Effect of Tongxinluo on basic fibroblast growth factor mRNA after cerebral ischemia/reperfusion injury

INTRODUCTION Neural stem cells (NSCs) are characterized by self-renewing and mul- ti-differentiated potencies. Exogenous factors, such as growth factors and cytokines, have great effects on the proliferation and differentia- tion of NSCs. Some researches …     

Differential induction of nerve growth factor and basic fibroblast growth factor mRNA in neonatal and aged rat brain

Stimulation of glucocorticoid or β-adrenergic receptors (BAR) has been shown to increase nerve growth factor (NGF) biosynthesis in adult rat brain. Little is known about the role of these receptors in the regulation of NGF expression in neonatal and aged brain. We have examined the effect of the synthetic glucocorticoid dexamethasone (DEX) and the BAR agonist clenbuterol (CLE) on the levels of NGF mRNA in neonatal (8 day old), adult (3 month old) and aged (24 month old) rats. By 3 h, DEX (0.5 mg/kg, s.c.) evoked a comparable increase in NGF mRNA in the cerebral cortex and hippocampus in both 8-day and 3-month-old rats. In contrast, CLE (10 mg/kg, i.p.) failed to change NGF mRNA levels in neonatal rats, while increasing (2–3-fold) NGF mRNA levels in the cerebral cortex of adult rats. In 24-month-old rats, both DEX and CLE elicited only a modest increase in NGF mRNA. This increase was, however, anatomically and temporally similar to that observed in adult animals. The weak effect of DEX or CLE was not related to a down-regulation of receptor function because both DEX and CLE were able to elicit a comparable increase in the mRNA levels for basic fibroblast growth factor (FGF2) in neonatal, adult and aged rat brain. Our data demonstrate that induction of NGF expression by neurotransmitter/hormone receptor activation varies throughout life and suggest that pharmacological agents might be useful tools to enhance trophic support in aging.     

Epidermal growth factor exerts neuronotrophic effects on dopaminergic and GABAergic CNS neurons: comparison with basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) have been described to exert neuronotrophic effects on central nervous system neurons in culture. To study the selectivity of trophic actions of these growth factors, neurotransmitter-identified populations of embryonic rat mesencephalon were used. At 20 days in vitro, EGF (3 ng/ml) promoted survival and neurite outgrowth from these neurons. The neuritogenic effect of bFGF (3 ng/ml) was, however, more robust. Quantitative analysis with the neurofilament monoclonal antibody RR97 and ELISA confirmed the differential response, bFGF being 2-2.5 times more effective at all concentrations tested (ED100: 3-10 ng/ml for both EGF and bFGF). At 10 days in vitro, EGF displayed no trophic activity--even at 30 ng/ml. Treatment of mesencephalic cultures with EGF (3 ng/ml) for 20 days stimulated [3H]dopamine and [14C]GABA uptakes about 4-fold. While bFGF (3 ng/ml) also stimulated GABA uptake some 4-fold, dopamine uptake was increased almost 20-fold. Thus, EGF is also capable of enhancing the transmitter traits of selected central neuronal populations; however, the actions of bFGF appear to preferentially address dopaminergic cells.     

Localization of basic fibroblast growth factor,a mitogen and angiogenic factor,in human brain tumors

Summary Fibroblast growth factor (FGF) is a potent angiogenic factor and a mitogen for a variety of mesoderm-and neuroectoderm-derived cell types (e.g., fibroblasts, endothelial cells, astrocytes, oligodendrocytes). After application of a monospecific polyclonal antiserum, we localized basic FGF on frozen sections of 73 human brain tumors using immunohisto-chemistry. FGF was present in a variable number of tumor cells (16/16 astrocytomas, 5/5 ependymomas, 0/3 benign and 4/7 anaplastic oligodendrogliomas, 11/12 glioblastomas, 11/11 meningiomas, 6/6 neurilemmomas, 0/3 pituitary adenomas, 2/2 choroid plexus papillomas, 0/1 neurocytoma, 2/2 benign fibrous histiocytomas, 2/5 metastatic carcinomas). FGF was detected in vascular cells of 59 tumors and in fibroblasts of connective tissue stroma from all papillomas and metastases. These results tend to indicate FGF involvement in the malignant progression of gliomas due to an autocrine or paracrine action. Histopathological aspects of malignant gliomas (e.g., pseudopalisading or pathological vessels) could be related to FGF activity.     

