Na+-dependent high-affinity glutamate transport in macrophages

Excessive accumulation of glutamate in the CNS leads to excitotoxic neuronal damage. However, glutamate clearance is essentially mediated by astrocytes through Na+-dependent high-affinity glutamate transporters (excitatory amino acid transporters (EAATs)). Nevertheless, EAAT function was recently shown to be developmentally restricted in astrocytes and undetectable in mature astrocytes. This suggests a need for other cell types for clearing glutamate in the brain. As blood monocytes infiltrate the CNS in traumatic or inflammatory conditions, we addressed the question of whether macrophages expressed EAATs and were involved in glutamate clearance. We found that macrophages derived from human blood monocytes express both the cystine/glutamate antiporter and EAATs. Kinetic parameters were similar to those determined for neonatal astrocytes and embryonic neurons. Freshly sorted tissue macrophages did not possess EAATs, whereas cultured human spleen macrophages and cultured neonatal murine microglia did. Moreover, blood monocytes did not transport glutamate, but their stimulation with TNF-alpha led to functional transport. This suggests that the acquisition of these transporters by macrophages could be under the control of inflammatory molecules. Also, monocyte-derived macrophages overcame glutamate toxicity in neuron cultures by clearing this molecule. This suggests that brain-infiltrated macrophages and resident microglia may acquire EAATs and, along with astrocytes, regulate extracellular glutamate concentration. Moreover, we showed that EAATs are involved in the regulation of glutathione synthesis by providing intracellular glutamate. These observations thus offer new insight into the role of macrophages in excitotoxicity and in their response to oxidative stress.     

Effects of lipopolysaccharide on glial phenotype and activity of glutamate transporters: Evidence for delayed up-regulation and redistribution of GLT-1

Excitatory amino acid transporters (EAATs) are responsible for homeostasis of extracellular L-glutamate, and the glial transporters are functionally dominant. EAAT expression or function is altered in acute and chronic neurological conditions, but little is known about the regulation of EAATs in reactive astroglia found in such neuropathologies. These studies examined the effects of the bacterial endotoxin lipopolysaccharide (LPS) on glial EAATs in vitro. The effects of LPS (1 microg/ml, 24-72 h) on EAAT activity and expression were examined in primary cultures of mouse astrocytes. [(3)H]D-aspartate uptake increased to 129% of control by 72 h treatment with LPS. Saturation analysis revealed that apparent K(m) was unchanged whilst V(max) was significantly increased to 172% of control by 72 h LPS treatment. Biotinylation and Western blotting indicated that cell-surface expression of GLT-1 was significantly elevated (146% control) by LPS treatment whereas GLAST expression was unchanged. Confocal analyses revealed that LPS treatment resulted in cytoskeletal changes and stellation of astrocytes, with rearrangement of F-actin (as shown by phalloidin labelling). Immunocytochemistry revealed clustering of GLAST, and increased expression and redistribution of GLT-1 to the cell-surface following treatment with LPS. Similar experiments were conducted in microglia, where LPS (50 ng/ml) was found to up-regulate expression of GLT-1 at 24 and 72 h in concert with cytoskeletal changes accompanying activation. These findings suggest an association of cytoskeletal changes in glia with EAAT activity, with the predominant adaptation involving up-regulation and redistribution of GLT-1.     

天冬酰胺内肽酶和胶质细胞在创伤性脑损伤中的激活

摘要 目的：创伤性脑损伤（traumatic brain injury, TBI）缺乏安全有效的治疗手段,亟须寻找新的干预靶点。天冬酰胺内肽酶 (asparaginyl endopeptidase, AEP)在免疫和神经系统疾病中起重要作用,本研究观察了小鼠TBI模型中AEP的激活和变化,探讨AEP对脑损伤和修复的意义。方法：控制性皮层撞击法在小鼠右脑半球制作TBI损伤,在造模后的不同时间点,测定受损脑组织内的乳酸含量和AEP的活性变化,免疫荧光化学染色观察TBI之后3天的胶质细胞活化,以及AEP在其中的表达。结果：TBI造成乳酸在受损脑组织内逐渐堆积,导致小胶质细胞和星形胶质细胞的反应性活化和增生,AEP的上调和激活出现在TBI的继发性脑损伤阶段,AEP在小胶质细胞和星形胶质细胞内均出现上调。结论：AEP有可能参与调控TBI引发的胶质细胞活化,在神经损伤和修复中发挥重要作用。     

