Monocyte chemoattractant protein-1 expressed in neurons and astrocytes during focal ischemia in mice

Focal cerebral ischemia elicits an inflammatory response characterized by the infiltration and accumulation of leukocytes, as well as the secretion of inflammatory mediators (Clark et al., Brain Res. Bull., 35 (1994) 387-392; Garcia et al., Am. J. Pathol., 144 (1994) 188-199; Wang et al., J. Neurochem. 71 (1998) 1194-1204). Leukocytes eliminate microbial invaders and necrotizing tissue debris, and can also turn against surrounding healthy tissue and exacerbate tissue injury (Furie and Randolph, Am. J. Pathol., 146 (1995) 1287-1301; Kochanek and Hallenbeck, Stroke 23 (1992) 1367-1379). Inflammatory mediators are considered to play an important role in attracting and stimulating leukocytes (Weiss, N. Engl. J. Med., 320 (1989) 365-376). Monocyte chemoattractant protein-1 (MCP-1) functions as an inflammatory mediator, whose source and role in focal cerebral ischemia is worth studying. MCP-1, a potent chemoattractant factor, may play an important role in ischemia-induced inflammatory response. The aim of the present study is to determine the time course and cell type of MCP-1 protein expression after permanent focal ischemia in mice. ELISA and immunohistochemical staining were used to detect the expression of MCP-1 protein after 0 h, 2 h, 4 h, 12 h, 1 day, 2 days, 3 days, 5 days and 7 days of middle cerebral artery occlusion (n=3-5 in each group). Double-labeled fluorescent staining was used to examine the cellular localization of MCP-1. The results demonstrated that MCP-1 expression was mainly observed in the ischemic core after 12 h of middle cerebral artery occlusion, then gradually increased and extended to the ischemic perifocal area. MCP-1 expression peaked at 2 days and 3 days, and gradually decreased after 5 days of MCAO. Double-labeled immunostaining for MCP-1 and neuron specific enolase (NSE) or glial fibrillary acidic protein (GFAP) showed that MCP-1 positive neurons were observed as early as 12 h of ischemia, while MCP-1 positive astrocytes were observed after 2 days of ischemia. These results support the functional role of MCP-1 in ischemic brain injury and reveal a distinct temporal and spatial expression of MCP-1 in cells believed to be neurons and astrocytes.     

Monocyte chemoattractant protein-1 regulation of blood-brain barrier permeability.

The present study was designed to elucidate the effects of the chemokine monocyte chemoattractant protein (MCP-1) on blood-brain barrier (BBB) permeability. Experiments were conducted under in vitro conditions (coculture of brain endothelial cells and astrocytes) to study the cellular effects of MCP-1 and under in vivo conditions (intracerebral and intracerebroventricular administration of MCP-1) to study the potential contribution of MCP-1 to BBB disruption in vivo. Our results showed that MCP-1 induces a significant increase in the BBB permeability surface area product for fluorescein isothiocyanate (FITC)-albumin under in vivo conditions, particularly during prolonged (3 or 7 days) exposure (0.096+/-0.008 versus 0.031+/-0.005 microL/g min in controls at 3 days, P<0.001). Monocyte chemoattractant protein-1 also enhanced (17-fold compared with control) the permeability of the in vitro BBB (coculture) model. At the cellular level, MCP-1 causes alteration of tight junction (TJ) proteins in endothelial cells (redistribution of TJ proteins determined by Western blotting and loss of immunostaining for occludin, claudin-5, ZO-1, ZO-2). Monocyte chemoattractant protein-1-induced alterations in BBB permeability are mostly realized through the CCR2 receptor. Absence of CCR2 diminishes any effect of MCP-1 on BBB permeability in vitro and in vivo. The permeability surface area product for FITC-albumin after 3 days exposure to MCP-1 was 0.096+/-0.006 and 0.032+/-0.007 microL/g min, in CCR2+/+ and CCR2-/- mice, respectively (P<0.001). Monocytes/macrophages also participate in MCP-1-induced alterations in BBB permeability in vivo. Monocytes/macrophages depletion (by clodronate liposomes) reduced the effect of MCP-1 on BBB permeability in vivo approximately 2 fold. Our results suggest that, besides its main function of recruiting leukocytes at sites of inflammation, MCP-1 also plays a role in 'opening' the BBB.     