脑室内注射碱性成纤维细胞生长因子治疗大鼠创伤生脑外伤的实验研究

目的 探讨脑室内注射碱性成纤维细胞生长因子(bFGF)对创伤性脑外伤大鼠是否具有治疗作用. 方法 24只成年SD大鼠按随机数字表法分为治疗组和对照组,采用改良的Feeney氏自由落体撞击方法建立大鼠创伤性脑外伤模型,致伤后24 h分别给予脑室内注射bFGF和等量的生理盐水;采用行为测试试验(前肢放置试验、平衡试验)评分观察肢体功能恢复情况;采用免疫组化法,以5溴脱氧嘧啶尿苷(Brdu)标记神经干细胞,观察并比较两组大鼠致伤后第3、7、14天侧脑室室管膜下区(SVZ)、海马齿状回及损伤区域Brdu阳性细胞的表达. 结果 治疗组前肢放置试验评分在第3～12天、平衡试验在第3～11天的评分低于对照组,比较差异有统汁学意义(P<0.05).治疗组双侧SVZ、海马齿状回和损伤区域出现的Brdu阳性细胞数较对照组明显增多,差异有统计学意义(P<0.05).两组损伤侧Brdu阳性细胞数均高于损伤对侧,比较差异有统计学意义(P<0.05).行为学评分与Brdu阳性细胞数之间呈负相关关系. 结论 脑室内注射bFGF有助于创伤性脑外伤大鼠模型内源性神经干细胞的增殖,并能促进其肢体功能的恢复.     

外源性碱性成纤维细胞生长因子对缺血大鼠脑内源性碱性成纤维细胞生长因子表达的影响

目的 研究外源性碱性成纤维细胞生长因子(bFGF)缩小局灶性脑缺血梗死灶的机制。方法 用免疫组化ABC法检测在局灶性脑缺血模型上给予生理盐水或bFGF后早期生长反应蛋白-1(Egr-1)，bFGF，碱性成纤维细胞生长因子受体(bFGFR)的动态表达。结果 给药组在3h～3d各时间段梗死灶均有不同程度的缩小。对照组和给药组Egr-1表达均表现为3～6h的增强过程，但给药组更强于对照组。对照组12h见有bFGF表达增强，而bFGFR表达3h到达高峰，6h起下降，12h时bFGFR的表达已恢复至正常水平(出现了配体和受体表达时相上不匹配)。给药组bFGF表达提前且增强，3h即见有bFGF表达增强，6h时出现第一峰，从而与bFGFR 3～6h的表达增强过程相吻合。结论 外源性bFGF能缩小梗死灶，该神经保护作用是通过Egr-1蛋白高表达使内源性bFGF的表达增高且提前，从而与bFGFR的表达增强过程重叠而实现的。     

Increased levels of basic fibroblast growth factor (bFGF) following focal brain injury

Focal injury to the mammalian central nervous system (CNS) results in a cascade of cellular responses - including glial and capillary proliferation and neural sprouting - that contribute to the repair of neural tissue and to the recovery of neurological function. Fibroblast growth factors (FGFs) are heparin-binding polypeptides with potent trophic effects on CNS glia, endothelia, and neurons; both acidic and basic forms are found in the mammalian CNS. We used heparin-affinity chromatography coupled to Balb/c 3T3 mitogenic assay to show a marked increase in levels of bioactive FGFs in tissue surrounding focal cortical lesions of the mature rat brain at one week after injury. Heparin-affinity HPLC showed that this increase was due to a large increase in levels of basic FGF (bFGF), and a much smaller increase in levels of acidic FGF (aFGF) after injury. Increased bFGF bioactivity was paralleled by increased levels of immunoreactive bFGF, as assessed by Western blotting techniques. Increased bFGF levels may play an important role in the cascade of cellular reactions occurring after focal brain injury.     

