TNF alpha and Fas mediate tissue damage and functional outcome after traumatic brain injury in mice.

Tumor necrosis factor-alpha (TNFalpha) and Fas are induced after traumatic brain injury (TBI); however, their functional roles are incompletely understood. Using controlled cortical impact (CCI) and mice deficient in TNFalpha, Fas, or both (TNFalpha/Fas-/-), we hypothesized that TNFalpha and Fas receptor mediate secondary TBI in a redundant manner. Compared with wild type (WT), TNFalpha/Fas-/- mice had improved motor performance from 1 to 4 days (P<0.05), improved spatial memory acquisition at 8 to 14 days (P<0.05), and decreased brain lesion size at 2 and 6 weeks after CCI (P<0.05). Protection in TNFalpha/Fas-/- mice from histopathological and motor deficits was reversed by reconstitution with recombinant TNFalpha before CCI, and TNFalpha-/- mice administered anti-Fas ligand antibodies had improved spatial memory acquisition versus similarly treated WT mice (P<0.05). Tumor necrosis factor-alpha/Fas-/- mice had decreased the numbers of cortical cells with plasmalemma damage at 6 h (P<0.05 versus WT), and reduced matrix metalloproteinase-9 activity in injured brain at 48 and 72 h after CCI. In immature mice subjected to CCI, genetic inhibition of TNFalpha and Fas conferred beneficial effects on histopathology and spatial memory acquisition in adulthood (both P<0.05 versus WT), suggesting that the beneficial effects of TNFalpha/Fas inhibition may be permanent. The data suggest that redundant signaling pathways initiated by TNFalpha and Fas play pivotal roles in the pathogenesis of TBI, and that biochemical mechanisms downstream of TNFalpha/Fas may be novel therapeutic targets to limit neurological sequelae in children and adults with severe TBI.     

Changes of Fas and Fas ligand immunoreactivity after compression trauma to rat spinal cord

This immunohistochemical study evaluated Fas and Fas ligand (FasL) in the rat nervous system and their changes in the spinal cord subjected to compression. Normal spinal cord showed a low level of Fas and FasL immunoreactivity in the white matter except in the corticospinal tracts. Fas and FasL immunoreactivity seemed to be located in axons and their myelin sheaths. Other regions of the nervous system did not show immunoreactivity to Fas and FasL. Moderate and severe compression injury of the spinal cord resulted in a reduction of Fas and FasL immunoreactivity in the white matter of injured T8–9 segments at 4 h and a complete loss at 1 day after trauma. This was seen even in the remaining white matter. In contrast, increased immunoreactivity to Fas and FasL was present in the cranial T7, caudal T10 (moderate injury) and T12 (severe injury) segments at day 4 with most intense staining were seen at day 9 after trauma. Increased Fas and FasL immunoreactivity may have pathophysiological implications for the development of secondary injuries after trauma to the spinal cord. Fas-FasL interactions may for instance be involved in apoptosis of oligodendrocytes which occurs as a delayed phenomenon after trauma to the spinal cord. The integrity of myelin sheaths may in this way be jeopardized by apoptosis of oligodendrocytes. Received: 30 June 1999 / Revised: 8 October 1999 / Accepted: 12 October 1999     

Widespread and lateralized effects of acute traumatic brain injury on norepinephrine turnover in the rat brain

Norepinephrine (NE) has been implicated in recovery of function following traumatic brain injury (TBI). While bilateral decreases in brain NE turnover occur at 6–24 h after TBI, it is unknown what effects unilateral TBI might have on brain NE turnover over the first few minutes after injury. Here male Sprague-Dawley rats had unilateral contusions of either the right or left somatosensory cortex produced by an air driven piston. At 30min after TBI, brain NE turnover was assessed by measuring the ratio of 3-methoxy-4-hydroxyphenylglycol (MHPG) to NE levels in various brain regions. Both right and left TBI produced 32–103% increases in NE turnover at the injury site and in the ipsilateral cerebral cortex surrounding, rostral and caudal to the injury as compared to the contralateral, uninjured site or to the homologous sites in uninjured controls. NE turnover was also altered selectively in some brain areas not affected by right TBI. Left TBI decreased NE turnover by 29% in the frontal cortex contralateral to the injury and by 24% bilaterally in the hypothalamus while increasing locus coeruleus NE turnover by 72% compared to uninjured controls. Thus, unilateral cortical TBI produced predominantly ipsilateral increases in cortical NE turnover but variable, bilateral changes in NE turnover in subcortical areas which were dependent upon the side of injury. These subcortical differences may explain some of the lateralized effects of cortical injury on post-injury behavior.     