De novo expression of the hemoglobin scavenger receptor CD163 by activated microglia is not associated with hemorrhages in human brain lesions

The main function of CD163 (hemoglobin scavenger receptor) is to bind the hemoglobin-haptoglobin complex, thereby mediating extravasal hemolysis. However, CD163 also has an antiinflammatory function. After CD163-mediated endocytosis, hemoglobin is catabolized further by hemeoxygenase 1 (HO-1). Previously, we found expression of HO-1 to be restricted to microglia/macrophages at sites of hemorrhages in human traumatic and ischemic brain lesions. We now investigated if CD163 expression is also correlated with hemorrhages in brain lesions. Methods. Autopsy brain tissue from 44 cases with hemorrhagic brain lesions (32 traumatic brain injuries/TBI, 12 intracerebral bleedings/ICB), 56 non-hemorrhagic brain lesions (30 ischemias, 26 hypoxias) and 6 control brains were investigated. The post injury survival times ranged from a few minutes to 60 months. Results. In controls, single perivascular monocytes expressed CD163, but only single CD163+ microglia were found in 3/6 cases. CD163+ cells in the parenchyma (activated microglia/macrophages) increased significantly within 24 hours after trauma and ischemia and within 1-7 days following ICB or hypoxia. Overall, significantly lower and higher levels of parenchymal CD163+ cells occurred in hypoxia and ischemia, respectively. Perivascular CD163+ cells also increased significantly in all pathological conditions. In areas remote from circumscribed brain lesions (TBI, ICB, ischemia), significant changes were only found in ICB and ischemia. Conclusions. De novo expression of CD163 by activated microglia/macrophages and CD163+ infiltrating monocytes are neither restricted to nor predominant in hemorrhagic brain lesions. Thus, the antiinflammatory function of CD163 probably predominates, both in hemorrhagic and non-hemorrhagic brain lesions and points to possible immunomodulatory treatment strategies targeting CD163.     

Changes of the GPR17 receptor, a new target for neurorepair, in neurons and glial cells in patients with traumatic brain injury

Unveiling the mechanisms participating in the damage and repair of traumatic brain injury (TBI) is fundamental to develop new therapies. The P2Y-like GPR17 receptor has recently emerged as a sensor of damage and a key actor in lesion remodeling/repair in the rodent brain, but its role in humans is totally unknown. Here, we characterized GPR17 expression in brain specimens from seven intensive care unit TBI patients undergoing neurosurgery for contusion removal and from 28 autoptic TBI cases (and 10 control subjects of matched age and gender) of two university hospitals. In both neurosurgery and autoptic samples, GPR17 expression was strong inside the contused core and progressively declined distally according to a spatio-temporal gradient. Inside and around the core, GPR17 labeled dying neurons, reactive astrocytes, and activated microglia/macrophages. In peri-contused parenchyma, GPR17 decorated oligodendrocyte precursor cells (OPCs) some of which had proliferated, indicating re-myelination attempts. In autoptic cases, GPR17 expression positively correlated with death for intracranial complications and negatively correlated with patients’ post-traumatic survival. Data indicate lesion-specific sequential involvement of GPR17 in the (a) death of irreversibly damaged neurons, (b) activation of microglia/macrophages remodeling the lesion, and (c) activation/proliferation of multipotent parenchymal progenitors (both reactive astrocytes and OPCs) starting repair processes. Data validate GPR17 as a target for neurorepair and are particularly relevant to setting up new therapies for TBI patients.     

Neuronal Expression of Splice Variants of “Glial” Glutamate Transporters in Brains Afflicted by Alzheimer’s Disease: Unmasking an Intrinsic Neuronal Property

