脑梗死患者外周血与颈动脉斑块内脂蛋白相关磷脂酶A2表达的相关性

目的探讨外周血与颈动脉斑块内脂蛋白相关磷脂酶A2(Lp-PLA2)表达的相关性。方法选择颈内动脉系动脉粥样硬化性脑梗死患者37例为研究对象,采用彩色多普勒超声对斑块类型进行划定,采用免疫增强比浊法测定血浆和斑块组织提取物中Lp-PLA2含量后行相关性分析。结果 F检验显示,斑块类型对血浆Lp-PLA2水平有影响(P0.05)。进一步采用SNK-q检验显示,Lp-PLA2水平变化关系:无斑块组稳定粥样斑块组不稳定粥样斑块组,两两比较差异均有统计学意义(P0.05)。脑梗死患者外周血与颈动脉斑块内Lp-PLA2表达相关性对比发现,2个部位该蛋白的表达呈正相关(r=0.831,P=0.008)。结论 Lp-PLA2可能是颈动脉不稳定粥样斑块的临床标志物,外周血与颈动脉斑块内Lp-PLA2表达呈线性正相关。     

The symptomatic carotid plaque

BACKGROUND: The natural histories of equally severe symptomatic and asymptomatic carotid stenoses are very different, which suggests dichotomy in plaque behavior. The vascular biology of the symptomatic carotid plaque is presented in this review. SUMMARY OF REVIEW: Histology studies comparing asymptomatic and symptomatic plaques were identified from MEDLINE. Reports in which stenosis severity was not stated or not similar for symptomatic and asymptomatic patients were excluded. In vitro studies and reports from the coronary circulation were reviewed with regard to the vascular biology of the plaque. Histology studies comparing carotid plaques removed from symptomatic and asymptomatic patients reveal characteristic features of unstable plaques: surface ulceration and plaque rupture (48% of symptomatic compared with 31% of asymptomatic, P<0.001), thinning of the fibrous cap, and infiltration of the cap by greater numbers of macrophages and T cells. In vitro studies suggest that macrophages and T cells release cytokines and proteinase, which stimulate breakdown of cap collagen and smooth muscle cell apoptosis and thereby promote plaque rupture. CONCLUSIONS: Infiltration of inflammatory cells to the surface of carotid plaques may be a critical step in promoting plaque rupture and resultant embolization or carotid occlusion. Further understanding of cell recruitment and behavior in carotid atherosclerosis may allow better detection of unstable plaques and therapeutic methods of plaque stabilization.     

Increased expression of endothelial lipase in symptomatic and unstable carotid plaques

The aim of this study was to evaluate endothelial lipase (EL) protein expression in advanced human carotid artery plaques (HCAP) with regard to plaque (in)stability and the incidence of symptoms. HCAP were collected from 66 patients undergoing carotid endarterectomy (CEA). The degree of plaque (in)stability was estimated by ultrasound and histology. In HCAP sections, EL expression was determined by immunostaining and the intensity was assessed on a semi-quantitative scale (low: <25%, high: >25% positive cells). Monocytes and macrophages in adjacent HCAP sections were stained with a CD163 specific antibody. High EL staining was more prevalent in histologically unstable plaques (in 33.3% of fibrous plaques, 50% of ulcerated non-complicated plaques and 79.2% of ulcerated complicated plaques; χ(2) test, p?=?0.004) and in the symptomatic group (70.8 vs. 42.9% in the asymptomatic group; χ(2) test, p?=?0.028). The majority of EL immunostaining was found in those HCAP regions exhibiting a strong CD163 immunostaining. EL in HCAP might be a marker and/or promoter of plaque instability and HCAP-related symptomatology.     

Four-dimensional ultrasonographic characterization of plaque surface motion in patients with symptomatic and asymptomatic carotid artery stenosis.

