Microglial-macrophage synthesis of tumor necrosis factor after focal cerebral ischemia in mice is strain dependent.

Commonly used inbred mouse strains display substantial differences in sensitivity to focal cerebral ischemia. Such differences can often be ascribed to differences in vascular anatomy. The authors investigated whether a contributing factor could be strain-related differences in cellular synthesis of the pleiotropic and potential neurotoxic cytokine tumor necrosis factor (TNF) in the border zone of and within the focal cerebral infarct. In all mouse strains investigated they found that TNF was synthesized by infarct and periinfarct infiltrating Mac-1 immunopositive microglia-macrophages. BALB/c mice, which developed the largest infarcts, contained significantly fewer TNF-producing microglia-macrophages compared with SJL and C57BL/6 mice at both 12 and 24 hours after permanent occlusion of the distal part of the middle cerebral artery. SJL mice developed larger infarcts than C57BL/6 mice, whereas the number of TNF-producing microglia-macrophages per infarct volume unit was comparable. Western blotting data confirmed the increased TNF levels in SJL mice compared with BALB/c and C57BL/6 mice. Furthermore, mice with 12-hour postischemic survival consistently contained two-to threefold more TNF-producing microglia-macrophages than mice with 24-hour survival. The data show that the magnitude of the cellular TNF response to cerebral ischemia is strain dependent, while the time-profile and the cellular sources of TNF are similar irrespective of genetic background. Furthermore, the lack of correlation between infarct size and cellular TNF response suggests that the functionally important TNF is produced in the very early phase (minutes to a few hours) after induction of ischemia, just as it raises the possibility that different mouse strains display different sensitivities to TNF.     

Microglia and macrophages are the major source of tumor necrosis factor in permanent middle cerebral artery occlusion in mice.

The proinflammatory cytokine tumor necrosis factor (TNF) is known to be expressed in brain ischemia; however, its cellular and temporal appearance is not fully settled. In this study, nonradioactive in situ hybridization for murine TNF mRNA was performed on brain sections from adult C57x129 mice at 6 hours, 12 hours, 24 hours, 2 days, 5 days, or 10 days (six to eight mice per group) after induction of permanent focal cerebral ischemia. Cortical infarct volumes were estimated, and TNF mRNA-expressing cells were counted within the infarct and infarct border using Cast-Grid analysis. At 12 hours, a peak of 19.2 +/- 5.1 TNF mRNA-expressing cells/mm2 was counted, contrasting two to three times lower values at 6 and 24 hours (6.4 +/- 4.6 and 9.2 +/- 3.4 cells/mm2, respectively) and <2 cells/mm2 at 48 hours and later stages. The TNF mRNA-expressing cells were distributed along the entire rostrocaudal axis of the cortical infarcts and occasionally within the caudate putamen. At all time points, TNF mRNA colocalized with Mac-1-positive microglia/macrophages but not with Ly-6G (Gr-1)-positive polymorphonuclear leukocytes. Similarly, combined in situ hybridization for TNF mRNA and immunohistochemistry for glial fibrillary acidic protein at 12 and 24 hours revealed no TNF mRNA-expressing astrocytes at these time points. Translation of TNF mRNA into bioactive protein was demonstrated in the neocortex of C57B1/6 mice subjected to permanent middle cerebral artery occlusion. In summary, this study points to a time-restricted microglial/macrophage production of TNF in focal cerebral ischemia in mice.     

Enlarged infarct volume and loss of BDNF mRNA induction following brain ischemia in mice lacking FGF-2

