNF) expression in the spinal cord in DNIR, suggesting that BDNF‐mediated neurotrophin signaling might be one of the contributing factors to the beneficial effect of brief ES on DNIR. In conclusion, the present findings indicate the potential of using brief ES as a useful method to improve functional recovery for delayed repair of peripheral nerve lesions.     

Transient axonal glycoprotein-1 induces apoptosis- related gene expression without triggering apoptosis in U251 glioma cells

Previous studies show that transient axonal glycoprotein-1, a ligand of amyloid precursor pro- tein, increases the secretion of amyloid precursor protein intracellular domain and is involved in apoptosis in Alzheimer＇s disease. In this study, we examined the effects of transient axonal glyco- protein-1 on U251 glioma cells. 3-（4,5-Dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide assay showed that transient axonal glycoprotein-1 did not inhibit the proliferation of U251 cells, but promoted cell viability. The terminal deoxynucleotidyl transferase dUTP nick end labeling assay showed that transient axonal glycoprotein-1 did not induce U251 cell apoptosis. Real-time PCR revealed that transient axonal glycoprotein-1 substantially upregulated levels of amyloid precursor protein intracellular C-terminal domain, and p53 and epidermal growth factor recep- tor mRNA expression. Thus, transient axonal glycoprotein-1 increased apoptosis-related gene expression in U251 cells without inducing apoptosis. Instead, transient axonal glycoprotein-1 promoted the proliferation of these glioma cells.     

Bumetanide promotes neural precursor cell regeneration and dendritic development in the hippocampal dentate gyrus in the chronic stage of cerebral ischemia

Bumetanide has been shown to lessen cerebral edema and reduce the infarct area in the acute stage of cerebral ischemia. Few studies focus on the effects of bumetanide on neuroprotection and neurogenesis in the chronic stage of cerebral ischemia. We established a rat model of cerebral ischemia by injecting endothelin-1 in the left cortical motor area and left corpus striatum. Seven days later, bumetanide 200 μg/kg/day was injected into the lateral ventricle for 21 consecutive days with a mini-osmotic pump. Results demonstrated that the number of neuroblasts cells and the total length of dendrites increased, escape latency reduced, and the number of platform crossings increased in the rat hippocampal dentate gyrus in the chronic stage of cerebral ischemia. These findings suggest that bumetanide promoted neural precursor cell regeneration, dendritic development and the recovery of cognitive function, and protected brain tissue in the chronic stage of ischemia.     

FGF和EGF对神经干细胞增殖及分化的影响

胚胎和成年哺乳动物脑内均存在的神经干细胞,成纤维细胞生长因子和表皮生长因子对神经干细胞的增殖及分化有一定的影响,ＦＧＦ和ＥＧＦ及其受体在胚胎期和成年期表达各异。ＦＧＦ和ＥＧＦ能促进神经干细胞增殖,在不同的条件下对分化和作用不同。     

Description of distributed features of the nestin‐containing cells in brains of adult mice: A potential source of neural precursor cells

The distribution of neural precursor cells (NPCs) in adult mice brain has so far not been described. Therefore, we investigated the distribution of NPCs by analyzing the nestin‐containing cells (NCCs) in distinct brain regions of adult nestin second‐intron enhancer‐controlled LacZ reporter transgenic mice through LacZ staining. Results showed that NCCs existed in various regions of adult mouse brain. In cerebellum, the greatest number of NCCs existed in cortex of the simple lobule, followed by cortex of the cerebellar lobule. In olfactory bulb, NCCs were most numerous in the granular cell layer, followed by the mitral cell layer and the internal plexiform, glomerular, and external plexiform layers. In brain nuclei (nu), NCCs were most numerous in the marginal nu, followed by the brainstem and diencephalon nu. NCCs in sensory nu of brainstem were more numerous than in motor nu, and NCCs in the dorsal of sensory nu were more numerous than in the ventral part. In brain ventricle systems, NCCs were largely distributed in the center of and external to the lateral ventricle, the inferior part of the third ventricle, the dorsal and inferior parts of the fourth ventricle, and the gray matter around the cerebral aqueduct. NCCs in the left vs. right brain were not significantly different. These data collectively indicate that NCCs were extensively distributed in the cerebellum and olfactory bulb, the partial nu of the marginal system, the partial brain nu adjacent to brain ventricle systems, the subependymal zone, and the cerebral cortex around the marginal lobe and were a potential source of NPCs. © 2009 Wiley‐Liss, Inc.     