BACKGROUND AND PURPOSE: In vitro studies of atherosclerotic plaque fracture mechanics suggest that analysis of local variations in surface deformability may provide information on relative vulnerability to plaque fissuring or rupture. We investigated plaque surface deformations in patients with symptomatic and asymptomatic carotid artery disease using 4-dimensional ultrasonography and techniques for measuring optical flow. METHODS: Four-dimensional ultrasound examinations of carotid artery plaques were performed in 23 asymptomatic and 22 symptomatic patients with 50% to 90% stenosis of the internal carotid artery. Plaque surface motion during 1 cardiac cycle was computed with a hierarchical model-based motion estimator. Results were compared with plaque echogenicity and surface structure. RESULTS: Of the 45 patients examined, plaque surface motion estimates were obtained for 18 asymptomatic and 13 symptomatic patients. There were no significant differences in echogenicity or surface structure of asymptomatic and symptomatic plaques (P>0.05). Results of motion estimation showed that asymptomatic plaques had surface motion vectors of equal orientation and magnitude to those of the internal carotid artery, whereas symptomatic plaques demonstrated evidence of inherent plaque movement. There was no significant difference in maximal plaque velocity between symptomatic and asymptomatic plaques (P<0.14). Maximal discrepant surface velocity (MDSV) in symptomatic plaques was 3.85+/-1.26 mm/s (mean+/-SD), which was significantly higher (P<0.001) than MDSV of asymptomatic plaques with 0.58+/-0.42 mm/s (mean+/-SD). CONCLUSIONS: ++MDSV of carotid artery plaques is significantly different in asymptomatic and symptomatic disease. Further studies are warranted to determine whether plaque surface motion patterns can identify vulnerable plaques in patients with carotid artery stenosis.     

Cx36, Cx43 and Cx45 in mouse and rat cerebellar cortex: species‐specific expression,compensation in Cx36 null mice and co‐localization in neurons vs. glia

Electrical synapses formed by connexin36 (Cx36)‐containing gap junctions between interneurons in the cerebellar cortex have been well characterized, including those formed between basket cells and between Golgi cells, and there is gene reporter‐based evidence for the expression of connexin45 (Cx45) in the cerebellar molecular layer. Here, we used immunofluorescence approaches to further investigate expression patterns of Cx36 and Cx45 in this layer and to examine localization relationships of these connexins with each other and with glial connexin43 (Cx43). In mice, strain differences were found, such that punctate labelling for Cx36 was differentially distributed in the molecular layer of C57BL/6 vs. CD1 mice. In mice with EGFP reporter representing Cx36 expression, Cx36‐puncta were localized to processes of stellate cells and other cerebellar interneurons. Punctate labelling of Cx45 was faint in the molecular layer of wild‐type mice and was increased in intensity in mice with Cx36 gene ablation. The vast majority of Cx36‐puncta co‐localized with Cx45‐puncta, which in turn was associated with the scaffolding protein zonula occludens‐1. In rats, Cx45‐puncta were also co‐localized with Cx36‐puncta and additionally occurred along Bergmann glial processes adjacent to Cx43‐puncta. The results indicate strain and species differences in Cx36 as well as Cx45 expression, possible compensatory processes after loss of Cx36 expression and localization of Cx45 to both neuronal and Bergmann glial gap junctions. Further, expression of both Cx43 and Cx45 in Bergmann glia of rat may contribute to the complex properties of junctional coupling between these cells and perhaps to their reported coupling with Purkinje cells.     

脑梗死患者颈动脉粥样硬化斑块形成的研究

目的 探讨脑梗死(CI)患者颈动脉粥样硬化斑块形成的状况.方法 对138例CI患者行颈动脉彩色多普勒超声检查,观察其颈动脉斑块形成、性质及部位,并与正常对照组比较.结果 CI组138例中120例(87.7%)检出颈动脉粥样硬化斑块175块,正常对照组140人检出斑块为36人(25.7%)(P＜0.01).CI组中不稳定性斑块比率(80.8%)显著高于稳定性斑块(19.2%)(P＜0.01);斑块位于颈总动脉(CCA)(81.1%)显著高于颈内动脉(ICA)(18.9%)(P＜0.01);位于CCA分叉处(66.3%)又显著高于主干(14.9%)(P＜0.05).结论 CI患者颈动脉粥样硬化斑块的发生率高,多位于CCA分叉处,且大多为不稳定性斑块.提示CI与颈动脉粥样硬化有密切关系.     

