Upregulation of TACE/ADAM17 after ischemic preconditioning is involved in brain tolerance.

A short ischemic event (ischemic preconditioning [IPC]) can result in a subsequent resistance to severe ischemic injury (ischemic tolerance [IT]). Although tumor necrosis factor-alpha (TNF-alpha) contributes to the brain damage, its expression and neuroprotective role in models of IPC have also been described. However, the role of TNF-alpha convertase (TACE) in IPC and IT is not known. Using in vitro models, the authors previously demonstrated that TACE is upregulated after ischemic brain damage. In the present study, the authors used a rat model of transient middle cerebral artery occlusion as IPC to investigate TACE expression, its involvement in TNF-alpha release, and its role in IT. Western blot analysis showed that TACE expression is increased after IPC. Ischemic preconditioning caused TNF-alpha release, an effect that was blocked by the selective TACE inhibitor BB-1101 (10 mg. kg(-1). day(-1); SHAM, 1,050 +/- 180; IPC, 1,870 +/- 290; IPC + BB, 1,320 +/- 260 ng/mg; n = 4, < 0.05). Finally, IPC produced a reduction in infarct volume, which was inhibited by treatment with BB-1101 and with anti-TNF-alpha (10 microg/5 doses; SHAM + permanent middle cerebral artery occlusion [pMCAO], 335 +/- 20; IPC + pMCAO, 244 +/- 14; IPC + BB + pMCAO, 300 +/- 6; IPC + anti-TNF + pMCAO, 348 +/- 22 mm3; n = 6-10, < 0.05). Taken together, these data demonstrate that TACE is upregulated after IPC, plays a major role in TNF-alpha shedding in IPC, and has a neuroprotective role in IT.     

Peri-sciatic administration of recombinant rat TNF-alpha induces mechanical allodynia via upregulation of TNF-alpha in dorsal root ganglia and in spinal dorsal horn: the role of NF-kappa B pathway

Previous studies have shown that tumor necrosis factor-alpha (TNF-alpha) and TNF receptor 1 (TNFR1) in dorsal root ganglia (DRG) and in spinal dorsal horn are upregulated after nerve injury and that many TNF-alpha-containing neurons overexpress TNFR1. In the present study, we found that peri-sciatic administration of rat recombinant TNF-alpha (rrTNF) at the concentrations of 10, 100 and 1000 pg/ml (daily for 2 days) induced mechanical allodynia in bilateral hindpaws, lasting for about 20 days. The immunoreactivity (IR) of TNF-alpha and TNFR1 in the ipsilateral (but not in the contralateral) L4 and L5 DRGs increased significantly on day 1 and day 3 after administration of rrTNF, respectively. Double immunofluorescence staining revealed that in DRGs the increased TNF-alpha-IR was mainly in neuronal cells and with a lesser extent in satellite glial cells, while the upregulation of TNFR1-IR was almost restricted at neuronal cells. TNF-alpha-IR but not TNFR1-IR also increased in bilateral lumbar spinal dorsal horn from day 3 to day 14, which was observed in astrocytes, microglias and neurons. In addition, a progressive infiltration of monocyte/macrophages and T lymphocytes in the ipsilateral L5 DRG and sciatic nerve was observed, starting on day 2 following administration of rrTNF. Intrathecal delivery of PDTC (8.2 ng in 10 microl volume), a nuclear factor-kappa B (NF-kappaB) inhibitor, 30 min before each rrTNF administration blocked mechanical allodynia completely and inhibited the upregulation of TNF-alpha-IR and TNFR1-IR substantially. The results suggest that peri-sciatic administration of rrTNF may induce mechanical allodynia by an autocrine mechanism via activation of the NF-kappaB pathway.     

Obligatory role of inducible nitric oxide synthase in ischemic preconditioning.