Early expressions of hypoxia-inducible factor 1alpha and vascular endothelial growth factor increase the neuronal plasticity of activated endogenous neural stem cells after focal cerebral ischemia

Endogenous neural stem cells become "activated" after neuronal injury, but the activation sequence and fate of endogenous neural stem cells in focal cerebral ischemia model are little known. We evaluated the relationships between neural stem cells and hypoxia-inducible factor-1α and vascular endothelial growth factor expression in a photothromobotic rat stroke model using immunohistochemistry and western blot analysis. We also evaluated the chronological changes of neural stem cells by 5-bromo-2′-deoxyuridine(BrdU) incorporation. Hypoxia-inducible factor-1α expression was initially increased from 1 hour after ischemic injury, followed by vascular endothelial growth factor expression. Hypoxia-inducible factor-1α immunoreactivity was detected in the ipsilateral cortical neurons of the infarct core and peri-infarct area. Vascular endothelial growth factor immunoreactivity was detected in bilateral cortex, but ipsilateral cortex staining intensity and numbers were greater than the contralateral cortex. Vascular endothelial growth factor immunoreactive cells were easily found along the peri-infarct area 12 hours after focal cerebral ischemia. The expression of nestin increased throughout the microvasculature in the ischemic core and the peri-infarct area in all experimental rats after 24 hours of ischemic injury. Nestin immunoreactivity increased in the subventricular zone during 12 hours to 3 days, and prominently increased in the ipsilateral cortex between 3–7 days. Nestin-labeled cells showed dual differentiation with microvessels near the infarct core and reactive astrocytes in the peri-infarct area. BrdU-labeled cells were increased gradually from day 1 in the ipsilateral subventricular zone and cortex, and numerous BrdU-labeled cells were observed in the peri-infarct area and non-lesioned cortex at 3 days. BrdU-labeled cells rather than neurons, were mainly co-labeled with nestin and GFAP. Early expressions of hypoxia-inducible factor-1α and vascular endothelial growth factor after ischemia made up the microenvironment to increase the neuronal plasticity of activated endogenous neural stem cells. Moreover, neural precursor cells after large-scale cortical injury could be recruited from the cortex nearby infarct core and subventricular zone.     

丁苯酞对大鼠脑缺血再灌注损伤后的Fas／FasL蛋白表达的影响

目的研究大鼠脑缺血再灌注损伤后Fas和FasI。蛋白的表达和丁笨酞保护作用的机制。方法线栓法制备大鼠大脑中动脉缺血再灌注模型,SD大鼠随机分为正常对照组、假手术组、缺血再灌注组和丁苯酞组;应用尼氏染色显示存活神经元,末端转移酶介导的缺口末端标记（TUNEI。）方法检测程序化死亡细胞;免疫组织化学法观察脑缺血再灌注后6h、1d、3d、7d四个时间点脑组织中Fas和FasI．蛋白的表达变化。结果丁苯酞组皮层存活神经元数量多于缺血再灌注组,TUNEI．阳性细胞数减少,丁苯酞组在再灌注后各时间点的Fas和FasL阳性细胞数较缺血再灌注组少,差异均有统计学意义。结论丁苯酞可能通过抑制Fas／FasL蛋白的表达,从而减少神经元凋亡,减轻缺血再灌注后脑组织的损伤。     

Co-expression of Fas and Fas ligand in malignant glial tumors and cell lines

Fas ligand (FasL) is involved in tumor evasion from the immune system. We analyzed 22 human gliomas for expression of FasL and its receptor, Fas. Positive FasL and Fas immunoreactivity was detected in 13 out of 22 tumors by Western blotting and in 15 out of 22 tumors by immunohistochemistry. Immunohistochemistry also showed that Fas and FasL expression was confined to tumor cells. Co-expression of these molecules was confirmed by Western blotting and immunohistochemistry in 4 of 7 glioma cell lines. Co-expression of FasL and Fas within tumor cells suggests that their contribution in vivo to the process of immune system evasion and tumor cell apoptosis is complex and probably involves additional factors.     