Association between arterial bifurcation anatomy and angiographic plaque ulceration among 4,627 carotid stenoses

Stability of atheromatous plaques is influenced by local mechanical and haemodynamic factors, such as plaque motion and shear stress. However, although blood vessel anatomy is an important determinant of haemodynamics, particularly at bifurcations, there have been no previous clinical studies of the association between arterial anatomy and plaque ulceration. We therefore studied arterial anatomy and plaque ulceration using angiograms of 4,627 carotid bifurcations with atheromatous disease from the European Carotid Surgery Trial (ECST). We studied the vessel diameter and area ratios that have been shown in flow models to affect local haemodynamics and shear stress, and which are known to vary widely between and within individuals (internal to common, external to common, external to internal carotid artery and outflow/inflow area). Angiographic plaque surface morphology was defined as ulcerated or not ulcerated. To avoid any potential bias due to selective inclusion of patients in the ECST, we studied the contralateral, and usually asymptomatic, as well as the symptomatic carotid artery. To correct for the effects of systemic factors that might influence plaque stability, we also studied the relationship between the degree of asymmetry of bifurcation anatomy within individuals and the presence of plaque ulceration. Despite considerable inter-individual variation in carotid anatomy, we found no association between the prevalence of angiographic plaque ulceration and any of the anatomical parameters studied in either symptomatic or contralateral carotid arteries. There were also no associations between ipsilateral bifurcation anatomy and plaque ulceration in individuals with unilateral plaque ulceration. Carotid arterial anatomy does not appear to be an important determinant of plaque stability. Other factors that influence local haemodynamics, such as the anatomy and composition of the plaque itself may be more important.     

Immunohistochemical analysis of MMP-9 and COX-2 expression in carotid atherosclerotic plaques among patients undergoing carotid endarterectomy: A prospective study

BackgroundMatrix metalloproteinase-9 protein (MMP-9) and cyclooxygenase-2 (COX-2) proteins may have a role in remodelling of atherosclerotic plaques. We analysed and compared the radiological, histological and immunohistochemical characteristics of carotid atherosclerotic plaques between symptomatic and asymptomatic patients who underwent carotid endarterectomy (CEA).MethodsThis prospective single-blinded study included 31 patients (70 [64-75] years, 58% males, 42% symptomatic) who underwent CEA and a total of 155 carotid plaque sections that were analysed. Preoperative assessment and multimodality diagnostic imaging with magnetic resonance imaging (MRI) or computed tomography angiography (CTA), histological and immunohistochemical analyses of carotid plaques including the expression of MMP-9 and COX-2 proteins were performed.ResultsSymptomatic and asymptomatic patients did not significantly differ in respect to preoperative characteristics. Unstable plaques were detected in 12/13 (92.3%, p = 0.020) symptomatic patients using MRI or CTA. There was no perioperative mortality and perioperative outcomes were comparable in both groups. A significantly higher expression of MMP-9 in macrophages was observed among symptomatic patients (p = 0.020). ROC curve analysis showed statistically significant associations of both the higher intensity of COX-2 staining in CD68 PG-M1 positive macrophages (area under the curve [AUC]=0.701, p = 0.014) and higher MVD (AUC=0.821, p < 0.001) within the plaque with cerebrovascular symptoms. The expression of COX-2 and the intensity of COX-2 staining in macrophages within the unstable carotid plaques detected by preoperative MRI or CTA were significantly higher (76.1% vs. 40.0%, p = 0.038; 76.2% vs. 30.0%, p = 0.01, respectively).ConclusionsAdvanced non-invasive multimodality diagnostic imaging including MRI or CTA is reliable in differentiating unstable from stable carotid plaques. High expression of MMP-9 and COX-2 in macrophages within the symptomatic plaque is associated with increased risk of cerebrovascular complications.Trial RegistrationThis study has been registered at the ISRCTN registry (ID ISRCTN46536832), isrctn.org Identifier: https://www.isrctn.com/ISRCTN46536832     