FGF-2, a potent multifunctional and neurotrophic growth factor, is widely expressed in the brain and upregulated in cerebral ischemia. Previous studies have shown that intraventricularly or systemically administered FGF-2 reduces the size of cerebral infarcts. Whether endogenous FGF-2 is beneficial for the outcome of cerebral ischemia has not been investigated. We have used mice with a null mutation of the fgf2 gene to explore the relevance of endogenous FGF-2 in brain ischemia. Focal cerebral ischemia was produced by occlusion of the middle cerebral artery (MCAO). We found a 75% increase in infarct volume in fgf2 knock-out mice versus wild type littermates (P < 0.05). This difference in the extent of ischemic damage was observed after 24 h, and correlated with decreased viability in fgf2 mutant mice following MCA occlusion. Increased infarct volume in fgf2 null mice was associated with a loss of induction in hippocampal BDNF and trkB mRNA expression. These findings indicate that signaling through trkB may contribute to ameliorating brain damage following ischemia and that bdnf and trkB may be target genes of FGF-2. Together, our data provide the first evidence that endogenous FGF-2 is important in coping with ischemic brain damage suggesting fgf2 as one crucial target gene for new therapeutic strategies in brain ischemia.     

Reelin-deficient mice show impaired neurogenesis and increased stroke size

Reelin (Reln) is a protein involved in migration of newborn neurons during development. Reln mutations produce the reeler phenotype in mice, which is characterized by a defect in brain lamination, and autosomal recessive lissencephaly in humans. Reln expression persists in adult brain, but little is known about its function. We used reeler mice to investigate the effects of Reln deficiency on neurogenesis and the response to injury in the adult brain. Newborn neurons were decreased in number in the dentate gyrus and rostral migratory stream of reeler, compared to wild-type, mice. This was due, at least in part, to impaired cell migration. In addition, reeler mice showed increased susceptibility to ischemic brain injury. Cerebral infarcts from middle cerebral artery occlusion were larger in reeler than in wild-type mice, and associated neurobehavioral abnormalities were more severe. The brains of reeler mice also showed larger excitotoxic lesions after the intracerebral injection of N-methyl-D-aspartate. Finally, despite the fact that reeler mice had larger cerebral infarcts, the ischemia-induced enhancement of neurogenesis observed in wild-type mice was attenuated. These findings suggest that, in addition to its neurodevelopmental effects, Reln deficiency continues to influence neurogenesis and ischemic neuronal injury in the adult brain.     

Expression of glial cell line-derived neurotrophic factor induced by transient forebrain ischemia in rats.

This study examined the expression of glial cell line-derived neurotrophic factor (GDNF) mRNA and the cellular localization of GDNF production in rats subjected to transient forebrain ischemia induced by four-vessel occlusion. Transient forebrain ischemia induced GDNF mRNA expression in the hippocampus from 3 h to 3 days after the ischemic episode, with peak expression at 6 h. The GDNF mRNA increase in the cerebral cortex was similar to that in the hippocampus, whereas no increase in GDNF mRNA was observed in the striatum and brainstem. Western blot analysis showed that GDNF in the hippocampal CA1 region was increased slightly from 3 to 24 h after the ischemia, and then subsequently declined to below the baseline level. In the hippocampus, GDNF was evenly produced in pyramidal neurons of both sham-operated rats and normal rats, as determined by immunohistochemistry. Interestingly, we found that ischemia-induced reactive astrocytes, as well as surviving neurons, produced GDNF in 3-7 days after the ischemia. On the other hand, in other regions, such as the cerebral cortex, striatum, and brainstem, there was no change in GDNF-positive cells secondary to ischemia. These findings suggest that expression of GDNF mRNA is regulated in part via ischemia-induced neuronal degeneration. They also suggest that ischemia-induced reactive astrocytes may produce GDNF to protect against neuronal death. Therefore, GDNF may play an important role in ischemia-induced neuronal death in the brain.     

Ischemia-induced development of cerebral edema in awake and anesthetized gerbils

General anesthesia is often used to immobilize experimental animals prior to the induction of cerebral ischemia. However, anesthetics are known to alter many of the biochemical and physiological parameters used for the assessment of stroke-induced brain damage. We examined the effects of bilateral carotid artery ligations on mortality and the development of cerebral edema in unanesthetized gerbils. We found that increasing the length of the ischemic episode resulted in increased mortality, both during the ischemic period and during cerebral reperfusion. The duration of the ischemic episode was also correlated with the rate and degree of the development of cerebral edema. Both of these estimates of ischemia-induced brain damage were significantly reduced by the pretreatment of the animals with pentobarbital. Based on the variable effects of different anesthetics on CNS activities, and the observed effects of barbiturate anesthesia on ischemia-induced mortality and edema development in the present model, we suggest that it may be inappropriate to anesthetize experimental animals when investigating certain aspects of stroke-induced brain damage.     