Anomalies in glutamate homeostasis may contribute to the pathological processes involved in Alzheimer’s disease (AD). Glutamate released from neurons or glial cells is normally rapidly cleared by glutamate transporters, most of which are expressed at the protein level by glial cells. However, in some patho-physiological situations, expression of glutamate transporters that are normally considered to be glial types, appears to be evoked in populations of distressed neurons. This study analysed the expression of exon-skipping forms of the three predominant excitatory amino acid (glutamate) transporters (EAATs1-3) in brains afflicted with AD. We demonstrate by immunocytochemistry in temporal cortex, the expression of these proteins particularly in limited subsets of neurons, some of which appeared to be dys-morphic. Whilst the neuronal expression of the “glial” glutamate transporters EAAT1 and EAAT2 is frequently considered to represent the abnormal and ectopic expression of such transporters, we suggest this may be a misinterpretation, since neurons such as cortical pyramidal cells normally express abundant mRNA for these EAATs (but little if any EAAT protein expression). We hypothesize instead that distressed neurons in the AD brain can turn on the translation of pre-existent mRNA pools, or suppress the degradation of alternately spliced glutamate transporter protein, leading to the “unmasking” of, rather than evoked expression of “glial” glutamate transporters in stressed neurons. Special issue article in honor of Dr. Graham Johnston.     

Hetero-oligomerization of neuronal glutamate transporters

Excitatory amino acid transporters (EAATs) mediate the uptake of glutamate into neuronal and glial cells of the mammalian central nervous system. Two transporters expressed primarily in glia, EAAT1 and EAAT2, are crucial for glutamate homeostasis in the adult mammalian brain. Three neuronal transporters (EAAT3, EAAT4, and EAAT5) appear to have additional functions in regulating and processing cellular excitability. EAATs are assembled as trimers, and the existence of multiple isoforms raises the question of whether certain isoforms can form hetero-oligomers. Co-expression and pulldown experiments of various glutamate transporters showed that EAAT3 and EAAT4, but neither EAAT1 and EAAT2, nor EAAT2 and EAAT3 are capable of co-assembling into heterotrimers. To study the functional consequences of hetero-oligomerization, we co-expressed EAAT3 and the serine-dependent mutant R501C EAAT4 in HEK293 cells and Xenopus laevis oocytes and studied glutamate/serine transport and anion conduction using electrophysiological methods. Individual subunits transport glutamate independently of each other. Apparent substrate affinities are not affected by hetero-oligomerization. However, polarized localization in Madin-Darby canine kidney cells was different for homo- and hetero-oligomers. EAAT3 inserts exclusively into apical membranes of Madin-Darby canine kidney cells when expressed alone. Co-expression with EAAT4 results in additional appearance of basolateral EAAT3. Our results demonstrate the existence of heterotrimeric glutamate transporters and provide novel information about the physiological impact of EAAT oligomerization.     

EAAT expression by macrophages and microglia: still more questions than answers

Glutamate is the main excitatory amino acid, but its presence in the extracellular milieu has deleterious consequences. It may induce excitotoxicity and also compete with cystine for the use of the cystine–glutamate exchanger, blocking glutathione neosynthesis and inducing an oxidative stress-induced cell death. Both mechanisms are critical in the brain where up to 20% of total body oxygen consumption occurs. In normal conditions, the astrocytes ensure that extracellular concentration of glutamate is kept in the micromolar range, thanks to their coexpression of high-affinity glutamate transporters (EAATs) and glutamine synthetase (GS). Their protective function is nevertheless sensitive to situations such as oxidative stress or inflammatory processes. On the other hand, macrophages and microglia do not express EAATs and GS in physiological conditions and are the principal effector cells of brain inflammation. Since the late 1990s, a number of studies have now shown that both microglia and macrophages display inducible EAAT and GS expression, but the precise significance of this still remains poorly understood. Brain macrophages and microglia are sister cells but yet display differences. Both are highly sensitive to their microenvironment and can perform a variety of functions that may oppose each other. However, in the very particular environment of the healthy brain, they are maintained in a repressed state. The aim of this review is to present the current state of knowledge on brain macrophages and microglial cells activation, in order to help clarify their role in the regulation of glutamate under pathological conditions as well as its outcome.     