颈动脉超声在无症状脑梗死患者中的应用分析

目的分析无症状脑梗死患者的颈动脉超声特点及应用价值。方法选取2015-12—2016-11于我院接受治疗的无症状脑梗死患者40例(观察组),症状性脑梗死40例(症状组),另选取健康体检者32例为对照组,回顾性分析其颈动脉彩超特征。结果斑块检出率、IMT厚度、Crouse斑块积分及斑块形态3组组间比较差异均有统计学意义(P0.05);狭窄程度观察组与对照组比较差异有统计学意义(P0.05),观察组与症状组比较差异无统计学意义(P0.05);3组颈内动脉血流参数比较差异具有统计学意义(P0.05),颈总动脉血流参数观察组与症状组比较差异有统计学意义(P0.05),观察组与对照组比较差异无统计学意义(P0.05)。结论存在颈部动脉硬化及斑块但尚未明确诊断无症状脑梗死者,颈动脉彩超观察斑块的形态和性质尤为重要;诊断明确的无症状脑梗死患者,应常规筛查颈动脉超声评估斑块情况,定期监测颈动脉斑块及血流参数变化,给予早期干预,预防疾病进一步发展。     

Symptomatic and asymptomatic high-grade carotid stenoses in Doppler color-flow imaging.

We examined 63 patients with 31 symptomatic and 44 asymptomatic carotid stenoses with Doppler color-flow imaging (DCFI); conventional Doppler duplex had shown a hemodynamic obstruction (greater than or equal to 80% stenosis) in all patients. Analysis of plaque surface morphology demonstrated more ulcerated plaques in symptomatic (43%) than asymptomatic (23%) stenoses. Although the frequency of homogeneous and heterogeneous plaques was not different, calcific lesions were more frequent in asymptomatic (46% versus 29%), and echolucent plaques, probably indicating mural thrombi, were more frequent in symptomatic (29% versus 11%) stenosis. Color-coded hemodynamic patterns, such as jet flow, poststenotic turbulence, or reversed flow, were not different in symptomatic and asymptomatic stenoses. Comparison of DCFI with 30 angiograms showed agreement in plaque surface analysis in 70%. DCFI measurements of area reduction in cross sections correlated with angiography in 85%, while DCFI tended to underestimate the degree of stenosis from diameter reduction in longitudinal cuts. The advanced DCFI technique identified distinct morphologic features but no hemodynamic patterns, separating symptomatic from asymptomatic high-grade carotid stenoses.     

Connexin35/36 gap junction proteins are expressed in photoreceptors of the tiger salamander retina

Photoreceptors in the vertebrate retina are electrically coupled with one another. Such coupling plays important roles in visual information processing. Physiological properties of rod-rod and rod-cone coupling have been best studied in the salamander retina, yet the cellular and molecular basis of these electrical synapses has not been established. Recently, connexin35/36 (Cx35/36) gap junction proteins were found to be highly expressed in brain and retina, suggesting that it may mediate photoreceptor coupling. To test this idea, we examined the cellular distribution of Cx35/36 in the salamander retina. Western blot analysis showed the expression of Cx35/36 proteins, and confocal microscopy revealed characteristic punctate Cx35/36 immunoreactivity in both synaptic layers. In addition, Cx35/36-positive plaques were detected in the outer nuclear layer (ONL) between neighboring rods, and these plaques outlined the mosaic of the rod network at a level distal to the external limiting membrane. Moreover, although Cx35/36 plaques were detected between some cones and their adjacent rods, the number and size of these plaques was smaller, and their staining intensity was diminished compared with the plaques between adjacent rods. Furthermore, Lucifer yellow injection together with confocal microscopy revealed that Cx35/36-puncta were colocalized with finlike structures of rod cell membrane, with the ultrastructure of gap junctions between paired rod fins having been found by electron microscopy. Therefore, our findings demonstrate that Cx35/36 expression in photoreceptors is primarily located between rods and to a lesser extent between rods and cones, suggesting that Cx35/36 may participate in electrical coupling between rods and between rods and cones in the salamander retina.     