Sublethal insults can induce a transient tolerance toward subsequent lethal ischemia, a phenomenon termed ischemic preconditioning (IPC). In the myocardium, nitric oxide derived from 'inducible' nitric oxide synthase (iNOS or NOS II) plays a critical role in the expression of IPC produced by sublethal ischemia. Here, we investigated whether iNOS is involved in IPC in brain. Ischemic preconditioning was produced in mice by three episodes of 1-min bilateral common carotid artery (BCCA) occlusion, each followed by 5 mins of reperfusion. After 24 h, mice underwent middle cerebral artery (MCA) occlusion for 20 mins. Intraischemic cerebral blood flow was monitored during both in BCCA and MCA occlusion (MCAO) by laser-Doppler flowmetry. Mice were killed 3 days after MCAO, and infarct volume was determined in thionine-stained sections. Infarct volume was significantly reduced 24 h after IPC (70%; P<0.05). Treatment with the iNOS inhibitor aminoguanidine (400 mg/kg), abolished the IPC-induced protection. Furthermore, IPC failed to induce ischemic tolerance in iNOS-null mice. In wild-type mice, IPC increased the resistance to Ca(2+)-mediated depolarization in isolated brain mitochondria. However, in iNOS-null mice IPC failed to induce such resistance. We conclude that iNOS is required for the full expression of IPC and that such effect is coupled to an increased resistance of mitochondria to injury. Thus, iNOS-derived nitric oxide, in addition to its deleterious effects on the late stages of ischemic brain damage, can also be beneficial by promoting ischemic tolerance through signaling, ultimately resulting in mitochondrial protection.     

Age-related changes to tumor necrosis factor receptors affect neuron survival in the presence of beta-amyloid

Inflammation including local accumulations of tumor necrosis factor alpha (TNF-alpha) is a part of Alzheimer's disease pathology and may exacerbate age-related neurodegeneration. Most studies on TNF-alpha and TNF neuronal receptors are conducted by using embryonic neurons. Few studies consider age-related deficits that may occur in neurons. Age-related changes in susceptibility to TNF-alpha through TNF receptor 1 (TNFR1) and receptor 2 (TNFR2) expression could increase susceptibility to beta-amyloid (1-42, Abeta42). Evidence is conflicting about which receptor mediates survival and/or apoptosis. We determined how aging affects receptor expression in cultured adult rat cortical neurons. Old neurons were more susceptible to Abeta42 toxicity than middle-aged neurons, and the addition of TNF-alpha was neuroprotective in middle-aged neurons, but exacerbated the toxicity from Abeta42 in old neurons. These pathologic and protective responses in old and middle-aged neurons, respectively, correlated with higher starting TNFR1 and TNFR2 mRNA levels in old vs. middle-aged neurons. Middle-aged neurons treated with TNF-alpha plus Abeta42 did not show an increase in either TNFR1 or TNFR2 mRNA, but old neurons showed an up-regulation in TNFR2 mRNA and not TNFR1 mRNA. Despite these mRNA changes, surface immunoreactivity of both TNFR1 and TNFR2 increased with the dose of TNF-alpha in middle-aged neurons. However, middle-aged neurons treated with TNF-alpha plus Abeta42 showed an up-regulation in both TNFR1 and TNFR2 surface expression, whereas old neurons failed to up-regulate surface expression of either receptor. These findings support the hypothesis that age-related changes in TNF-alpha surface receptor expression contribute to the neuronal loss associated with inflammation in Alzheimer's disease.     

Brain ischemic preconditioning is abolished by antioxidant drugs but does not up-regulate superoxide dismutase and glutathion peroxidase

The present work examined the hypothesis that brain ischemic tolerance induced by ischemic preconditioning (IPC) is triggered by an initial oxidative stress and is associated with an increase in antioxidant enzyme activities as one end-effector of the neuroprotection. Wistar rats were preconditioned by a single 3-min occlusion of the middle cerebral artery. After a various duration of reperfusion (30 min, 24, 72 or 168 h), rats were subjected to a 60-min focal ischemia and sacrificed 24 h later. Cerebral infarcts were significantly reduced when performed during the 24- to 72-h time window after IPC. The pretreatment with the protein synthesis inhibitor, cycloheximide (1 mg/kg, i.p., 30 min prior to IPC), completely suppressed the neuroprotection. The free radical scavenger, dimethylthiourea (DMTU; 300 mg/kg, i.p., 30 min prior to IPC) and the antioxidant ebselen (10 mg/kg, oral cramming, 2 h before and 12 h after IPC) also abolished the IPC-induced protection of the brain. Nevertheless, IPC did not induce any delayed changes in antioxidant enzyme (superoxide dismutase, glutathion peroxidase) activities nor in the neuronal expression of Mn and Cu/Zn superoxide dismutase. These results indicate that an initial oxidative stress could be involved as a trigger of IPC, while antioxidant enzymes do not play a key role as end-effectors in such a neuroprotection.     

