Cellular markers of neuroinflammation and neurogenesis after ischemic brain injury in the long-term survival rat model

MRI was employed to follow the neurodegenerative foci and the localization of inflammatory cells by magnetically labeled CD4+ or CD8+ lymphocytes in the ischemia/reperfusion long-lived rats (9 and 13 months after 10 min of cardiac arrest). MRI of ischemic rats showed: (1) blood–brain barrier (BBB) leakage in the area of the dorsal hippocampus and brainstem-hindbrain level in basal cerebellum, (2) unlike anti-CD8 magnetic antibodies anti-CD4 ultra small paramagnetic iron oxide particles (USPIO) antibodies revealed hypointense areas in the brainstem-interbrain region and caudoputamen not found in animals that were not injected with USPIO antibodies, and (3) dilation in the retrosplenial area. Immunocytochemistry revealed microglial activation in the hippocampus and striatum, with indications of activation in thalamic lateral dorsal nuclei and the subventricular zone. In the CA1 and CA3 regions, it was noted that OX42- and ED1-positive granules appear in neuronal somata. Immunostaining of lymphocytes with TCR confirmed the T-cell presence in ischemic brain parenchyma of the hippocampus and striatum. The above observations thus point to a persistent dysfunction of BBB that in long-term may still lead to infiltration of T cells that are predominantly of helper (CD4+) type. Such inflammatory processes are backed by microglial activity even up to 1 year after ischemia/reperfusion. Moreover, in these animals an augmented expression of neurogenesis markers and neuroblast migration was also revealed in the subventricular zone. Thus, a balance of degenerative processes and inflammatory surveillance with neurogenesis could determine the long-term outcome of global ischemia survival or the previously proposed formation of amyloid plaques and Alzheimer’s-type dementia.     

大鼠脑缺血区局部炎症反应的实验探查

目的:研究脑缺血区血管细胞粘附分子-1(VCAM-1)表达和单核/巨噬细胞浸润与脑缺血的病理联系。方法:运用免疫组化染色方法和局部脑缺血/再灌流模型探查40只SD大鼠脑缺血区VCAM-1阳性血管和单核/巨噬细胞的数量变化及其变化发生的时程。结果:大鼠脑缺血区微血管内皮细胞VCAM-1表达发生在脑缺血1h,并在16h的再灌流期间,其表达逐渐增加,显示明显的时间依赖性变化。单核/巨噬细胞在脑缺血区的浸润发生在脑缺血1h/再灌流2h,并随再灌流时间的延长,其数量逐渐增加,在再灌流16h,其数量最多,其浸润也显示明显的时间依赖性变化。脑缺血区血管内皮细胞VCAM-1表达的时相与单核/巨噬细胞浸润的时相基本一致。结论:脑缺血诱导缺血性血管内皮细胞表达VCAM-1和诱导单核/巨噬细胞在脑缺血区浸润。此结果提示VCAM-1和单核/巨噬细胞可能参与缺血性脑损伤的病理过程。     