Role of matrix metalloproteinases in apoptosis after transient focal cerebral ischemia in rats and mice

The involvement of matrix metalloproteinases (MMPs) in cerebral ischemia-induced apoptosis was investigated in a model of transient focal cerebral ischemia in rats treated intracerebroventricularly (i.c.v.) with 4-((3-(4-phenoxylphenoxy)propylsulfonyl)methyl)-tetrahydropyran-4-carboxylic acid N-hydroxy amide, a broad spectrum non-peptidic hydroxamic acid MMP inhibitor, and in MMP-9-deficient mice. Our results showed that MMP inhibition reduced DNA fragmentation by 51% (P < 0.001) and cerebral infarct by 60% (P < 0.05) after ischemia. This protection was concomitant with a 29% reduction of cytochrome c release into the cytosol (P < 0.005) and a 54% reduction of calpain-related alpha-spectrin degradation (P < 0.05), as well as with an 84% increase in the immunoreactive signal of the native form of poly(ADP) ribose polymerase (P < 0.01). By contrast, specific targeting of the mmp9 gene in mice did reduce cerebral damage by 34% (P < 0.05) but did not modify the apoptotic response after cerebral ischemia. However, i.c.v. injection of MMP-9-deficient mice with the same broad-spectrum inhibitor used in rats significantly reduced DNA degradation by 32% (P < 0.05) and contributed even further to the protection of the ischemic brain. Together, our pharmacological and genetic results indicate that MMPs other than MMP-9 are actively involved in cerebral ischemia-induced apoptosis.     

Estrogen inhibits Fas-mediated apoptosis in experimental stroke

Estrogen is protective in experimental cerebral ischemia, yet the mechanism remains unclear. Fas-mediated apoptosis has been shown to be induced after cerebral ischemia and significantly contribute to ischemic brain damage. In this study, we tested if estrogen is protective against cerebral ischemia by suppressing Fas-mediated apoptosis. 17β-estradiol-treated and untreated ovariectomized (OVX) female mice were subjected to 2 h middle cerebral artery occlusion (MCAO). Expression of Fas and Fas-associated death domain (FADD) were measured at 3, 6 and 12 h of reperfusion by RT-PCR and Western blot, respectively. Post-ischemic activities of caspase-8 and -3 activities, the two downstream effectors of Fas-induced apoptosis, were also assayed at same time points by ELISA. Finally, Fas antibody-induced cell death in primary cortical neurons was assayed by fluorescence activated cell sorter (FACS) in the presence and absence of estradiol. Our data showed that estradiol-treated OVX female mice sustained smaller infarct compared to untreated OVX mice. Ischemia upregulated Fas and FADD expression, and increased caspase-8 and -3 activities in OVX female mouse cortex, which were significantly attenuated by estradiol. Estradiol also significantly inhibited Fas antibody-induced neuronal cell apoptosis. Our data suggests that inhibition of ischemia-induced Fas-mediated apoptosis is an important mechanism of neuroprotection by estrogen in cerebral ischemia.     

Inhibition of MEK/ERK 1/2 pathway reduces pro-inflammatory cytokine interleukin-1 expression in focal cerebral ischemia