脑创伤后bcl—2蛋白的神经保护作用

目的 探讨液压脑损伤后凋亡抑制基因bcl—2的变化规律及bcl—2基因在创伤性脑损伤后细胞凋亡中的作用。方法 应用免疫组化观察大鼠中型液压脑损伤伤前及伤后6h、12h、1d、3d、7dbcl—2蛋白表达情况,应用TUNEIL和电镜观察伤后细胞死亡的形态。结果 免疫反应阳性细胞主要位于伤侧大脑半球皮质、皮层下白质、海马CAl、CA3及齿状回的神经元和神经胶质细胞,以海马CA3区最为显。在高倍镜下,表达Bcl—2蛋白的神经细胞胞核形态正常,很少见到凋亡或坏死的形态特征。伤后早期(6h),打击侧海马CA3区Bcl—2蛋白表达显下降;Bcl—2早期改变出现在伤后6h,比细胞凋亡提前表现;伤后l—3h,Bcl—2的表达下降相对缓慢。结论 bcl—2蛋白在抑制脑创伤后细胞凋亡中起重要作用,bcl—2可能是一种可诱导的神经保护因子。     

大鼠轻型颅脑损伤后星形胶质细胞与神经元的病理改变

目的 探讨轻型颅脑损伤（TBI）后神经元及星形胶质细胞改变的病理生理过程。方法 将24只成年SD大鼠随机分为轻型TBI组（n=18）和假手术组（n=6）,轻型TBI组又分为伤后3 h（n=6）、伤后24 h（n=6）、伤后72 h（n=6）三亚组。采用液压冲击法制作轻型TBI模型。采用胶质纤维酸性蛋白（GFAP）染色检测星形胶质细胞,采用Fluoro-Jade B（FJ-B）荧光染色检测变性神经元。结果 与假手术组相比,轻型TBI后3 h、24 h、72 h邻近顶叶皮质、海马CA2/3区GFAP阳性细胞数量均明显减少（P<0.05）;缺失区周围星形胶质细胞肿胀增生明显。FJ-B阳性神经元在损伤后3 h无明显增加（P>0.05）,伤后24 h皮层区FJ-B阳性神经元显著增加（P<0.05）,伤后72 h海马区FJ-B阳性神经元显著增加（P<0.05）。伤后72 h伤侧皮层区与海马区GFAP阳性细胞数和FJ-B阳性细胞数呈显著负相关（r=-0.8285,P<0.05）。结论 轻型TBI后星形胶质细胞超急性期（3 h）即出现损害和胶质反应,神经元则在急性期（24 h）至亚急性期（72 h）出现明显损害,星形胶质细胞缺失程度可以反应神经元损伤程度。     

Expression of cellular FLICE inhibitory proteins (cFLIP) in normal and traumatic murine and human cerebral cortex.

Cellular Fas-associated death domain-like interleukin-1-beta converting enzyme (FLICE) inhibitory proteins (cFLIPs) are endogenous caspase homologues that inhibit programmed cell death. We hypothesized that cFLIPs are differentially expressed in response to traumatic brain injury (TBI). cFLIP-alpha and cFLIP-delta mRNA were expressed in normal mouse brain-specifically cFLIP-delta (but not cFLIP-alpha) protein was robustly expressed. After controlled cortical impact (CCI), cFLIP-alpha expression increased initially then decreased to control levels at 12 h, increasing again at 24-72 h (P<0.05). cFLIP-delta expression was decreased in brain homogenates by 12 h after CCI, then increased again at 24 to 72 h (P<0.05). cFLIP-delta immunostaining was markedly reduced in injured cortex, but not hippocampus, at 3 to 72 h after CCI. In cortex, reduced cFLIP-delta staining was found in TUNEL-positive cells, but in hippocampus TUNEL-positive cells expressed cFLIP-delta immunoreactivity. cFLIP-delta was increased in a subset of reactive astrocytes in pericontusional cortex and hippocampus at 48 to 72 h. Low levels of both cFLIP isoforms were detected in human cortical tissue with no TBI, from four patients undergoing brain surgery for epilepsy and <24 h post mortem from three patients without CNS pathologic assessment. In cortical tissue surgically removed <18 h after severe TBI (n=3), cFLIP-alpha expression was increased relative to epilepsy controls (P<0.05) but not relative to post-mortem controls. The data suggest differential spatial and temporal regulation of cFLIP-alpha and cFLIP-delta expression that may influence the magnitude of cell death and further implicate programmed mechanisms of cell death after TBI.     