Generation of hydrogen peroxide during brief oxygen-glucose deprivation induces preconditioning neuronal protection in primary cultured neurons

Although reactive oxygen species (ROS) have been implicated in ischemic preconditioning (IPC)-induced neuronal protection, several key questions concerning ROS remain to be elucidated. The purpose of this study is to obtain direct evidence for the formation of specific ROS species generated by IPC, and to determine the specific species that is responsible for the observed neuronal protection. Primary cultured cortex neurons from rat embryos were preconditioned with 10 min of oxygen-glucose deprivation (OGD), which increased the intracellular levels of superoxide and hydrogen peroxide. This preconditioning markedly induced neuronal protection against 2-hr OGD stimuli. Preconditioning with exogenous ROS by the administration of xanthine/xanthine oxidase (X/XO), or hydrogen peroxide was also found to induce IPC-like neuronal protection. Administration of hydrogen peroxide scavengers, such as catalase, glutathione, or the thiol reductant N-(2-mercaptopriopionyl)-glycine, all reduced the increase in the intracellular hydrogen peroxide levels, which effectively eliminated IPC- or exogenous ROS-induced neuronal protection. In contrast, administration of the membrane-permeable superoxide dismutase mimic Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride was able to block the increase of intracellular superoxide levels during IPC, but did not abolish either IPC- or exogenous X/XO preconditioning-induced neuronal protection. These findings strongly suggest that IPC enhances the generation of superoxide, which is then converted to hydrogen peroxide, and that hydrogen peroxide is likely the main trigger involved in the mechanism of IPC-induced neuronal protection.     

Endotoxin preconditioning protects against the cytotoxic effects of TNFalpha after stroke: a novel role for TNFalpha in LPS-ischemic tolerance.

Lipopolysaccharide (LPS) preconditioning provides neuroprotection against subsequent cerebral ischemic injury. Tumor necrosis factor-alpha (TNFalpha) is protective in LPS-induced preconditioning yet exacerbates neuronal injury in ischemia. Here, we define dual roles of TNFalpha in LPS-induced ischemic tolerance in a murine model of stroke and in primary neuronal cultures in vitro, and show that the cytotoxic effects of TNFalpha are attenuated by LPS preconditioning. We show that LPS preconditioning significantly increases circulating levels of TNFalpha before middle cerebral artery occlusion in mice and show that TNFalpha is required to establish subsequent neuroprotection against ischemia, as mice lacking TNFalpha are not protected from ischemic injury by LPS preconditioning. After stroke, LPS preconditioned mice have a significant reduction in the levels of TNFalpha (approximately threefold) and the proximal TNFalpha signaling molecules, neuronal TNF-receptor 1 (TNFR1), and TNFR-associated death domain (TRADD). Soluble TNFR1 (s-TNFR1) levels were significantly increased after stroke in LPS-preconditioned mice (approximately 2.5-fold), which may neutralize the effect of TNFalpha and reduce TNFalpha-mediated injury in ischemia. Importantly, LPS-preconditioned mice show marked resistance to brain injury caused by intracerebral administration of exogenous TNFalpha after stroke. We establish an in vitro model of LPS preconditioning in primary cortical neuronal cultures and show that LPS preconditioning causes significant protection against injurious TNFalpha in the setting of ischemia. Our studies suggest that TNFalpha is a twin-edged sword in the setting of stroke: TNFalpha upregulation is needed to establish LPS-induced tolerance before ischemia, whereas suppression of TNFalpha signaling during ischemia confers neuroprotection after LPS preconditioning.     

局灶性缺血预处理对脑梗死大鼠神经生长因子表达的影响

目的:研究局灶性缺血预处理对脑梗死大鼠神经生长因子(nerve growthfactor,NGF)表达的影响,探讨缺 血预处理诱导脑缺血耐受机制。方法:SD大鼠随机分为3组。预缺血组和假手术组在大脑中动脉缺血(MCAO)前3天 分别接受10min的预缺血或假手术,MCAO 2h后再灌注22h处死;对照组两次均为假手术,比较各组神经功能评分、梗 死体积及NGF的表达。结果:预缺血组神经功能评分、梗死体积较假手术组减少(P<0．05),NGF表达明显高于其余两 组(P<0．01)。结论:局灶性缺血预处理可诱导脑缺血耐受的产生,其作用机制可能与NGF的表达改变有关。     