Monocyte chemoattractant protein-1 deficiency is protective in a murine stroke model.

Inflammatory processes have been implicated in the pathogenesis of brain damage after stroke. In rodent stroke models, focal ischemia induces several proinflammatory chemokines, including monocyte chemoattractant protein-1 (MCP-1). The individual contribution to ischemic tissue damage, however, is largely unknown. To address this question, the authors subjected MCP-1-deficient mice (MCP-1-/-) to permanent middle cerebral artery occlusion (MCAO). Measurement of basal blood pressure, cerebral blood flow, and blood volume revealed no differences between wild-type (wt) and MCP-1-/- mice. MCAO led to similar cerebral perfusion deficits in wt and MCP-1-/- mice, excluding differences in the MCA supply territory and collaterals. However, compared with wt mice, the mean infarct volume was 29% smaller in MCP-1-/- mice 24 hours after MCAO (P = 0.022). Immunostaining showed a reduction of phagocytic macrophage accumulation within infarcts and the infarct border in MCP-1-/- mice 2 weeks after MCAO. At the same time point, the authors found an attenuation of astrocytic hypertrophy in the infarct border and thalamus in MCP-1-/- mice. However, these effects on macrophages and astrocytes in MCP-1-/- mice occurred too late to suggest a protective role in acute infarct growth. Of note: at 6 hours after MCAO, MCP-1-/- mice produced significantly less interleukin-1beta in ischemic tissue; this might be related to tissue protection. The results of this study indicate that inhibition of MCP-1 signaling could be a new acute treatment approach to limit infarct size after stroke.     

小檗碱对大鼠脑缺血后MCP-1表达的影响

目的探讨小檗碱处理对大鼠脑缺血后单核细胞趋化蛋白-1（MCP-1）表达的影响及小檗碱对脑缺血的神经保护作用。方法建立大鼠短暂性全脑缺血模型，采用尼氏体亚甲蓝染色观察脑缺血后大鼠脑海马CA1区神经元存活情况；采用免疫荧光染色方法检测脑缺血后大鼠缺血脑组织中MCP-1的表达情况。结果（1）与假手术组比较，脑缺血组大鼠脑海马CA1区神经元明显缺失，而小檗碱处理组大鼠脑海马CA1区神经元存活数明显多于缺血对照组；（2）与假手术组比较，脑缺血组大鼠脑缺血区MCP-1表达显著增多，而小檗碱处理显著降低了大鼠脑缺血区MCP-1的阳性表达。结论脑缺血引起MCP-1表达上调，提示MCP-1可能参与脑缺血损伤。小檗碱可抑制缺血脑组织MCP-1的表达，推测其可能经此途径减轻脑缺血的炎症反应而发挥一定的神经保护作用。     

Anti-monocyte chemoattractant protein-1 gene therapy protects against focal brain ischemia in hypertensive rats.

Monocyte chemoattractant protein-1 (MCP-1) is expressed in the ischemic cortex after focal brain ischemia and appears to exacerbate ischemic damage. The authors examined the effect of gene transfer of dominant negative MCP-1, called 7ND, 90 minutes after induction of focal brain ischemia in hypertensive rats. Adenoviral vectors encoding mutant MCP-1 (Ad7ND; n = 11), or Escherichia coli beta-galactosidase (AdlacZ; n = 17) as control were injected into the lateral ventricle of male spontaneously hypertensive rats. Both AdlacZ (n = 12) and Ad7ND (n = 6) administration provided transgene expression as early as 6 hours after injection and the expression further increased on day 1, followed by a sustained detection on day 5. Five days after ischemia, infarct volume (75 +/- 13 mm, n = 5, mean +/- SD) significantly reduced to 72% of control (104 +/- 22 mm3, n = 5, P < 0.05) by 7ND gene transfer. Numbers of leukocytes in the vessels (48.3 +/- 32.9/cm2) and macrophage/monocyte infiltration (475.2 +/- 125.5/mm2) of the infarct area in the Ad7ND group were significantly less than those measured in the AdlacZ group (143.8 +/- 72.1/cm2 and 671.8 +/- 125.5/mm2, P < 0.05, respectively). In summary, the postischemic gene transfer of dominant negative MCP-1 attenuated the infarct volume and infiltration of inflammatory cells, suggesting potential usefulness of the anti-MCP-1 gene therapy.     