对颈动脉粥样硬化溃疡斑块内超声造影的定量研究

目的 了解颈动脉粥样硬化溃疡斑块内新生血管的血流动力学定量特点。方法 将颈动脉粥样硬化易损斑块患者按有无临床缺血症状分为症状组与非症状组,比较两组患者溃疡斑块发生率;对易损斑块进行超声造影检查,比较溃疡斑块和非溃疡斑块的造影剂到达时间、达峰时间、斑块内新生血管增强密度和斑块峰值强度/颈动脉管腔峰值强度比值差异。分析两组斑块血流动力学特点及斑块内新生血管的定量特征。结果 入组41例患者,共有48个易损斑块。症状组溃疡斑块发生率为68.2%,无症状组溃疡斑块发生率为38.5%,差异有统计学意义（P <0.05）。超声造影检查显示溃疡斑块组造影剂到达时间、斑块内新生血管增强密度和斑块峰值强度/颈动脉管腔峰值强度比值较非溃疡斑块组差异有统计学意义（7.21±2.17 s vs 10.32±3.17 s,P <0.05;15.1±6.4 dB vs 11.3±5.1 dB,P <0.05;0.64±0.17 vs 0.47±0.21,P <0.05)。结论 缺血症状组溃疡斑块的发生率较高;溃疡斑块组的新生血管密度高于非溃疡斑块组。     

Site of carotid plaque ulceration in relation to direction of blood flow: an angiographic and pathological study

BACKGROUND: Rupture of atherosclerotic plaque is the main cause of acute coronary syndromes and carotid territory ischaemic stroke. Haemodynamic stress is important in early plaque formation and may affect the stability of mature plaques. There is some evidence that macrophage infiltration and plaque rupture tend to localise to the proximal (upstream) part of the plaque where shear stress is highest. However, previous studies have been too small to assess this reliably. We studied the site of ulceration in a large number of carotid plaques. METHODS: We studied angiograms of 3007 symptomatic carotid stenoses, and the pathological appearance of 119 carotid plaques (77 asymptomatic), to identify the presence and position of plaque ulceration. RESULTS: Angiographic ulceration, which was present in 421 patients (14%), was more likely to be PROXIMAL than DISTAL to the point of maximum stenosis (OR = 16.6, 95% CI = 11.6-26.9, p < 0.001). This trend increased with severity of stenosis (p = 0.002). Pathological examination of the 119 carotid plaques also showed that ulceration was more likely to occur proximal to the point of maximum stenosis (OR = 6.1, 95% CI = 2.8-13.6, p < 0.001). CONCLUSIONS: Ulceration of carotid plaques, visible on angiography or on pathological examination, is seen most often in the proximal (upstream) part where shear stress is highest.     

Coupling of astrocyte connexins Cx26, Cx30, Cx43 to oligodendrocyte Cx29, Cx32, Cx47: Implications from normal and connexin32 knockout mice

Oligodendrocytes in vivo form heterologous gap junctions with astrocytes. These oligodendrocyte/astrocyte (A/O) gap junctions contain multiple connexins (Cx), including Cx26, Cx30, and Cx43 on the astrocyte side, and Cx32, Cx29, and Cx47 on the oligodendrocyte side. We investigated connexin associations at A/O gap junctions on oligodendrocytes in normal and Cx32 knockout (KO) mice. Immunoblotting and immunolabeling by several different antibodies indicated the presence of Cx32 in liver and brain of normal mice, but the absence of Cx32 in liver and brain of Cx32 KO mice, confirming the specificity and efficacy of the antibodies, as well as allowing the demonstration of Cx32 expression by oligodendrocytes. Oligodendrocytes throughout brain were decorated with numerous Cx30-positive puncta, which also were immunolabeled for both Cx32 and Cx43. In Cx32 KO mice, astrocytic Cx30 association with oligodendrocyte somata was nearly absent, Cx26 was partially reduced, and Cx43 was present in abundance. In normal and Cx32 KO mice, oligodendrocyte Cx29 was sparsely distributed, whereas Cx47-positive puncta were densely localized on oligodendrocyte somata. These results demonstrate that astrocyte Cx30 and oligodendrocyte Cx47 are widely present at A/O gap junctions. Immunolabeling patterns for these six connexins in Cx32 KO brain have implications for deciphering the organization of heterotypic connexin coupling partners at A/O junctions. The persistence and abundance of Cx43 and Cx47 at these junctions after Cx32 deletion, together with the paucity of Cx29 normally present at these junctions, suggests Cx43/Cx47 coupling at A/O junctions. Reductions in Cx30 and Cx26 after Cx32 deletion suggest that these astrocytic connexins likely form junctions with Cx32 and that their incorporation into A/O gap junctions is dependent on the presence of oligodendrocytic Cx32.     