胚胎干细胞源性神经前体细胞移植脑缺血大鼠局部细胞的免疫反应

背景：神经前体细胞的免疫原性各家研究结果不一,尤其是体内移植后的机体免疫反应模式需要进一步研究。 目的：体外观察神经前体细胞组成型及诱导型主要组织相容性抗原表达情况;体内观察神经前体细胞移植入大鼠脑缺血组织后局部免疫细胞活化情况,探讨神经前体细胞的移植排斥可能性及模式。 方法：自pCX-hrGFP ES-D3胚胎干细胞诱导分化神经前体细胞,流式细胞术体外检测主要组织相容性抗原Ⅰ,Ⅱ类分子表达及γ-干扰素诱导前后表达变化。实验分3组,磷酸盐缓冲液组、神经前体细胞组分别于大脑中动脉缺血大鼠模型造模后经侧脑室给予磷酸盐缓冲液注射及神经前体细胞移植,假手术组不造模。免疫组化法观察纹状区ED1+、CD4+、CD8+细胞浸润情况;淋巴细胞再刺激增殖实验观测神经前体细胞诱导移植大鼠颈部淋巴细胞的增殖指数。 结果与结论：神经前体细胞组成型高表达主要组织相容性抗原Ⅰ类分子,几乎不表达主要组织相容性抗原Ⅱ类分子;经γ-干扰素诱导后,主要组织相容性抗原Ⅰ类分子进一步上调,主要组织相容性抗原Ⅱ类分子亦有轻度上调,提示神经前体细胞有可能引起机体免疫反应。移植实验表明,与假手术组相比,磷酸盐缓冲液组及神经前体细胞组均表现强烈的ED1+、CD4+、CD8+细胞浸润(P < 0.05),说明脑缺血损伤本身能导致局部免疫细胞活化;神经前体细胞组比磷酸盐缓冲液组有更强的ED1+、CD4+细胞浸润(P < 0.05),提示神经前体细胞移植可能导致局部免疫更进一步活化,且以CD4+T细胞反应为主。磷酸盐缓冲液组及神经前体细胞组神经前体细胞诱导下的增殖指数值均较假手术组升高(P < 0.01),但前两组增殖指数值比较差异无显著性意义(P > 0.05),提示脑组织局部炎症导致颈部淋巴细胞增殖性增加,而离体神经前体细胞不足以单独刺激致敏淋巴细胞增殖。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

Migration of enhanced green fluorescent protein expressing bone marrow-derived microglia/macrophage into the mouse brain following permanent focal ischemia

Brain ischemia induces a marked response of resident microglia and hematopoietic cells including monocytes/macrophages. The present study was designed to assess the distribution of microglia/macrophages in cerebral ischemia using bone marrow chimera mice known to express enhanced green fluorescent protein (EGFP). At 24 h after middle cerebral artery occlusion (MCAO), many round-shaped EGFP-positive cells migrated to the ischemic core and peri-infarct area. At 48-72 h after MCAO, irregular round- or oval-shaped EGFP/ionized calcium-binding adapter molecule 1 (Iba 1)-positive cells increased in the transition zone, while many amoeboid-shaped or large-cell-body EGFP/Iba 1-positive cells were increased in number in the innermost area of ischemia. At 7 days after MCAO, many process-bearing ramified shaped EGFP/Iba 1-positive cells were detected in the transition to the peri-infarct area, while phagocytic cells were distributed in the transition to the core area of the infarction. The distribution of these morphologically variable EGFP/Iba 1-positive cells was similar up to 14 days from MCAO. The present study directly showed the migration and distribution of bone marrow-derived monocytes/macrophages and the relationship between resident microglia and infiltrated hematogenous element in ischemic mouse brain. It is important to study the distribution of intrinsic and extrinsic microglia/macrophage in ischemic brain, since such findings may allow the design of appropriate gene-delivery system using exogenous microglia/macrophages to the ischemic brain area.     

Toll-like receptor 4 (TLR4), but not TLR3 or TLR9, knock-out mice have neuroprotective effects against focal cerebral ischemia

Toll-like receptors (TLRs) are signaling receptors in the innate immune system that is a specific immunologic response to systemic bacterial infection. We investigated whether cerebral ischemia induced by the middle cerebral artery occlusion (MCAO) for 2 h differed in mice that lack a functional TLR3, TLR4, or TLR9 signaling pathway. TLR4, but not TLR3 or TLR9, knock-out (KO) mice had significantly smaller infarct area and volume at 24 h after ischemia-reperfusion (I/R) compared with wild-type mice. In addition, TLR4 KO mice improved in neurological deficits after I/R compared with wild-type mice. Moreover, we investigated the expression of TLR4 in the ischemic brain with immunohistochemistry. The number of TLR4-positive cells gradually increased from 1 h after MCAO to 22 h after I/R. We also examined the localization of TLR4 in the ischemic area. TLR4 was localized with CD11b-positive microglial cells in the ischemic striatum and the number of CD11b-positive microglial cells was smaller in TLR4 KO mice than in wild-type mice. In addition, we investigated the translocation of NF-κB among TLR3, 4, and 9 KO mice after I/R injury using western blotting. NF-κB's p65 subunit was decreased in TLR4 KO mice compared to wild-type mice, but not TLR3 or 9 KO mice. These data suggest that TLR4 knockout, but not TLR3 or TLR9 knockout, may play a neuroprotective role in ischemic brain injury induced by MCAO in mice.     