Developmental and regional expression of basic fibroblast growth factor mRNA in the rat central nervous system

A cDNA clone encoding rat basic fibroblast growth factor (bFGF) was used as a hybridization probe to determine the level of bFGF mRNA during rat brain development as well as in different adult rat brain regions. In the rat brain, a 3.7kb bFGF mRNA was detected together with lower levels of two minor bFGF mRNA species of 1.8kb and 1.5kb, respectively. The 3.7kb bFGF mRNA was detected in the rat brain already at embryonic day 16, the earliest time point tested. The embryonic brain contained 1.5 to 2 times higher levels of the 3.7kb bFGF mRNA than the adult brain. The amount of the 3.7kb bFGF mRNA in the adult rat brain was approximately 50 times higher than the level of beta-nerve growth factor mRNA in the rat brain. bFGF mRNA was found in all 12 brain regions tested in the adult rat brain with the highest level in colliculi, cerebral cortex, thalamus, and olfactory bulb. The lowest levels were found in pons and medulla oblongata. All three bFGF mRNA species showed the same regional distribution in the brain. In contrast to nerve growth factor mRNA, the level of bFGF mRNA in the neonatal hippocampus was slightly decreased 10 days after a cholinergic denervation by transection of the fimbria-fornix.     

Immunolocalization of basic fibroblast growth factor and its receptor in adult goldfish retina.

The neural retina of teleost fish can regenerate following surgical or neurotoxic lesions. As a first attempt to uncover the factors important for the regenerative response, we used immunocytochemistry to demonstrate the presence of basic fibroblast growth factor (bFGF) and its receptor in the goldfish retina. The bFGF-immunoreactivity was present throughout the retina, but was most intense in photoreceptor cells, especially cones, and Müller glia. Immunoreactivity for the bFGF receptor was strongest in the axon terminals of photoreceptors, both rods and cones. This pattern of immunolocalization is especially interesting since the proliferating cells that are thought to be responsible for generating the neural regenerate are located among the photoreceptor axon terminals. These proliferating cells have been identified as rod precursors because in the intact retina they give rise only to rod photoreceptors. When the neural retina is damaged, however, rod precursors are thought to be the source of proliferating neuroepithelial cells responsible for generating the retinal regenerate. The role played by bFGF in normal neurogenesis, cell differentiation, and/or neuronal regeneration in the fish retina has yet to be determined.     

Basic fibroblast growth factor and epidermal growth factor exert differential trophic effects on CNS neurons

Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) are potent mitogenic proteins capable of inducing cell division in a wide variety of cell types. In addition to their mitogenic properties, both proteins have recently been shown to enhance survival and process outgrowth from neurons of central nervous system origin. The full spectrum of neuronal subtypes responding to these factors has not been elucidated. In the present study, EGF was found to enhance survival and process outgrowth of primary cultures of cerebellar neurons of neonatal rat brain. This effect was dose-dependent and was observed with EGF concentrations as low as 100 pg/ml. In marked contrast, bFGF was ineffective in enhancing survival or neurite elongation from cerebellar neurons when tested in the range of 0.1 to 10.0 ng/ml. However, within this concentration range, bFGF did prove effective in stimulating an increase in [3H]thymidine incorporation into primary cultures of cerebellar astrocytes, demonstrating that bFGF was active and that cells in the cerebellum do respond to bFGF. These results suggest that EGF or an EGF-like peptide may act as a neurite elongation and maintenance factor for cerebellar neurons. EGF has now been shown to support striatal, cortical, and cerebellar neurons, suggesting that this factor may have trophic activity throughout the central nervous system. bFGF, in contrast, appears to exert its effects on limited populations of neurons.     