A beta-lactam antibiotic dampens excitotoxic inflammatory CNS damage in a mouse model of multiple sclerosis

In multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE), impairment of glial "Excitatory Amino Acid Transporters" (EAATs) together with an excess glutamate-release by invading immune cells causes excitotoxic damage of the central nervous system (CNS). In order to identify pathways to dampen excitotoxic inflammatory CNS damage, we assessed the effects of a beta-lactam antibiotic, ceftriaxone, reported to enhance expression of glial EAAT2, in "Myelin Oligodendrocyte Glycoprotein" (MOG)-induced EAE. Ceftriaxone profoundly ameliorated the clinical course of murine MOG-induced EAE both under preventive and therapeutic regimens. However, ceftriaxone had impact neither on EAAT2 protein expression levels in several brain areas, nor on the radioactive glutamate uptake capacity in a mixed primary glial cell-culture and the glutamate-induced uptake currents in a mammalian cell line mediated by EAAT2. Moreover, the clinical effect of ceftriaxone was preserved in the presence of the EAAT2-specific transport inhibitor, dihydrokainate, while dihydrokainate alone caused an aggravated EAE course. This demonstrates the need for sufficient glial glutamate uptake upon an excitotoxic autoimmune inflammatory challenge of the CNS and a molecular target of ceftriaxone other than the glutamate transporter. Ceftriaxone treatment indirectly hampered T cell proliferation and proinflammatory INFgamma and IL17 secretion through modulation of myelin-antigen presentation by antigen-presenting cells (APCs) e.g. dendritic cells (DCs) and reduced T cell migration into the CNS in vivo. Taken together, we demonstrate, that a beta-lactam antibiotic attenuates disease course and severity in a model of autoimmune CNS inflammation. The mechanisms are reduction of T cell activation by modulation of cellular antigen-presentation and impairment of antigen-specific T cell migration into the CNS rather than or modulation of central glutamate homeostasis.     

Immunohistochemical investigations of excitotoxicity in experimental spinal cord trauma

The concept of "excitotoxicity" assumes that high concentration of glutamate (main excitatory neuromediator) acting through specific receptors leads to damage of cells due to an influx of calcium ions. Proteins called "excitatory amino acid transporters" (EAATs), present in astroglia, play important role in the removal of glutamate. We investigated the expression of GluR2 (glutamate receptor), EAAT1, and EAAT2 by immunohistochemistry in formalin-fixed, paraffin-embedded rat spinal cords, previously subjected to experimental mechanical trauma. In the injured spinal cords, an elevated immunoreactivity of GluR2 was noted even 10 min after trauma and was still observed 2 days after injury. Strong immunoreactivity was observed not only in many cells in gray matter but also in some cells in white matter (probably glial cells). In the injured spinal cords, we observed stronger (as compared with controls) expression of EAATs in the white matter, especially 6 hours after injury. The results support the role of excitotoxicity in mechanical trauma of spinal cord suggesting a possibility of long lasting elevated expression of glutamate receptor. It may help to understand and to explain beneficial action of "anti-glutamate" drugs, reported by other investigators.     

Differential regulation of metallothionein-I and metallothionein-II mRNA expression in the rat brain following traumatic brain injury

In this study, we investigated the expression of metallothionein (MT)-I and MT-II in the rat brain following traumatic brain injury (TBI). In the early stage, significant induction of MT-I and MT-II were observed in various regions including ventricle walls, pia mater, and dentate gyrus. At 12-24 h after TBI, strong induction of MT-I mRNA was observed in cerebral cortical layer II/III, amygdala, and piriform cortex where neurons reside. On the other hand, MT-II appeared to be expressed mainly in glial cells localized in the cerebral cortex and hippocampal formation. Three days after TBI, MTs were observed in the vimentin-positive astrocytes in the penumbra as revealed by double immunohistochemistry. The differences in expression of MT-I and MT-II in different brain regions and cell types (neuron vs. glial cells) suggests that multiple regulatory mechanisms are involved in the control of MT expression following brain injury.     

Microglia are the major source of TNF-α and TGF-β1 in postnatal glial cultures; regulation by cytokines,lipopolysaccharide, and vitronectin

Damage to the central nervous system (CNS) leads to increased production of TNF-α and TGF-β1 cytokines that have pro- or anti-inflammatory actions, respectively. To define whether astrocytes or microglia express these cytokines, prior studies have used mixed glial cultures (MGC) to represent astrocytes, thought these results are inevitably complicated by the presence of contaminating microglia within MGC. To clarify the cellular source of these cytokines, here we employed a recently described method of preparing microglia-free astrocyte cultures, in which neural stem cells (NSC) are differentiated into astrocytes. Using ELISA to quantify cytokine production in three types of glial culture: MGC, pure microglia or pure astrocytes, this showed that microglia but not astrocytes, produce TNF-α, and that this expression is increased by LPS, IFN-γ, and to a lesser extent by vitronectin, but decreased by TGF-β1. In contrast, TGF-β1 was produced by microglia and astrocytes, though at 10-fold higher levels by microglia. TGF-β1 expression in microglia was increased by vitronectin and to a lesser extent by TNF-α and LPS, but astrocyte TGF-β1 expression was not regulated by any factor tested. In summary, our data reveal that microglia, not astrocytes are the major source of TNF-α and TGF-β1 in postnatal glial cultures, and that microglial production of these antagonistic cytokines is tightly regulated by cytokines, LPS, and vitronectin.     