大鼠慢性局灶脑缺血区CD8抗原表达的免疫组织化学研究

本文目的在于探查免疫分子 CD8抗原在脑缺血区表达的状况 ,进而探讨免疫因素与慢性局灶脑缺血的病理联系。采用大脑中动脉阻塞的局灶脑缺血模型和免疫组织化学 ABC方法观察 3 6只成年雄性 SD大鼠脑缺血区 CD8抗原表达的细胞种类、分布形式和表达时程。结果证明 :慢性局灶脑缺血诱导脑缺血区 CD8抗原表达 ,其阳性细胞呈无突起的圆形和分支状二类。圆形细胞在脑缺血 3 d时 ,其数量显著增加 (P<0 .0 5 ) ,位于大脑皮质梗塞灶的边缘 ,阳性细胞于脑缺血 1周达到高峰 (P<0 .0 1) ,此时 ,大部分阳性细胞已迁移到梗塞灶中央区并显示为大小和形状不同的无突起的细胞。分支状的 CD8阳性细胞出现在大脑皮质梗塞灶周围和缺血侧尾壳核 ,其数量在脑缺血 3 d显著增加 (P<0 .0 5 ) ,于脑缺血 1周 (尾壳核 )和 2周 (大脑皮质 )时达到高峰 (P<0 .0 1) ,随后缓慢下降 ,分支状的 CD8阳性细胞主要分布于大脑皮质梗塞灶的“半影区”和尾壳核的背外侧区。结果表明 :慢性局灶脑缺血诱导 CD8阳性 T淋巴细胞在脑缺血区浸润以及小胶质细胞的反应和激活 ,提示免疫因素与慢性局灶脑缺血有关联     

Immunohistochemical characterization of immune cell infiltration in paediatric and adult Langerhans cell histiocytosis

Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia commonly affecting children with frequent somatic mutations in MAPK pathway genes including BRAF^V600E and MAP2K1. Some studies suggest that LCH cells can recruit and modulate inflammatory cells, which could provide reciprocal survival signals. To characterize the immune profile of infiltrating inflammatory cells, and to clarify their participation in LCH pathogenesis, a detailed immunohistochemical analysis was performed. Fifteen (10 children, 5 adults) LCH cases were assessed through macrophage (CD68 and CD163), mature dendritic cell (mDC; CD83 and CD208), regulatory T cell (Treg; CD4, CD25 and FOXP3) and cytotoxic lymphocyte (CL; CD56, CD57, perforin and granzyme B) immunomarkers. Moreover, lymphocytic and LCH markers were also analysed. All cases were S100, CD1a, CD207 and CD4-positive. Bcl-2 and cyclin D1 expression was observed in 13 of 15 cases. In the immune microenvironment, M2-polarized macrophages and Tregs were the predominant cell populations, followed by significantly (P < .005) smaller levels of mDCs and CLs. Additionally, the number of CD3 + cells was significantly higher than that of CD20 + cells. In the CD3 + cell population, there were a significantly higher number of CD4 + cells than CD8 + cells. While there were no differences when comparing the paediatric and adult populations, FOXP3 + cells were significantly higher in patients with multisystem involvement and treated with chemotherapy, than single-site cases and those without chemotherapy. Our results suggest that M2-polarized macrophages and Treg infiltration can promote LCH development and survival, probably through pro-tumoral, immunosuppressive and/or cytokine-mediated mechanisms. This work highlights the need for further exploration of immune-targeted therapy for LCH.     