缺血预处理（IPC）对大鼠局灶性脑梗死后HSP70和FOS表达的影响

目的 :研究缺血预处理 (IPC)对局灶性脑梗死后HSP70和FOS表达的影响 ,探讨IPC的脑保护作用机制。方法 :利用线栓法建立局灶性脑缺血 大脑中动脉闭塞模型 (MCAO)。MCAO 10min作为IPC ,IPC后 48h制作永久性大脑中动脉梗死 (PMCAO)模型。了解IPC对PMCAO后大脑神经功能和脑组织学损害的影响 ,免疫组化法研究PMCAO后 3hFOS表达变化以及 2 4h后HSP 70表达变化。结果 :IPC显著减轻PMCAO后大鼠神经功能损害和组织学损害 ,减少PMCAO后FOS、HSP 70的表达。结论 :IPC对其后PMCAO有明显的保护作用 ,能诱导脑缺血耐受 (IT)的产生 ,脑IT的神经保护作用与脑梗死后HSP 70和FOS表达改变密切相关。     

Ischemic preconditioning reduces ischemic brain injury by suppressing nuclear factor kappa B expression and neuronal apoptosis

Ischemic stroke induces a series of complex pathophysiological events including blood-brain barrier disruption, inflammatory response and neuronal apoptosis. Previous studies demonstrate that ischemic preconditioning attenuates ischemic brain damage via inhibiting blood-brain barrier disruption and the inflammatory response. Rats underwent transient (15 minutes) occlusion of the bilateral common carotid artery with 48 hours of reperfusion, and were subjected to permanent middle cerebral artery occlusion. This study explored whether ischemic preconditioning could reduce ischemic brain injury and relevant molecular mechanisms by inhibiting neuronal apoptosis. Results found that at 72 hours following cerebral ischemia, myeloperoxidase activity was enhanced, malondialdehyde levels increased, and neurological function was obviously damaged. Simultaneously, neuronal apoptosis increased, and nuclear factor-κB and cleaved caspase-3 expression was significantly increased in ischemic brain tissues. Ischemic preconditioning reduced the cerebral ischemia-induced inflammatory response, lipid peroxidation, and neurological function injury. In addition, ischemic preconditioning decreased nuclear factor-κB p65 and cleaved caspase-3 expression. These results suggested that ischemic preconditioning plays a protective effect against ischemic brain injury by suppressing the inflammatory response, reducing lipid peroxidation, and neuronal apoptosis via inhibition of nuclear factor-κB and cleaved caspase-3 expression.     

Endogenous neuroprotection in the retina

Ischemic preconditioning (IPC) protects the rat retina against the injury that ordinarily follows severe ischemia. The retina is protected against the damage following severe ischemia for up to 72h after the application of IPC. However, there is no early preconditioning, i.e. protective effects starting within hours of preconditioning. The IPC stimulus consists of a brief, non-damaging period of ischemia. It results in complete preservation of retinal structure and function following ischemia, and is thus the most robust neuroprotection demonstrated in the retina to date. Release of adenosine, de novo protein synthesis, and mediators such as protein kinase C and K(+) ATP channels are required for IPC protection. Both the adenosine A1 and A2a receptors are involved. However, the molecular mechanisms for neuroprotection have not been completely described. It appears that both increased expression of protective proteins and decreased expression of pro-apoptotic proteins are involved. In addition, IPC prevents hypoperfusion following severe ischemia. Further study of the IPC phenomenon could lead to an enhanced understanding of the mechanisms of ischemic damage and its prevention in the retina.     

The effect of rapid preconditioning on the microglial, astrocytic and neuronal consequences of global cerebral ischemia

Previous studies indicated preconditioning of the brain with sublethal ischemic insults separated by many hours, protected tissues from a subsequent lethal insult. We recently reported neuroprotection by a rapid preconditioning paradigm where a sublethal ischemic insult preceded test ischemia by only 30 min. We hypothesize that neuroprotection caused by the rapid ischemic preconditioning (IPC) will result in lowered microglial, reactive astrocytes and increased normal neuronal cell counts. Wistar rats underwent normothermic (36.5–37 °C) global cerebral ischemia, produced by bilateral carotid artery ligation after lowering mean systemic blood pressure. The preconditioning ischemic insult lasted 2 min and was associated with a sufficient amount of time to provoke anoxic depolarization. After a 30-min reperfusion period, 10-min test ischemia was produced, and histopathology was assessed 3 and 7 days later. Normal neuronal cell counts for control rats at 3 days survival were significantly lower (by 58%) than in IPC animals. Although there was a trend toward protection in IPC rats at 7 days, the difference in normal neuronal cell count between the IPC and control groups was not significant. IPC rats at 3 days but not 7 days of survival showed a significantly lower microglial cell count (by 56%) than control rats. These results showed that the protection induced through IPC at 3 days of survival produced lower numbers of microglia, while maintaining normal neuronal cells. No significant differences between control and IPC groups were found in astrocytic cell count at any time of reperfusion in any region of the hippocampus studied. The beneficial effects of IPC may, therefore, involve anti-inflammatory processes that target microglial activation after cerebral ischemia. Received: 1 July 1998 / Revised: 30 September 1998 / Accepted: 21 October 1998     

Epsilon protein kinase C mediated ischemic tolerance requires activation of the extracellular regulated kinase pathway in the organotypic hippocampal slice.