碱性成纤维生长因子和神经生长因子联合应用培养成年大鼠海马神经干细胞向神经元样细胞的分化

背景：影响神经干细胞向神经元分化的因素很多,各种营养因子可以不同程度地刺激神经干细胞向神经元分化,如何使神经干细胞大量分化为神经元是研究的热点问题。 目的：观察联合应用碱性成纤维生长因子和神经生长因子对成年大鼠海马神经干细胞为神经元的影响。 方法：无菌条件下分离大鼠脑海马组织,传至第4代克隆球直径约为200 μm时,滴加DMEM/F12+2% B27+20 μg/L表皮生长因子+20 μg/L碱性成纤维细胞生长因子,进行单细胞克隆培养,传代的神经干细胞分成空白对照组、碱性成纤维细胞生长因子组、神经生长因子组、碱性成纤维细胞生长因子+神经生长因子组。观察传代后的克隆球进行神经干细胞免疫细胞化学染色鉴定,计数神经元特异性烯醇化酶阳性细胞率,检测神经干细胞向神经元的分化情况。 结果与结论：①单细胞克隆培养后,克隆球细胞表达巢蛋白,诱导分化后神经元特异性烯醇化酶、胶质纤维酸性蛋白均呈阳性表达。②与空白对照组神经干细胞分化为神经元的比例比较,碱性成纤维细胞生长因子组、神经生长因子组、碱性成纤维细胞生长因子组+神经生长因子组均明显提高(P < 0.05),且碱性成纤维细胞生长因子组+神经生长因子组神经元的比例最高(P < 0.05)。提示,碱性成纤维细胞生长因子可以提高神经生长因子诱和神经生长因子均可促进神经干细胞向神经元分化,且二者联合应用效果更佳。     

功能性电刺激促进急性脑梗死大鼠脑部内源性神经干细胞增殖的研究

目的 研究功能性电刺激(FES)对急性脑梗死大鼠行为学和内源性神经干细胞(NSC)增殖的影响.探讨FES治疗改善脑梗死后神经功能的机制. 方法 54只成年雄性SD大鼠按随机数字表法分为FES治疗组、安慰刺激组和假手术组(每组各18只).行大脑中动脉阻断(MCAO)制作局灶性脑梗死模型后第3天,FES治疗组开始接受FES治疗(10 min/d,每天1次),安慰刺激组阻断动脉但不予电刺激.在FES治疗后3、7、14d评价大鼠行为学功能(平衡木行走测评、转棒上行走测评、网屏试验),免疫组织化学法观察大鼠海马齿状回和室管膜下区NSCs巢蛋白(nestin)的表达水平.Western blot法检测梗死侧脑组织nestin总蛋白表达量. 结果 FES治疗组网屏试验评分在治疗后14 d时低于安慰刺激组,比较差异有统计学意义(P<0.05).FES治疗组大鼠海马齿状回和室管膜下区nestin阳性细胞数和梗死侧脑组织nestin总蛋白表达量在治疗后7d、14d时均高于安慰刺激组和假手术组,比较差异有统计学意义(P<0.05).结论 FES能促进急性脑梗死大鼠脑部内源性NSCs的增殖并改善大鼠行为学功能,这可能是FES治疗改善脑梗死后神经功能的机制之一.     

Short-term environmental enrichment exposure induces proliferation and maturation of doublecortin-positive cells in the prefrontal cortex

Previous studies have demonstrated that doublecortin-positive immature neurons exist pre- dominantly in the superficial layer of the cerebral cortex of adult mammals such as guinea pigs, and these neurons exhibit very weak properties of self-proliferation during adulthood under physiological conditions. To verify whether environmental enrichment has an impact on the proliferation and maturation of these immature neurons in the prefrontal cortex of adult guinea pigs, healthy adult guinea pigs were subjected to short-term environmental enrichment. Animals were allowed to play with various cognitive and physical stimulating objects over a period of 2 weeks, twice per day, for 60 minutes each. Immunofluorescence staining results indicated that the number of doublecortin-positive cells in layer II of the prefrontal cortex was significantly increased after short-term environmental enrichment exposure. In addition, these doublecortin-positive cells co-expressed 5-bromo-2-deoxyuridine （a marker of cell prolifera- tion）, c-Fos （a marker of cell viability） and NeuN （a marker of mature neurons）. Experimental findings showed that short-term environmental enrichment can induce proliferation, activation and maturation of doublecortin-positive cells in layer II of the prefrontal cortex of adult guinea pigs.     

Hyperbaric oxygen treatment promotes neural stem cell proliferation in the subventricular zone of neonatal rats with hypoxic-ischemic brain damage

Hyperbaric oxygen therapy for the treatment of neonatal hypoxic-ischemic brain damage has been used clinically for many years, but its effectiveness remains controversial. In addition, the mechanism of this potential neuroprotective effect remains unclear. This study aimed to investigate the influence of hyperbaric oxygen on the proliferation of neural stem cells in the subventricular zone of neonatal Sprague-Dawley rats (7 days old) subjected to hypoxic-ischemic brain damage. Six hours after modeling, rats were treated with hyperbaric oxygen once daily for 7 days. Immunohistochemistry revealed that the number of 5-bromo-2′-deoxyuridine positive and nestin positive cells in the subventricular zone of neonatal rats increased at day 3 after hypoxic-ischemic brain damage and peaked at day 5. After hyperbaric oxygen treatment, the number of 5-bromo-2′- deoxyuridine positive and nestin positive cells began to increase at day 1, and was significantly higher than that in normal rats and model rats until day 21. Hematoxylin-eosin staining showed that hyperbaric oxygen treatment could attenuate pathological changes to brain tissue in neonatal rats, and reduce the number of degenerating and necrotic nerve cells. Our experimental findings indicate that hyperbaric oxygen treatment enhances the proliferation of neural stem cells in the subventricular zone of neonatal rats with hypoxic-ischemic brain damage, and has therapeutic potential for promoting neurological recovery following brain injury.