The inflammatory cell influx and cytokines changes during transition from acute inflammation to fibrous repair around implanted materials

The inflammatory and fibrous responses in a subcutaneous rat model were evaluated around degradable polyurethane urea (PUUR; Artelon), with titanium and tissue culture polystyrene (PS) discs having different surface chemical properties but similar surface topography. Cytokines, viability, cellular response, differentiation of cells and fibrous capsule formation and vascularization was investigated after 1, 7 and 21 days of implantation. The exudates retrieved from the pockets were analysed with respect to the total cell numbers, the proportions of cell types, the differentiation of monocytes/macrophages (ED1, ED2), the DNA content and the viability (LD, Trypan blue). Tumour necrosis factor alpha ((h)TNF-alpha) and interleukin-10 ((h)IL-10) were quantified by ELISA. The number of blood vessels, blood vessel luminal area, blood vessel distribution and the fibrous capsule thickness were analysed. The highest number of cells in the exudates around all implants was detected during the early phase of healing (1-7 days). The proportion of ED2-positive cells in the exudates increased from 2-8% at 1 day to 43-56% at 21 days. The levels of TNF-alpha were low with a decrease at 7 days. After 21 days high amounts of IL-10 in the exudates were detected, in particular around PUUR. This study shows that the transition from inflammation to repair (1-21 days) around PUUR, Ti and PS materials was characterized by a decrease in inflammatory cell influx, an increasing proportion of ED2-expressing macrophages, a biphasic TNF-alpha secretion, an increase of IL-10 and a fibrous capsule formation similar to all materials tested.     

AngⅡ在脑缺血大鼠大脑皮质中的表达及依达拉奉对其干预的影响

目的:研究大鼠局灶性脑缺血后AngⅡ在脑组织中的表达变化规律及应用自由基清除剂-依达拉奉干预治疗对脑组织中AngⅡ表达的影响,探讨脑缺血后脑内AngⅡ表达的生物学作用及依达拉奉对缺血性脑损伤的保护作用。方法:采用微创开颅法建立大鼠大脑中动脉闭塞(MCAO)模型,分为正常对照组、假手术组、脑缺血组和药物干预组。应用免疫组织化学及尼氏染色方法分别观察脑缺血后和依达拉奉干预后AngⅡ在脑内的表达和神经元的变化。结果:在缺血半暗区可见大量AngⅡ阳性细胞,以缺血后1周数目最多,且免疫阳性反应最强,与对照组相比有显著性差异(P<0.05);尼氏染色显示,缺血半暗区可见大量变性坏死神经元,其中以1周组数量最多,与对照组相比有统计学意义(P<0.05);经依达拉奉干预后半暗区AngⅡ阳性细胞数量、光密度值及变性坏死神经元数量明显减少,与对照组比较有统计学差异,以治疗后3 d最为显著(P<0.01)。结论:①大鼠局灶性脑缺血后缺血半暗区AngⅡ表达增强,可能与缺血后神经元的病理变化有一定联系;②脑损伤后,依达拉奉可能通过抑制AngⅡ的表达而减少神经元的坏死,发挥脑保护作用。     

Immunohistochemical characterization of glomerular inflammatory cells and expression of adhesion molecules in anti-glomerular basement membrane (anti-GBM) glomerulonephritis induced in WKY rats with monoclonal anti-GBM antibodies of different subclasses