Vascular endothelial growth factor and matrix metalloproteinase 9 expression in human carotid atherosclerotic plaques: relationship with plaque destabilization via neovascularization

BACKGROUND: The aim of this study was to evaluate the potential impact of vascular endothelial growth factor (VEGF) on carotid plaque destabilization in association with matrix metalloproteinase 9 (MMP-9) production. METHODS: Expression of VEGF and MMP-9 were determined immunohistochemically in 53 human endarterectomized atherosclerotic carotid plaques. The relationship to carotid plaque characteristics, clinical data and histological morphology was investigated. RESULTS: VEGF and MMP-9 had parallel overexpression in the inflammatory cells, especially in the neovascularized plaque lesions and around the cholesterol crystals. Strong expression of VEGF was evident in symptomatic patients (p < 0.057), in high-degree stenosis (p = 0.005), and in patients with ischemic infarct in brain scan (p = 0.021). No relation was proved between molecule expression and plaque ultrasonic characteristics. CONCLUSIONS: An intense expression of VEGF and MMP-9 in carotid plaques is related to plaque instability, high degree of stenosis and presence of symptomatic carotid occlusive disease.     

Differential connexin expression,gap junction intercellular coupling,and hemichannel formation in NT2/D1 human neural progenitors and terminally differentiated hNT neurons

Connexin-mediated gap junctions and open hemichannels in nonjunctional membranes represent two biologically relevant mechanisms by which neural progenitors can coordinate their response to changes in the extracellular environment. NT2/D1 cells are a teratocarcinoma progenitor line that can be induced to differentiate terminally into functional hNT neurons and NT-G nonneuronal cells. Clinical transplants of hNT neurons and experimental grafts of NT2/D1 progenitors or hNT neurons have been used in cell-replacement therapy in vivo. Previous studies have shown that NT2/D1 cells express connexin 43 (Cx43) and that NT2/D1 progenitors are capable of dye transfer. To determine whether NT2/D1 progenitors and differentiated hNT cultures express other connexins, Cx26, Cx30, Cx32, Cx36, Cx37, Cx43, and Cx46.6 mRNA and protein were analyzed. NT2/D1 progenitors express Cx30, Cx36, Cx37, and Cx43. hNT/NT-G cultures express Cx36, Cx37, and de novo Cx46.6. Cx26 and Cx32 were not expressed in NT2/D1 or hNT/NT-G cells. NT2/D1 progenitors formed functional gap junctions as assessed by dye coupling as well as open hemichannels in nonjunctional membranes as assessed by dye-uptake studies. Dye coupling was inhibited by the gap junction blocker 18alpha-glycyrrhetinic acid. Hemichannel activity was inhibited by the dual-specificity chloride channel/connexin hemichannel inhibitor flufenamic acid but not by the chloride channel inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. Both dye coupling and dye uptake were substantially reduced following differentiation of NT2/D1 progenitors. We conclude that the pattern of connexin expression in NT2/D1 cells changes over the course of differentiation corresponding with a reduction in biochemical coupling and hemichannel activity in differentiated cells.     

Plaque Rupture is a Determinant of Vascular Events in Carotid Artery Atherosclerotic Disease: Involvement of Matrix Metalloproteinases 2 and 9

Background and Purpose

Unstable carotid atherosclerotic plaques are characterized by cap rupture, leading to thromboembolism and stroke. Matrix metalloproteinases (MMPs) have been implicated in the progression of atherosclerosis and plaque rupture. The aim of this study was to assess the relationship between the expressions of MMP-2 and MMP-9 and carotid plaque instability.

Methods

Eighty atherosclerotic plaques were collected from 74 patients undergoing carotid endarterectomy. Clinical information was obtained from each patient, and plaque morphology was examined at the macroscopic and microscopic levels. The immunohistochemical expressions of MMPs were graded using semiquantitative scales.