It has been proposed that mitogen-activated protein kinase (MAPK) pathways may play a role in the regulation of pro-inflammatory cytokines, such as interlukine-1, during cerebral ischemia. Our previous study showed that extracellular-signal-regulated kinases 1 and 2 (ERK 1/2) were activated during focal cerebral ischemia in mice [J. Cereb. Blood Flow Metab. 20 (2000) 1320]. However, the effect of ERK 1/2 activation in focal cerebral ischemia is still unclear. In this study we reported that in vivo phospho-ERK 1/2 expression increased following 30 min of middle cerebral artery occlusion (MCAO) in the mouse brain in both the ischemic core and perifocal regions. Western blot analysis and immunohistochemistry demonstrated that pro-treatment with 1,4-diamino-2,3-dicyano-1,4-bis butadiene (U0126) [J. Biol. Chem. 273 (1998) 18623] could significantly inhibit mouse brain phospho-MEK 1/2 and phospho-ERK 1/2 expression after 1-2 h of MCAO (p<0.05). Compared to the control group of mice, brain infarct volume was significantly decreased after 24 h of MCAO in the U0126-treated mice (27+/-6 vs. 46+/-9 mm(2), p<0.05). Inhibition of the MEK/ERK 1/2 pathway also prevented downstream kinase Elk-1 phosphorylation, and further reduced cytokine IL-1beta mRNA, but not TNFalpha, IL-1alpha, or chemokine MIP-1alpha mRNA expression. Our data demonstrates that in vivo the close linking of MEK 1/2, ERK 1/2, Elk-1, and IL-1 mRNA expression in the cerebral ischemia animals suggests that ERK 1/2 pathway activation is important in pro-inflammatory cytokine IL-1beta signaling, which induces an inflammatory response and exacerbates ischemic brain injury. Inhibiting the ERK 1/2 pathway may therefore provide a novel approach for the reduction of ischemia-induced IL-1beta overexpression.     

Brain damage in a mouse model of global cerebral ischemia. Effect of NMDA receptor blockade

The importance of particular genes in neuronal death following global cerebral ischemia can readily be studied in genetically modified mice provided a reliable model of ischemia is available. For that purpose, we developed a mouse model of global cerebral ischemia that induces consistent damage to different regions of the brain and with a low mortality rate. Twelve minutes of ischemia was induced in C57BL/6 mice by bilateral common carotid artery occlusion under halothane anesthesia and artificial ventilation. Body and brain temperature were monitored and cortical cerebral blood flow in each hemisphere was measured by laser Doppler flowmeter before, during, and for 5 min after ischemia. Extensive damage was found in the striatum and marked cell damage was observed in the CA1 and CA2 regions of hippocampus and in thalamus. Mild damage was seen in the CA3 region, dentate gyrus and cortex. Hippocampal damage in the CA1 region is delayed and developed over 48 h. Intraischemic hypothermia of 33 degrees C provided a robust neuroprotection. The non-competitive N-methyl-D-aspartate receptor blocker, MK-801, did not provide protection in the hippocampus, cortex, striatum or thalamus when administered 30 min prior to ischemia or 2 h after the end of ischemia, but selectively mitigated damage in the hippocampus, when administered immediately following ischemia. This model of global cerebral ischemia may be useful in pharmacological and genomic studies of ischemic brain damage.     

Transgenic mice overexpressing truncated trkB neurotrophin receptors in neurons show increased susceptibility to cortical injury after focal cerebral ischemia

It has been suggested that the increased production of endogenous BDNF after brain insults supports the survival of injured neurons and limits the spread of the damage. In order to test this hypothesis experimentally, we have produced transgenic mouse lines that overexpress the dominant-negative truncated splice variant of BDNF receptor trkB (trkB.T1) in postnatal cortical and hippocampal neurons. When these mice were exposed to transient focal cerebral ischemia by occluding the middle cerebral artery for 45 min and the damage was assessed 24 h later, transgenic mice had a significantly larger damage than wild-type littermates in the cerebral cortex (204 +/- 32% of wild-type, P = 0.02), but not in striatum, where the transgene is not expressed. Our results support the notion that endogenously expressed BDNF is neuroprotective and that BDNF signaling may have an important role in preventing brain damage after transient ischemia.     

Increased susceptibility to ischemic brain injury in cyclooxygenase-1-deficient mice.