A β-Lactam Antibiotic Dampens Excitotoxic Inflammatory CNS Damage in a Mouse Model of Multiple Sclerosis

In multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE), impairment of glial “Excitatory Amino Acid Transporters” (EAATs) together with an excess glutamate-release by invading immune cells causes excitotoxic damage of the central nervous system (CNS). In order to identify pathways to dampen excitotoxic inflammatory CNS damage, we assessed the effects of a β-lactam antibiotic, ceftriaxone, reported to enhance expression of glial EAAT2, in “Myelin Oligodendrocyte Glycoprotein” (MOG)-induced EAE. Ceftriaxone profoundly ameliorated the clinical course of murine MOG-induced EAE both under preventive and therapeutic regimens. However, ceftriaxone had impact neither on EAAT2 protein expression levels in several brain areas, nor on the radioactive glutamate uptake capacity in a mixed primary glial cell-culture and the glutamate-induced uptake currents in a mammalian cell line mediated by EAAT2. Moreover, the clinical effect of ceftriaxone was preserved in the presence of the EAAT2-specific transport inhibitor, dihydrokainate, while dihydrokainate alone caused an aggravated EAE course. This demonstrates the need for sufficient glial glutamate uptake upon an excitotoxic autoimmune inflammatory challenge of the CNS and a molecular target of ceftriaxone other than the glutamate transporter. Ceftriaxone treatment indirectly hampered T cell proliferation and proinflammatory INFγ and IL17 secretion through modulation of myelin-antigen presentation by antigen-presenting cells (APCs) e.g. dendritic cells (DCs) and reduced T cell migration into the CNS in vivo. Taken together, we demonstrate, that a β-lactam antibiotic attenuates disease course and severity in a model of autoimmune CNS inflammation. The mechanisms are reduction of T cell activation by modulation of cellular antigen-presentation and impairment of antigen-specific T cell migration into the CNS rather than or modulation of central glutamate homeostasis.     

FTY720 attenuates accumulation of EMAP-II+ and MHC-II+ monocytes in early lesions of rat traumatic brain injury

FTY720 (Fingolimod) is a novel type of immunosuppressive agent inhibiting lymphocyte egress from secondary lymphoid tissues thereby causing peripheral lymphopenia. FTY720 can inhibit macrophage infiltration into inflammatory lesions under pathological conditions. FTY720 has been clinically evaluated for prophylaxis of allograft rejection and treatment of multiple sclerosis, showing promising immunosuppressive effects. A robust inflammatory response after traumatic brain injury (TBI) plays an important role in the secondary or delayed injuries of TBI. Here we have investigated by immunohistochemistry in a rat TBI model the effects of FTY720 on early cell accumulation into the inflammatory tissue response and on expression of major histo-compatibility complex class II (MHC-II) and endothelial-monocyte activating polypeptide II (EMAP-II). Accumulation of MHC-II(+) or EMAP-II(+) cells became significant 1 day after injury and continuously increased during the early time periods. Further, double-staining experiments confirmed that the major cellular sources of MHC-II were reactive macrophages, however MHC-II(+) cells only constituted a small subpopulation of reactive macrophages. Immediately after TBI, peripheral administration of FTY720 (1 mg/kg in 1 mL saline, every second day) significantly attenuated the accumulation of MHC-II(+) macrophages from Day 1 to 4 and significantly attenuated the accumulation of EMAP-II(+) macrophages/microglia at Day 4. Our findings show that FTY720 attenuates early accumulation of EMAP-II(+) and MHC-II(+) reactive monocytes following TBI, indicating that FTY720 might be a drug candidate to inhibit brain inflammatory reaction following TBI.     