Results

Macroscopic ulceration (84.6% versus 63.4%, p=0.042) and microscopic cap rupture (79.5% versus 51.2%, p=0.010) were more common in symptomatic than in asymptomatic patients. Immunoreactivities of MMP-2 and MMP-9 were increased in 40 and 36 atheromatous plaques, respectively. Macroscopic ulceration was strongly correlated with the expressions of MMP-2 (p<0.001) and MMP-9 (p=0.001). There were significant correlations between increased MMP-2 expression and cap rupture (p=0.002), intraplaque hemorrhage (p=0.039), and a thin fibrous cap (p=0.002), and between increased MMP-9 expression and cap rupture (p=0.010) and a large lipid core (p=0.013).

Conclusions

Plaque rupture was significantly associated with the development of vascular events in carotid atherosclerotic disease. MMP-2 and MMP-9 are strongly correlated with plaque instability.     

Disruption of oligodendrocyte gap junctions in experimental autoimmune encephalomyelitis

Gap junctions (GJs) are vital for oligodendrocyte survival and myelination. In order to examine how different stages of inflammatory demyelination affect oligodendrocyte GJs, we studied the expression of oligodendrocytic connexin32 (Cx32) and Cx47 and astrocytic Cx43 in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis (MS) induced by recombinant myelin oligodendrocyte glycoprotein. EAE was characterized by remissions and relapses with demyelination and axonal loss. Formation of GJ plaques was quantified in relation to the lesions and in normal appearing white matter (NAWM). During acute EAE at 14 days postimmunization (dpi) both Cx47 and Cx32 GJs were severely reduced within and around lesions but also in the NAWM. Cx47 was localized intracellularly in oligodendrocytes while protein levels remained unchanged, and this redistribution coincided with the loss of Cx43 GJs in astrocytes. Cx47 and Cx32 expression increased during remyelination at 28 dpi but decreased again at 50 dpi in the relapsing phase. Oligodendrocyte GJs remained reduced even in NAWM, despite increased formation of Cx43 GJs toward lesions indicating astrogliosis. EAE induced in Cx32 knockout mice resulted in an exacerbated clinical course with more demyelination and axonal loss compared with wild-type EAE mice of the same backcross, despite similar degree of inflammation, and an overall milder loss of Cx47 and Cx43 GJs. Thus, EAE causes persistent impairment of both intra- and intercellular oligodendrocyte GJs even in the NAWM, which may be an important mechanism of MS progression. Furthermore, GJ deficient myelinated fibers appear more vulnerable to CNS inflammatory demyelination.     

Connexin26 expression in brain parenchymal cells demonstrated by targeted connexin ablation in transgenic mice

Astrocytes are known to express the gap junction forming proteins connexin30 (Cx30) and connexin43 (Cx43), but it has remained controversial whether these cells also express connexin26 (Cx26). To investigate this issue further, we examined immunofluorescence labelling of glial connexins in wild-type vs. transgenic mice with targeted deletion of Cx26 in neuronal and glial cells (Cx26fl/fl:Nestin-Cre mice). The Cx26 antibodies utilized specifically recognized Cx26 and lacked cross reaction with highly homologous Cx30, as demonstrated by immunoblotting and immunofluorescence in Cx26-transfected and Cx30-transfected C6 glioma cells. Punctate immunolabelling of Cx26 with these antibodies was observed in leptomeninges and subcortical brain regions. This labelling was absent in subcortical areas of Cx26fl/fl:Nestin-Cre mice, but persisted in leptomeningeal tissues of these mice, thereby distinguishing localization of Cx26 between parenchymal and non-parenchymal tissue. In subcortical brain parenchyma, Cx26-positive puncta were often co-localized with astrocytic Cx43, and some were localized along astrocyte cell bodies and processes immunolabelled for glial fibrillary acidic protein. Cx26-positive puncta were also co-localized with punctate labelling of Cx47 around oligodendrocyte somata. Comparisons of Cx26 labelling in rodent species revealed a lower density of Cx26-positive puncta and a more restricted distribution in subcortical regions of mouse compared with rat brain, perhaps partly explaining reported difficulties in detection of Cx26 in mouse brain parenchyma using antibodies or Cx26 gene reporters. These results support our earlier observations of Cx26 expression in astrocytes and its ultrastructural localization in individual gap junction plaques formed between astrocytes as well as in heterotypic gap junctions between astrocytes and oligodendrocytes.