Cyclooxygenase-1 (COX-1), a rate-limiting enzyme in the synthesis of prostanoids, is involved in selected vasodilatatory responses of the cerebral circulation. Cyclooxygenase-1-null mice were used to determine whether COX-1 influences cerebral ischemic damage. The middle cerebral artery was occluded in COX-1 -/- and +/+ mice (n = 9/group), and lesion volume was determined in thionin-stained sections 24 or 96 hours later. Middle cerebral artery occlusion produced larger infarcts in COX-1 -/- mice, both at 24 (35 +/- 17%; P < 0.05) and 96 hours (41 +/- 16%; P < 0.05) after ischemia. The enlargement was not due to increased susceptibility to glutamate excitotoxicity, because microinjection of N-methyl-D-aspartate or kainate in the parietal cortex produced comparable lesions in COX-1 +/+ and -/- mice ( P > 0.05; n = 8/group). To examine the contribution of hemodynamic factors to the enlargement of the infarct, cerebral blood flow was monitored by laser-Doppler flowmetry in the ischemic territory (n = 6/group). Although the reduction in cerebral blood flow was comparable in the ischemic core ( P > 0.05), at the periphery of the ischemic territory the reduction was greater in COX-1 -/- mice (-58 +/- 4%) than in COX-1 +/+ mice (-34 +/- 5%; P < 0.05). It is concluded that mice lacking COX-1 are more susceptible to focal cerebral ischemia, an effect that can be attributed to a more severe cerebral blood flow reduction in vulnerable regions at the periphery of the ischemic territory. Thus, the vascular effects of COX-1 may contribute to maintain cerebral blood flow in the postischemic brain and, as such, play a protective role in ischemic brain injury.     

Knockout of silent information regulator 2 (SIRT2) preserves neurological function after experimental stroke in mice

Sirtuin-2 (Sirt2) is a member of the NAD⁺-dependent protein deacetylase family. Various members of the sirtuin class have been found to be involved in processes related to longevity, regulation of inflammation, and neuroprotection. Induction of Sirt2 mRNA was found in the whole hemisphere after experimental stroke in a recent screening approach. Moreover, Sirt2 protein is highly expressed in myelin-rich brain regions after stroke. To examine the effects of Sirt2 on ischemic stroke, we induced transient focal cerebral ischemia in adult male Sirt2-knockout and wild-type mice. Two stroke models with different occlusion times were applied: a severe ischemia (45 minutes of middle cerebral artery occlusion (MCAO)) and a mild one (15 minutes of MCAO), which was used to focus on subcortical infarcts. Neurological deficit was determined at 48 hours after 45 minutes of MCAO, and up to 7 days after induction of 15 minutes of cerebral ischemia. In contrast to recent data on Sirt1, Sirt2^−/− mice showed less neurological deficits in both models of experimental stroke, with the strongest manifestation after 48 hours of reperfusion. However, we did not observe a significant difference of stroke volumes or inflammatory cell count between Sirt2-deficient and wild-type mice. Thus we postulate that Sirt2 mediates myelin-dependent neuronal dysfunction during the early phase after ischemic stroke.     

淀粉前体蛋白基因的Swedish突变对光化学法所致脑缺血的影响

目的观察淀粉前体蛋白基因的Swedish突变(APP/SWE)对光化学法所致脑缺血的影响。方法在APP/SWE转基因鼠与非转基因鼠应用光化学法诱导脑梗塞形成,以激光多普勒流量计测定缺血半暗带区脑血流变化,以TTC方法测定脑梗塞体积。结果(1)在APP/SWE转基因鼠组和非转基因鼠组之间缺血半暗带区脑血流在各个时间点均无显著差异,从照射6 min开始,两组缺血半暗带区脑血流较基线显著降低。(2)缺血后2 d APP/SWE转基因鼠组总梗塞体积、皮层梗塞体积及脑水肿指数均显著小于非转基因鼠组。(3)缺血后7d两组总梗塞体积、皮层梗塞体积、海马梗塞体积及脑水肿指数均无显著差异。结论APP/SWE在光化学法所致脑缺血最初30 min对缺血程度没有影响,在缺血后2d可起到神经保护作用,减小梗塞体积,在缺血后7d神经保护作用消失。其机制可能与APP/SWE所致脑血管舒张功能障碍,限制自由基扩散有关。     

Endogenous opioids inhibit ischemia-induced generation of immature hippocampal neurons via the mu-opioid receptor