According to previous report, adhesion of CD8-positive cells and macrophages to glomerular endotherial cells through the lymphocyte function-associated antigen-1 (LFA-1)/intercellular adhesion molecule-1 (ICAM-1) pathway is crucial for the initiation and subsequent progression of anti-glomerular basement membrane (anti-GBM) antibody-induced glomerulonephritis (anti-GBM nephritis) in WKY rats. In the present study glomerular inflammatory cell infiltration and LFA-1/ICAM-1 expression were examined in anti-GBM nephritis induced in WKY rats with monoclonal anti-GBM antibodies of different subclasses: IgG1, IgG2a, and IgG2b. The IgG2a and IgG2b subclasses induced significant proteinuria from day 3 as compared with the IgG1 subclass. Glomerular infiltration of macrophages and CD8-positive cells after administration of IgG2a and IgG2b subclass antibodies was significantly elevated compared to that for the IgG1 subclass. The intensity of glomerular ICAM-1 immunostaining by the IgG2a and IgG2b subclass antibodies tended to be stronger than that by the IgG1 subclass. Glomerular LFA-1-positive cell infiltration by the IgG2a and IgG2b subclasses was significantly higher than that of the IgG1 subclass. These results demonstrate that monoclonal antibodies belonging to the IgG2a and IgG2b subclasses strongly induce glomerular infiltration of inflammatory cells and expression of adhesion molecules in rat anti-GBM nephritis.     

尾壳核与大脑皮质缺血区炎症细胞反应的比较

目的：观察尾壳核和大脑皮质缺血区炎症细胞的反应模式,探讨尾壳核对缺血反应敏感的病理机制。方法：采用局灶脑缺血模型和组化,免疫组化染色方法,观察尾壳核和大脑皮质缺血区ED1、OX6和CD3阳性细胞的反应。结果：HE染色显示缺血尾壳核的组织病理学变化呈明显的团块状坏死区,T淋巴细胞和单核/巨噬细胞却显示明显的团块状排列,同时,缺血尾壳核小胶质细胞亦发生强烈的激活反应,并高表达免疫分子MHCⅡ类抗原,炎症细胞在尾壳核与在大脑皮质缺血区的反应形式不同。结论：单核/巨噬细胞,小胶质细胞和T淋巴细胞在缺血尾壳核的反应和分布特征与该区的组织病理学变化明显一致。提示这些因素可能与尾壳核对缺血的敏感性有关。     

Differential expression of vasoactive intestinal peptide receptor 1 expression in inflammatory bowel disease

There is conflicting evidence regarding the significance of vasoactive intestinal peptide (VIP) in inflammatory bowel disease (IBD). Involvement of the VIP receptor in IBD has not been reported. We examined the expression and localization of the VIP receptor in IBD. We determined the location of VIP receptor 1 (VIPR1) immunohistologically in surgically resected intestinal samples from 10 controls, 15 patients with ulcerative colitis, and 10 patients with Crohn's disease. A fluorescein-linked immunohistological study was performed using anti-VIPR1 antibody, with double-staining with antibodies to CD3, CD19, and CD68. Correlations with interleukin (IL)-4 and TNF-alpha expression were also investigated. Results showed that the number of VIPR1-positive cells was significantly increased in the inflammatory mucosa. VIPR1 was expressed in CD3-, CD19-, and CD68-positive cells. The proportion of VIPR1-positive cells among CD3-positive cells was significantly higher in the lamina propria of patients with ulcerative colitis than in those with Crohn's disease and the controls. The proportion of VIPR1-positive cells among CD68-positive cells was significantly higher in patients with ulcerative colitis and Crohn's disease than in the controls. A correlation between the numbers of VIPR1- and IL-4-positive cells was found in patients with ulcerative colitis, and between the numbers of VIPR1- and TNF-alpha-positive cells in patients with Crohn's disease. In conclusion, VIPR1 was widely expressed in infiltrating inflammatory cells, especially CD3- and CD68-positive cells in ulcerative colitis mucosa and CD68-positive cells in Crohn's disease mucosa. The differential expression of VIPR1 in ulcerative colitis and Crohn's disease mucosa suggests that the VIP system plays different roles in the pathogenesis of IBD.     