Differential expression of fibroblast growth factor-2 and fibroblast growth factor receptor 1 in a scarring and nonscarring model of CNS injury in the rat

Injury to the adult brain results in abortive axon regeneration and the deposition of a dense fibrous glial scar. Therapeutic strategies to promote postinjury axon regeneration are likely to require antiscarring strategies. In neonatal brain wounds, scar material is not laid down and axons grow across the lesion site, either by de novo growth or regeneration. To achieve the therapeutic goal of recapitulating the nonscarring neonatal response in the injured adult, an understanding of how ontogenic differences in scarring reflect developmental diversities in the trophic response to injury is required. Fibrobast growth factor-2 (FGF-2) expression is developmentally regulated and has been implicated as a regulator of the wounding response of the adult rat central nervous system. We have investigated the expression of FGF-2 and fibroblast growth factor receptor 1 (FGFR1) after penetrating lesions to the cerebral cortex of 5 days post partum (dpp) (nonscarring) and 16 dpp and adult (scarring) rats. In situ hybridization, immunohistochemistry and Western blotting showed robust and sustained increases in FGF-2 and FGFR1 mRNA and protein in reactive astrocytes around the lesion in scarring rats, a response that was attenuated substantially in the nonscarring neonate. These results demonstrate that changes in astrocyte FGF-2 and FGFR1 expression are coincident with the establishment of a mature pattern of glial scarring after injury in the maturing central nervous system, but it is premature to infer a causal relationship without further experiments.     

Astrocytic basic fibroblast growth factor expression in dopaminergic regions after perinatal anoxia.

BACKGROUND: Perinatal anoxia leads to persistent behavioral and neurochemical alterations suggestive of sensitized dopaminergic function. Because astrocytic basic fibroblast growth factor (bFGF) activity in the midbrain dopaminergic cell body region is required for the development of enduring changes in dopaminergic function induced by stimulant drugs, we investigated the effects of intrauterine anoxia on astrocytic bFGF expression in dopaminergic regions at 2 weeks of age and after a stress manipulation in adults. METHODS: We examined bFGF immunoreactivity in dopaminergic regions of young and adult rats born by cesarean section, cesarean section + 15 min of intrauterine anoxia, or vaginally. bFGF immunoreactivity was also assessed before and after tail-pinch stress in adult animals exposed to the same perinatal interventions. RESULTS: Perinatal anoxia produced persistent decreases in basal bFGF immunoreactivity in the ventral tegmental area (VTA), but enhanced the effect of stress on VTA bFGF immunoreactivity. CONCLUSIONS: Perinatal anoxia has enduring effects on VTA bFGF immunoreactivity and influences adult neuroadaptations to stress. The mechanisms whereby perinatal anoxia alters dopaminergic function may be similar to those responsible for the development of sensitization to stimulant drugs and may involve bFGF.     

Localization of transforming growth factor-beta1 and receptor mRNA after experimental spinal cord injury

Transforming growth factor-beta1 (TGFbeta1) is a cytokine/growth factor found within the pathological central nervous system. TGFbeta1 has been shown to inhibit the release of cytotoxic molecules from microglia and macrophages, decrease astrocyte proliferation, and promote neuron survival. Because of the relevance of these actions to spinal cord injury, we examined TGFbeta1 and its receptors betaRI and betaRII mRNA levels and localization within the contused rat spinal cord using in situ hybridization. At the lesion site, TGFbeta1 mRNA peaked at 7 days postinjury and declined thereafter. Temporal and spatial localization of the betaRI and betaRII receptor mRNA closely mimicked that for TGFbeta1 in the epicenter. TGFbeta1, betaRI, and betaRII mRNAs also were elevated rostral and caudal to the injury, especially in regions known to contain activated microglia and degenerating axon profiles. Immunohistochemical staining of nearby sections confirmed that the highest levels of TGFbeta1 and receptor mRNA corresponded to regions filled with activated microglia and macrophages. The similar expression pattern of TGFbeta1, betaRI, and betaRII mRNA within the injured spinal cord suggests a local site of action. Since TGFbeta1 can act as an immunosuppressant as well as a stimulant for growth factors and neurite sprouting, it likely plays an important role, both temporally and spatially, in orchestrating postinjury events within the spinal cord.     

Overexpression of nerve growth factor and basic fibroblast growth factor in AIDS dementia complex.

Although neurotrophic factors are currently considered as treatment for neurodegenerative diseases, little is still known about their presence in the central nervous system under pathological conditions. We investigated the expression of the neurotrophic molecules NGF, bFGF, BDNF and IGF-1 in brain tissue of patients suffering from AIDS dementia complex. In contrast to IGF-1 and BDNF, NGF and bFGF mRNA levels were significantly elevated. Strong NGF immunoreactivity was found in perivascular areas and was colocalized with infiltrating macrophages, whereas intense bFGF staining was found in cells with characteristic astrocytic morphology. These data suggest that the induction of NGF and bFGF alone appears to be insufficient as a compensatory mechanism to prevent ADC.     