Monocyte chemoattractant protein-1 immunoreactivity in sensory ganglia and hindpaw after adjuvant injection

Monocyte chemoattractant protein-1 (MCP-1)/CCL2 is a member of the CC chemokine family that exhibits potent chemotactic activity for monocytes/macrophages. In the current study, the proportion of monocyte chemoattractant protein-1-immunoreactive (IR) neurons in the dorsal root ganglion (DRG) of rats was shown to increase markedly following adjuvant injection into the hindpaw. MCP-1-IR axon terminals were not found in the spinal cord or hindpaw of control or adjuvant-treated rats. Instead, the inflamed hindpaw dermis was infiltrated by numerous MCP-1-IR inflammatory cells. Following adjuvant injection, the majority of MCP-1-IR neurons in the DRG colocalized with IB4 binding. Our findings suggest that peripheral tissue inflammation induces increased MCP-1 expression in DRG neurons and this may be dependent upon glial cell line-derived neurotrophic factor.     

Fas (CD95/APO-1) plays a role in the pathophysiology of focal cerebral ischemia

The purpose of this study was to investigate the role of fas antigen, a member of the TNF receptor family, in cell death after focal cerebral ischemia. Focal ischemia was induced in the Sprague-Dawley rat. Evidence for apoptosis was determined by morphology as well as the presence of DNA fragmentation by the end labeling technique (TUNEL). Immunohistochemistry was performed to detect expression of both fas and fas ligand (fasL). In a separate set of experiments, two groups of mice were studied: lpr (that have a loss of function mutation for fas) and wild type. Infarct volume was measured at 24 hr as well as evidence for apoptosis. Twenty-four hours after ischemia, there was evidence for apoptosis based on morphological criteria as well as the TUNEL technique in the rat. Immunohistochemistry demonstrated increased expression of both fas and fasL in the ischemic region, with maximal staining occurring between 24-48 hr for both. Twenty-four hours after ischemia in the mice, there was evidence of apoptosis in both groups, however, the mutant mice (lpr) had significantly smaller infarcts as compared to the wild type. There was no difference in the cerebrovasculature of the two groups of mice. These data support the hypothesis that apoptosis plays a role in the pathophysiology of focal cerebral ischemia. Furthermore, these data suggest that fas-mediated apoptosis contributes to this process.     

Monocyte chemoattractant protein-1 correlates with subcortical brain injury in HIV infection

Various biomarkers have been suggested as associative or predictive of HIV-associated neurocognitive impairment. Plasma levels of monocyte chemoattractant protein 1 (MCP-1), tumor necrosis factor alpha (TNF-alpha), and hematocrit were evaluated for relationships with diffusion tensor imaging measurements of centrum semiovale, caudate, and putamen. MCP-1 levels correlated with tissue status (mean diffusivity) in all examined regions. Plasma markers were also significantly correlated with anisotropy measurements in centrum semiovale (TNF-alpha) and putamen (hematocrit).     

Monocyte chemoattractant protein-1 increases microglial infiltration and aggressiveness of gliomas

Macrophages are thought to represent a first line of defense in anti-tumor immunity. Despite infiltration by microglial cells, however, malignant gliomas are still highly aggressive tumors. We here identify monocyte chemoattractant protein-1 (MCP-1) as a critical chemoattractant for glioma-infiltrating microglial cells. MCP-1-transfected rat CNS-1 gliomas were massively infiltrated by microglial cells. Whereas MCP-1 did not promote the growth of CNS-1 cells in vitro, intracerebral CNS-1-transfected tumors grew more aggressively than control-transfected tumors. This provides the first functional evidence that MCP-1 recruits microglial cells to gliomas and promotes their growth in vivo. Microglial cells may support rather than suppress glioma growth.     