Fas and Fas ligand expression in Alzheimer's disease

The Fas/Fas ligand (L) signaling system has been implicated in the control of cell death and cell survival of T and B lymphocytes and in a variety of cell types under particular pathological conditions. In the present study we examined the expression of Fas and Fas-L, by Western blotting and immunohistochemistry, in the human frontal cortex and hippocampus of individuals with advanced Alzheimer's disease (AD) and age-matched controls. Expression levels of Fas and Fas-L, as seen in Western blots, are preserved in the frontal cortex but decreased in the hippocampus in AD when compared with age-matched controls. Yet Fas and Fas-L immunoreactivity is found in remaining AD neurons in the frontal cortex and hippocampus. Moreover, Fas and Fas-L are expressed equally in tangle-bearing and non-tangle-bearing neurons, as revealed with double-labeling immunohistochemistry to Fas or Fas-L and tau or phosphorylated neurofilament epitopes. Dystrophic neurites of senile plaques are not stained with Fas and Fas-L antibodies. A moderate increase in Fas and a strong increase in Fas-L immunoreactivity occur in reactive astrocytes in AD. Yet there is no relationship between Fas or Fas-L expression and increased nuclear DNA vulnerability as revealed with double-labeling immunohistochemstry and in situ end-labeling of nuclear DNA fragmentation. Although the Fas/Fas-L system may have some effect in the control of reactive astrocytosis in AD, the present results show no evidence that Fas/Fas-L signals participate in specific processes of the disease, including neurofibrillary degeneration, dystrophic neurite formation, and cell death.     

Reactive astrocytes upregulate Fas (CD95) and Fas ligand (CD95L) expression but do not undergo programmed cell death during the course of anterograde degeneration

Tissue homeostasis is determined by a balance between proliferation and apoptosis. Various lesions in the brain are accompanied by proliferation and subsequent death of glial cells, but the mechanisms that limit this expansion of glial populations remains unknown. One possible candidate is the death ligand, FasL, and its receptor Fas, because the expression of both proteins was reported on glial cells. To elucidate the expression and putative function of Fas and FasL on proliferative glial cells, we performed stereotactic lesion of the entorhinal cortex of adult rats. Such lesions induce proliferation of astrocytes and microglial cells in the hippocampal fields of anterograde degeneration. Subsequently, the total number of both cell types returns to pre-lesion counts. We found that Fas and FasL is strongly upregulated on astrocytes in the zone of anterograde degeneration with a peak 5 days postlesion (dpl) and a return to control levels at 10 dpl. However, evidence for astrocytic cell death was neither detected by TUNEL staining, immunocytochemistry for c-Jun, and apoptosis-specific protein (ASP), nor by staining for morphologic hallmarks of apoptotic or necrotic cell death at the light and electron microscopic level. Thus, increased expression of Fas and FasL is not accompanied by cell death of reactive astrocytes during anterograde degeneration.     

大鼠急性脊髓损伤后细胞凋亡以及相关蛋白表达的实验研究

目的 观察脊髓损伤（spinalcordinjury，SCI）后细胞凋亡和相关调控基因（FasL和Caspase．3）的表达情况。方法 通过建立静压型SCI模型（30g的重量，压迫T10节段10min），并用TUNEL染色（terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling）和免疫组化染色方法，了解Sprague-Dawley大鼠SCI后（伤后6、12、24、48和72h以及伤后7、14、21d）细胞凋亡以及相关调控基因（FasL与Caspase-3）表达的情况。结果 大鼠SCI后损伤节段（T10节段）6h出现TUNEL阳性细胞数增加，12h达到高峰，持续3天～1周后显著减少；损伤相邻节段（T9、T11节段）伤后12h出现TUNEL阳性细胞，伤后3dTUNEL阳性细胞数达峰值；SCI后FasL与Cas-pase-3表达均有不同程度的增高。结论 大鼠脊髓损伤后多节段、长时间存在着大量的细胞凋亡；细胞凋亡可能与FasL-Caspase-3途径有关。     

Immunosuppressant FK506 affects multiple signaling pathways and modulates gene expression in astrocytes

Brain injury is often associated with proliferation and hypertrophic response of glial cells (reactive gliosis). We have previously reported immunosuppressant effects on survival of glioma cells and adult reactive astrocytes. In the present study, we demonstrate growth-inhibitory effect of FK506 on cortical astrocytes from newborn rats. FK506 inhibits Erk and PI-3K/Akt signaling, two crucial pro-survival pathways. The levels of phosphorylated Akt and p42/44 Erk decline in few hours after FK506 addition. Furthermore, in FK506-treated astrocyte cultures the levels of mRNA encoding PDGF, bFGF, and CNTF decreased. Downregulation of growth factor expression by FK506 may play a role in the inhibition of mitogenic/hypertrophic responses. FasL mRNA level was elevated and interaction of FasL with Fas receptor expressed in astrocytes may trigger cell death. Interestingly, expression of BDNF increased in a dose-dependent manner in FK506-treated astrocytes. Upregulation of BDNF mRNA and protein level in astrocytes exposed to FK506 may underlie neuroprotective action of FK506.     