Ischemic preconditioning (IPC) promotes brain tolerance against subsequent ischemic insults. Using the organotypic hippocampal slice culture, we conducted the present study to investigate (1) the role of adenosine A1 receptor (A1AR) activation in IPC induction, (2) whether epsilon protein kinase C (epsilonPKC) activation after IPC is mediated by the phosphoinositol pathway, and (3) whether epsilonPKC protection is mediated by the extracellular signal-regulated kinase (ERK) pathway. Our results demonstrate that activation of A1AR emulated IPC, whereas blockade of the A1AR during IPC diminished neuroprotection. The neuroprotection promoted by the A1AR was also reduced by the epsilonPKC antagonist. To determine whether epsilonPKC activation in IPC and A1AR preconditioning is mediated by activation of the phosphoinositol pathway, we incubated slices undergoing IPC or adenosine treatment with a phosphoinositol phospholipase C inhibitor. In both cases, preconditioning neuroprotection was significantly attenuated. To further characterize the subsequent signal transduction pathway that ensues after epsilonPKC activation, mitogen-activated protein kinase kinase was blocked during IPC and pharmacologic preconditioning (PPC) (with epsilonPKC, NMDA, or A1AR agonists). This treatment significantly attenuated IPC- and PPC-induced neuroprotection. In conclusion, we demonstrate that epsilonPKC activation after IPC/PPC is essential for neuroprotection against oxygen/glucose deprivation in organotypic slice cultures and that the ERK pathway is downstream to epsilonPKC.     

Temporal kinetics and cellular phenotype of TNF p55/p75 receptors in experimental allergic encephalomyelitis

TNF-alpha and LT-alpha are thought to be involved in the immunopathology of CNS demyelinating diseases. Both cytokines induce cellular effects through 55-kDa type-1 receptors (R1) and 75-kDa type-2 receptors (R2). To date, no study has specifically identified the various cell populations that express TNF receptors (TNFR) in the inflammatory and demyelinating mouse model, EAE. Phenotyping the TNFR positive cells is important in determining when and where the ligands may be acting and playing a role in disease pathology. We observed an upregulation of TNF R1 and R2 mRNA in high endothelial venules (HEVs) in the lymph node and CNS before the onset of EAE (preclinical phase). This upregulation of TNFR expression in HEVs was followed by a rapid increase in leukocytes within the CNS after the onset of clinical disease. The temporal kinetics of these data suggest that HEVs become activated early, probably through the release of pro-inflammatory cytokines originating from circulating leukocytes. An increase in TNFR on HEVs would make these cells more susceptible to TNF-induced changes, such as increasing cellular adhesion molecules, thereby further facilitating the trafficking of leukocytes into the CNS parenchyma.     

Ischemic preconditioning preserves mitochondrial function after global cerebral ischemia in rat hippocampus.

Ischemic tolerance in brain develops when sublethal ischemic insults occur before "lethal" cerebral ischemia. Two windows for the induction of tolerance by ischemic preconditioning (IPC) have been proposed: one that occurs within 1 hour after IPC, and another that occurs 1 or 2 days after IPC. The authors tested the hypotheses that IPC would reduce or prevent ischemia-induced mitochondrial dysfunction. IPC and ischemia were produced by bilateral carotid occlusions and systemic hypotension (50 mm Hg) for 2 and 10 minutes, respectively. Nonsynaptosomal mitochondria were harvested 24 hours after the 10-minute "test" ischemic insult. No significant changes were observed in the oxygen consumption rates and activities for hippocampal mitochondrial complexes I to IV between the IPC and sham groups. Twenty-four hours of reperfusion after 10 minutes of global ischemia (without IPC) promoted significant decreases in the oxygen consumption rates in presence of substrates for complexes I and II compared with the IPC and sham groups. These data suggest that IPC protects the integrity of mitochondrial oxidative phosphorylation after cerebral ischemia.