Monocyte chemoattractant protein-1 is a pathogenic component in a model for a hereditary peripheral neuropathy

Macrophages are critically involved in the pathogenesis of genetically caused demyelination, as it occurs in models for inherited demyelinating neuropathies. It is presently unknown which factors link the Schwann cell-based myelin mutation to the activation of endoneurial macrophages. Here we identified the chemokine monocyte chemoattractant protein-1 (MCP-1) as a first and crucial factor upregulated in Schwann cells of mice heterozygously deficient for the myelin protein zero. The chemokine could be identified as an important mediator of macrophage immigration into peripheral nerves. Furthermore, a 50% reduction of chemokine expression by crossbreeding with MCP-1-deficient mice reduced the increase in macrophage numbers in the mutant nerves and lead to a robust amelioration of pathology. Surprisingly, the complete absence of MCP-1 aggravated the disease. Our findings show that reducing but not eliminating chemokine expression can rescue genetically caused demyelination that may be an interesting target in treating demyelinating diseases of the peripheral nervous system.     

Multiple molecular penumbras after focal cerebral ischemia.

Though the ischemic penumbra has been classically described on the basis of blood flow and physiologic parameters, a variety of ischemic penumbras can be described in molecular terms. Apoptosis-related genes induced after focal ischemia may contribute to cell death in the core and the selective cell death adjacent to an infarct. The HSP70 heat shock protein is induced in glia at the edges of an infarct and in neurons often at some distance from the infarct. HSP70 proteins are induced in cells in response to denatured proteins that occur as a result of temporary energy failure. Hypoxia-inducible factor (HIF) is also induced after focal ischemia in regions that can extend beyond the HSP70 induction. The region of HIF induction is proposed to represent the areas of decreased cerebral blood flow and decreased oxygen delivery. Immediate early genes are induced in cortex, hippocampus, thalamus, and other brain regions. These distant changes in gene expression occur because of ischemia-induced spreading depression or depolarization and could contribute to plastic changes in brain after stroke.     

局灶性脑缺血后脑内髓过氧化物酶活性观察

目的　探讨局灶性脑缺血后脑组织髓过氧化物酶（ＭＰＯ） 活性的测定方法,以及与缺血性损害的关系。方法　采用新型小鼠大脑中动脉线栓模型,检测不同缺血时间组梗塞体积及ＭＰＯ活性。结果　缺血１ ｈ 后再灌注２３ ｈ 组（ｔＭＣＡＯ）缺血灶体积明显小于缺血２４ ｈ 组（ｐＭＣＡＯ）;ＭＰＯ活性在各缺血组缺血侧明显高于对照侧和对照组（ Ｐ＜ ０－０５）,ｐＭＣＡＯ 组缺血侧基底节区ＭＰＯ 活性显著高于ｔＭＣＡＯ 组（ Ｐ＜ ０－０５） ,而两组缺血皮质区ＭＰＯ 活性则无显著差异。结论　本研究建立了局灶性脑缺血的ＭＰＯ活性测定方法,证明ＭＰＯ活性与缺血损伤间具有一定关系。     

大鼠局灶性脑缺血细胞因子介导的中性粒细胞化学吸附剂的动态观察

目的;探索细胞因子介导的中性粒细胞化学吸附剂（CINC）在脑缺血损伤中的作用。方法：用线栓法制备大鼠局灶性脑缺血模型,观察不同脑缺血时程血浆（sera）和脑组织内CINC浓度与髓质过氧化物酶（MPO）活性。结果：缺血组,血浆中CINC浓度的高峰在缺血6小时,脑组织内CINC浓度和髓质过氧化物酶（MPO）活性均呈低水平,其中CINC浓度高峰在缺血12小时,与对照组比较,差异均有极显著性意义（P＜0．01）;血浆中MPO活性随着缺血时间延长而逐渐升高,缺血6小时以上各组与对照组比较差异均有极显著性意义（P＜0．01）;脑组织内各组MPO活性差异无显著性意义（P＜0．05）。结论：缺血时,脑组织内表达的CINC可能是启动缺血性脑损伤的因素之一     