Excitotoxic mechanisms and the role of astrocytic glutamate transporters in traumatic brain injury

Glutamate excitotoxicity plays an important role in the development of secondary injuries that occur following traumatic brain injury (TBI), and contributes significantly to expansion of the total volume of injury. Acute increases in extracellular glutamate levels have been detected in both experimental brain trauma models and in human patients, and can lead to over-stimulation of glutamate receptors, resulting in a cascade of excitotoxic-related mechanisms culminating in neuronal damage. These elevated levels of glutamate can be effectively controlled by the astrocytic glutamate transporters GLAST (EAAT1) and GLT-1 (EAAT2). However, evidence indicate these transporters and splice variant are downregulated shortly following the insult, which then precipitates glutamate-mediated excitotoxic conditions. Lack of success with glutamate receptor antagonists as a potential source of clinical intervention treatment following TBI has resulted in the necessity for a better understanding of the mechanisms that underlie the process of excitotoxicity, including the function and regulation of glutamate transporters. Such new insight should improve the likelihood of development of novel avenues for therapeutic intervention following TBI.     

创伤性脑损伤中神经炎症相关细胞的研究进展

创伤性脑损伤(traumatic brain injury, TBI), 亦称颅脑损伤或头部外伤, 专指由外伤引起的脑组织损害。然而,从轻度到重度的TBI,改善TBI患者预后的治疗方法都十分匮乏。神经炎症可引起脑外伤后急性继发性损伤,并与慢性神经退行性疾病有关,因此,系统了解参与TBI后神经炎性反应的细胞显得尤为重要。主要对TBI中参与炎症反应的细胞（如小胶质细胞、星形胶质细胞、少突细胞、中性粒细胞和淋巴细胞）的启动以及相互作用的最新研究进展进行了综述,以期为临床研究提供新的策略。     

Modulation of Glutamate Transport and Receptor Binding by Glutamate Receptor Antagonists in EAE Rat Brain

The etiology of multiple sclerosis (MS) is currently unknown. However, one potential mechanism involved in the disease may be excitotoxicity. The elevation of glutamate in cerebrospinal fluid, as well as changes in the expression of glutamate receptors (iGluRs and mGluRs) and excitatory amino acid transporters (EAATs), have been observed in the brains of MS patients and animals subjected to experimental autoimmune encephalomyelitis (EAE), which is the predominant animal model used to investigate the pathophysiology of MS. In the present paper, the effects of glutamatergic receptor antagonists, including amantadine, memantine, LY 367583, and MPEP, on glutamate transport, the expression of mRNA of glutamate transporters (EAATs), the kinetic parameters of ligand binding to N-methyl-D-aspartate (NMDA) receptors, and the morphology of nerve endings in EAE rat brains were investigated. The extracellular level of glutamate in the brain is primarily regulated by astrocytic glutamate transporter 1 (GLT-1) and glutamate-aspartate transporter (GLAST). Excess glutamate is taken up from the synaptic space and metabolized by astrocytes. Thus, the extracellular level of glutamate decreases, which protects neurons from excitotoxicity. Our investigations showed changes in the expression of EAAT mRNA, glutamate transport (uptake and release) by synaptosomal and glial plasmalemmal vesicle fractions, and ligand binding to NMDA receptors; these effects were partially reversed after the treatment of EAE rats with the NMDA antagonists amantadine and memantine. The antagonists of group I metabotropic glutamate receptors (mGluRs), including LY 367385 and MPEP, did not exert any effect on the examined parameters. These results suggest that disturbances in these mechanisms may play a role in the processes associated with glutamate excitotoxicity and the progressive brain damage in EAE.     

胶质细胞谷氨酸转运体及其在脑缺血和脑缺血预适应中的变化

兴奋性氨基酸转运体(excitatory amino acid transporters,EAATs)是摄取细胞外液谷氨酸、保持细胞外谷氨酸低浓度的主要机制,已发现了五种EAATs,其中胶质细胞谷氨酸转运体在终止谷氨酸能神经传递、维持细胞外液谷氨酸浓度处于低水平方面发挥更重要作用。胶质细胞谷氨酸转运体的表达和功能受谷氨酸及其受体、垂体腺苷酸环化酶激活多肽、生长因子、内皮素、一氧化氮等许多因素的影响,其表达减少及功能降低与脑缺血损害的发生和发展密切相关,脑缺血预适应可通过调控其表达或改善其功能而诱导脑缺血耐受。