Fas and Fas-L expression in Huntington's disease and Parkinson's disease

The Fas/Fas-L signalling system plays a role in the control of cell death and the survival of lymphocytes, in the regulation of the immune system, and in the progression of autoimmune diseases. Studies in the nervous system have shown Fas/Fas-L activation in multiple sclerosis and in various paradigms leading to neuronal death. Enhanced Fas and Fas-L expression has also been documented in astrocytomas and glioma cell lines. However, little is known about the possible implication of Fas/Fas-L signals in primary human neurodegenerative diseases. In an attempt to gain understanding of the mechanisms commanding cell death and neurone loss in Huntington's disease (HD) and Parkinson's disease (PD), Fas and Fas-L expression has been examined in the brains of patients with HD and PD with Western blotting and immunohistochemistry. Fas and Fas-L expression levels are reduced in the caudate and putamen, but not in the parietal cortex, in HD, as revealed in Western blots. Moreover, Fas and Fas-L immunoreactivity is reduced in striatal neurones in HD. Fas and Fas-L immunoreactivity is also decreased in neurones of the substantia nigra pars compacta in PD. Reduced Fas and Fas-L expression is observed equally in Lewy body-bearing and non-Lewy body-bearing neurones. Yet increased Fas and Fas-L immunoreactivity occurs in normal astrocytes in control brains and in reactive astrocytes in diseased brains. The meaning of increased Fas and Fas-L expression in astrocytes is still unclear. However, the present results suggest that Fas/Fas-L signals are minimized in sensitive neurones in HD and PD.     

Retinal glial cell responses and Fas/FasL activation in rats with chronic ocular hypertension

Responses in the retina post injury provoke glial reactions that are not completely understood. This study investigated the reaction of retinal glial cells and the expression and localization of the Fas and Fas-ligand (FasL) in rats with chronic ocular hypertension. Experimental glaucoma was induced in one eye of 60 Sprague-Dawley rats by cauterizing three episcleral vessels. It caused a moderate intraocular pressure (IOP) elevation and significant retinal ganglion cell (RGC) loss for at least 6 weeks in all animals. Immunohistochemical analysis revealed that the expression of GFAP and OX-42 increased in the injured retinae. Fas/FasL immunoreactivity was elevated in the microglia, and we also observed an incremental increase in Fas associated death domain (FADD) immunoreactivity in Müller glial cells and RGCs in the IOP-elevated retinae. The activation of glial cells and upregulation of Fas and FasL suggest that glial cells may contribute to Fas-mediated cell death in the neurodegeneration process of chronic ocular hypertensive retinal insult.     

Regulation of Fas and FasL expression on rat Schwann cells

Although the PNS belongs to the immune privileged sites, it can become a target of immune attacks by invading T cells, causing inflammation and destruction. Yet the PNS also has a protective potential by eliminating the inflammatory cells via apoptosis. In analogy with other immune-protected sites, participation of the apoptosis-inducing Fas/FasL molecules could play an important role. To assess the possible involvement of the Fas/FasL system in T-cell apoptosis in the PNS of the rat, we characterized Fas and FasL expression on neonatal rat Schwann cells (SC) in vitro. Cells were stimulated in vitro with interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), or a combination of both. We observed upregulation of FasL expression under the influence of IFN-gamma, while adding TNF-alpha alone to the culture medium had no effect. IFN-gamma and TNF-alpha acted synergistically, leading to an increased FasL expression that reached its maximum 70 h after cytokine exposure, as shown by FACS analysis, SDS-PAGE, and Western blot. Fas expression on untreated SC showed fluctuating levels, while addition of IFN-gamma and TNF-alpha suppressed Fas expression completely. These findings are in accord with recently published results showing Fas and FasL expression on malignant human cells, derived from brain tumors and upregulation of FasL on astrocytes after exposure to IFN-gamma and TNF-alpha. Furthermore, FasL-expressing SC could be revealed by immunostaining of sciatic nerve from Lewis rats suffering from experimental autoimmune neuritis (EAN). We suggest that Fas/FasL expression on SC may contribute to the elimination of invading autoreactive T cells in the PNS in concert with other immune defense mechanisms.