Cognitive deficits after focal cerebral ischemia in mice

BACKGROUND AND PURPOSE: The interpretation of cognitive data in many experimental stroke studies is problematic because middle cerebral artery occlusion (MCAO) is associated with sensorimotor alterations that may become confounding factors in cognitive testing. The purpose of the current study was to determine if it is possible to measure MCAO-induced cognitive deficits by using short durations of ischemia that do not result in alterations in sensorimotor behavior in mice. METHODS: Male C57/Bl6 mice were subjected to 60 or 90 minutes of intraluminal MCAO or sham surgery. In the first cohort of animals (n=12/group), locomotor activity, balance, and coordination were evaluated 2 weeks after surgery. In a second cohort of animals (n=10/group), the effects of 60 minutes of MCAO on subsequent learning and memory were assessed with a step-down passive avoidance task beginning 1 week after surgery. In a third cohort of animals (n=8 to 10/group), training in a passive avoidance task was completed before 60 minutes of MCAO, then retention of the task was assessed 1 week after surgery. In all animals, infarction size was determined after 14 days of reperfusion with use of cresyl violet staining and quantitative image analysis. RESULTS: There was no significant difference in infarction volume in the cerebral cortex or caudoputamen after 60 versus 90 minutes of MCAO. However, there was a significant increase in latency to move 1 body length in the 90-minute MCAO group compared with the 60-minute MCAO and sham groups. In 2 additional cohorts of animals, 60-minute MCAO was associated with a deficit in the acquisition and retention of a passive avoidance task regardless of whether the task training occurred before or after MCAO. CONCLUSIONS: Long-term cognitive deficits can be induced in mice by using a short duration of MCAO (60 minutes) that does not result in concomitant sensorimotor deficits.     

Specific cardiological evaluation after focal cerebral ischemia

Purpose of this study was to define a subgroup of TIA/stroke patients who should be examined by transthoracal and transesophageal echocardiography or Holter-electrocardiography to identify those with cardiogenic brain embolism reliably; 300 consecutive patients with acute focal brain ischemia underwent a standardized diagnostic protocol for the evaluation of the etiology including, clinical examination by a cardiologist and routine electrocardiography, Holter-electrocardiography, transthoracal and transesophageal echocardiography. 188 patients had a potential cardiac source of embolism. In particular echocardiography was diagnostic in 163 patients, and Holter-electrocardiography 10; 159 of these 188 patients (84.6%) had competitive etiologies, predominantly large vessel atherosclerosis. In 136 patients cardiogenic brain embolism was assumed as quite definite or possible. To identify these patients reliably, transthoracal and transesophageal echocardiography would have been necessary in 89% of the entire group of patients (all with clinically cardiological abnormalities, pathological routine ECG, without vascular risk factors, or no atherosclerosis in duplex sonography), and Holter-electrocardiography in 54%.     

Up-regulation of neuropilin-1 in neovasculature after focal cerebral ischemia in the adult rat.

During development, neuropilin-1 is a receptor for semaphorin 3a-mediated axonal guidance and for vascular endothelial growth factor (VEGF) promotion of angiogenesis. The authors measured neuropilin-1 expression in the adult ischemic brain using Northern blot, in situ hybridization, and immunohistochemistry. Neuropilin-1 mRNA was significantly up-regulated as early as 2 hours and persisted at least 28 days after focal cerebral ischemia. Acute up-regulation of neuropilin-1 mRNA primarily localized to the ischemic neurons. A marked increase in both mRNA and protein of neuropilin-1 was detected in endothelial cells of cerebral blood vessels at the border and in the core of the ischemic lesion 7 days after ischemia, and neuropilin-1 gene expression persisted on these vessels for at least 28 days after ischemia. In these areas, neovascularization was detected using three-dimensional reconstructed images obtained from laser scanning confocal microscopy. Activated astrocytes also exhibited neuropilin-1 immunoreactivity during 7 to 28 days of ischemia. Double immunofluorescent staining showed colocalization of neuropilin-1 and VEGF to cerebral blood vessels and activated astrocytes. These data suggest that in addition to its role in axonal growth, up-regulation of neuropilin-1, in concert with VEGF and its receptors, may contribute to neovascular formation in the adult ischemic brain.