Identification of basic fibroblast growth factor as a cholinergic growth factor from human muscle

Dissociated embryonic chick ciliary ganglion cells in culture were used as a bioassay to isolate a cholinergic growth-promoting protein from extracts of autopsied adult human muscle. An active protein was purified after acid and salt precipitation of extract, cation exchange, molecular sieving, heparin affinity chromatography, and in some cases, SDS-PAGE. This protein increased levels of choline acetyltransferase activity and ACh synthesis with time in culture. The protein was identified as basic FGF by several criteria. It shared the high affinity for heparin and was the same approximate molecular weight, 18 kD, as basic FGF. Activity was removed from solution by antibodies specific for basic FGF. Recombinant human basic FGF was equally effective in stimulating CAT activity, but was not additive with our purified protein at saturating concentrations. Basic FGF was also found in extracellular matrix and conditioned medium from cultured embryonic chick muscle. The activity could be released from extracellular matrix by treatment with heparinase or high salt extraction. Basic FGF stimulates neurite outgrowth as well as the capacity for transmitter synthesis. Thus, basic FGF is present in embryonic and adult muscle and capable of acting as a growth regulator for cholinergic neurons.     

Wnt pathway in the formation of ischemic brain injury*★ Interventional pathway of basic fibroblast growth factor

BACKGROUND: Basic fibroblast growth factor (bFGF) can reduce neuronal apoptosis following ischemia/reperfusion (I/R) injury. Mechanism of the phenomenon should be elucidated. OBJECTIVE: The goal of this study was to observe the effect of bFGF on the expressions of Dickkopf-1(DKK-1) and β-catenin in the Wnt pathway in hippocampal tissue of rats following brain I/R injury, in order to investigate the role of Wnt pathway in the formation of ischemic brain injury. DESIGN: Randomized controlled experiment. SETTING: Shenyang Medical College. MATERIALS: Thirty healthy 3 months old male Wistar rats, weighing 300–350 g, were provided by the Experimental Animal Center of Shenyang Medical College. Thirty rats were randomized into sham-operation group, model group and treatment group. Goat anti-rat monoclonal antibody β-catenin was purchased from SANTA CRUZ Company. BFGF was developed by Beijing SL Pharmaceutical Co., Ltd. METHODS: This experiment was carried out in the Shenyang Medical College between November 2005 and May 2006. ①Focal brain I/R by suture-occluded method was modeled in rats in the treatment group and model group. Their middle cerebral artery was occluded 1 hour and reperfused for 24 hours. While in the sham-operation group, only the right common carotid artery and external carotid artery of rats were occluded for 90 minutes. ② The rats in the treatment group were intraperitoneally injected with 10 μg/kg bFGF, and those in the other groups were intraperitoneally injected with the same amount of saline. MAIN OUTCOME MEASURES: Following I/R 48 hours, the expressions of β-catenin and Dickkopf-1 mRNA in the neurons of hippocampal CA1 region by immunohistochemical SABC and RT-PCR. RESULTS: Following I/R 48 hours, the expressions of β-catenin and Dickkopf-1 mRNA in the neurons of hippocampal CA1 region was evaluated by means of immunohistochemical SABC and RT-PCR. ① Expression of DKK-1 mRNA in the sham-operation group was at low level, it was significantly higher in the model group compared to the sham-operation group; Expression of DKK-1 mRNA in the treatment group was significantly lower than that in the model group. ② Expression of β-catenin in the cerebral cortex and hippocampal cytoplasm of rats: The mean gray scale of β-catenin of model group was significantly lower than that of sham-operation group (74.27±2.65 vs. 111.36±5.39，P < 0.05); The mean gray scale of β-catenin of treatment group was significantly higher than that of model group (86.18±7.41 vs. 74.27±2.65, P < 0.05). CONCLUSION: bFGF may influence Wnt pathway by participating in the regulation of DKK-1 mRNA and β-catenin expressions, and thereby protect neurons.