It is established that hippocampal neurogenesis is dynamically regulated by physiological and pathological stimuli including learning, environmental complexity, mental disorders and brain lesion. Little is known about factors regulating adaptive changes in neurogenesis. Using µ-opioid receptor (MOP)-knockout mice we addressed whether endogenous opioids influence ischemia-induced enhancement of hippocampal neurogenesis. Permanent middle cerebral artery occlusion (MCAO) produced similar corticostriatal infarcts in MOP-knockout and wildtype mice. Analyses of BrdU/doublecortin-colabelled cells in the granule cell layer 14 days after MCAO showed that ischemic knockouts contained more immature neurons generated during days 9–11 than wildtypes. After 29 days, similar quantities of BrdU/NeuN-labelled cells were found in ischemic knockout and wildtype mice, suggesting that granule cells that were formed in excess during days 9–11 in the knockouts were eliminated by day 29. Neurogenesis was similar in knockout and wildtype mice subjected to sham operation. In addition to a transient increase in neurogenesis, MCAO caused a transient up-regulation of preprodynorphin and preproenkephalin mRNA expression in the granule cell layer. Our findings suggest that activated signalling via endogenous opioids and the MOP limits the enhanced generation of neuronal cells after ischemic corticostriatal lesions.     

Exercise preconditioning exhibits neuroprotective effects on hippocampal CA1 neuronal damage after cerebral ischemia

Recent evidence has suggested the neuroprotective effects of physical exercise on cerebral ischemic injury. However, the role of physical exercise in cerebral ischemia-induced hippocampal damage remains controversial. The aim of the present study was to evaluate the effects of pre-ischemia treadmill training on hippocampal CA1 neuronal damage after cerebral ischemia. Male adult rats were randomly divided into control, ischemia and exercise + ischemia groups. In the exercise + ischemia group, rats were subjected to running on a treadmill in a designated time schedule(5 days per week for 4 weeks). Then rats underwent cerebral ischemia induction th rough occlusion of common carotids followed by reperfusion. At 4 days after cerebral ischemia, rat learning and memory abilities were evaluated using passive avoidance memory test and rat hippocampal neuronal damage was detected using Nissl and TUNEL staining. Pre-ischemic exercise significantly reduced the number of TUNEL-positive cells and necrotic cell death in the hippocampal CA1 region as compared to the ischemia group. Moreover, pre-ischemic exercise significantly prevented ischemia-induced memory dysfunction. Pre-ischemic exercise mighct prevent memory deficits after cerebral ischemia through rescuing hippocampal CA1 neurons from ischemia-induced degeneration.     

Ischemia-induced ribosomal protein S3 expressional changes and the neuroprotective effect against experimental cerebral ischemic damage

Some ribosomal proteins are important regulators of development and DNA repair. However, few studies have been conducted on ribosomal protein S3 (rpS3) in the ischemic hippocampus. In the present study, we investigated ischemia-induced changes in rpS3 immunoreactivity, rpS3 mRNA, and protein levels in the hippocampal CA1 region of Mongolian gerbil after 5 min of transient forebrain ischemia. RpS3 immunoreactivity and its protein level were found to be significantly elevated at 6 hr after ischemia/reperfusion and then continuously decreased with time. RT-PCR analysis also showed that rpS3 mRNA levels were significantly elevated in CA1 at 6 hr after transient ischemia. In addition, during the course of this study, we developed a delivery vector (Pep-1) and its rpS3 fusion protein (Pep-1-rpS3) to elucidate the role of rpS3 in ischemia-induced damage. Pep-1-rpS3 administration to ischemic animals significantly and dose dependently increased neuronal survival in the stratum pyramidale of CA1. Moreover, Pep-1-rpS3 treatment reduced terminal deoxynucleotidyl dUTP nick-end labeling-positive CA1 pyramidal cell numbers in the stratum pyramidale. To elucidate how Pep-1-rpS3 ameliorates ischemic damage, changes in 4-hydroxy-2-nonnenal (HNE; an indicator of lipid peroxidation) immunoreactivity and protein levels were investigated. HNE levels and immunoreactivities in Pep-1-rpS3-treated ischemic animals were lower than in corresponding Pep-1-treated ischemic animals. These results indicate that rpS3 has a neuroprotective effect in the brain exposed to ischemia.