Development of the articular cavity in the rat temporomandibular joint with special reference to the behavior of endothelial cells and macrophages

Previous developmental studies on the temporomandibular joint (TMJ) have proposed several hypotheses on the formation of its articular cavity. However, detailed information is meager. The present study examined the formation process of the articular cavity in the rat TMJ by immunocytochemistry for CD31, RECA-1, and ED1, which are useful cellular markers for endothelial cells and monocyte/macrophage lineages, respectively. The upper articular cavity formation had begun by embryonic day 21 (E21) and was completed at postnatal day 1 (P1) in advance of the lower cavitation; the latter took place from P1 to P3. The occurrence and distribution pattern of the CD31-, RECA-1-, and ED1-positive cells differed between the upper and lower articular cavity-forming areas: the ED1-positive cells exclusively occurred in the area of the prospective upper articular cavity prior to its formation, while no ED1-positive cell appeared in the lower cavity-forming area. In contrast, the CD31- and RECA-1-positive endothelial cells were restricted to the lower cavity-forming area (never the prospective upper cavity) at E19 and diminished thereafter. Throughout the cavity formation, we failed to find any apoptotic cells in the cavity formation area, indicating no involvement of apoptosis in the cavity formation in TMJ. The present findings on the behaviors of endothelial cells and ED1-positive cells show a possibility of different mechanism in the cavity formation between the upper and lower articular cavities in the rat TMJ. The appearance of ED1-reactive cells and temporal vascularization may play crucial roles in the upper and lower articular cavity formation, respectively.     

Differential recruitment of CD8+ macrophages during Wallerian degeneration in the peripheral and central nervous system

The strong macrophage response occurring during Wallerian degeneration in the peripheral but not central nervous system has been implicated in tissue remodeling and growth factor production as key requirements for successful axonal regeneration. We have previously identified a population of CD8+ phagocytes in ischemic brain lesions that differed in its recruitment pattern from CD4+ macrophages/microglia found in other lesion paradigms. In the present study we show that crush injury to the sciatic nerve induced strong infiltration by CD8+ macrophages both at the crush site and into the degenerating distal nerve stump. At the crush site, CD8+ macrophages appeared within 24 hours whereas infiltration of the distal nerve parenchyma was delayed to the second week. CD8+ macrophages were ED1+ and CD11b+ but always MHC class II-. Most CD8+ macrophages coexpressed CD4 while a significant number of CD4+/CD8-macrophages was also present. Expression of the resident tissue macrophage marker ED2 was largely restricted to the CD4+/CD8- population. Following intraorbital crush injury to the optic nerve, infiltration of CD8+ macrophages was strictly confined to the crush site. Taken together, our study demonstrates considerable spatiotemporal diversity of CD8+ macrophage responses to axotomy in the peripheral and central nervous system that may have implications for the different extent of axonal regeneration observed in both systems.     

脑出血后的病理改变与T淋巴细胞反应

目的:探讨脑出血血肿周围局部病理改变及细胞免疫机制。方法:用免疫组化方法及组织HE染色法观察20只大鼠尾壳核脑出血24 h,3天、7天血肿周围区域的组织病变及CD3 、CD8 T淋巴细胞的分布形式和表达时程。结果:①在脑出血后24 h血肿周围即可见明显血管源性水肿,大量炎性细胞浸润,以中性粒细胞为主,少量散在淋巴细胞、小胶质细胞、星形细胞、少突胶质细胞弥漫浸润;出血3天、7天以淋巴细胞、巨噬细胞浸润为主,小胶质细胞、星形细胞、少突胶质细胞增生明显,双侧半球皮层下和血管周围出现小胶质细胞结节;②出血灶周围CD3 和CD8 T淋巴细胞浸润在出血后24 h已可见,出血后3天组和出血后7天组,CD3 和CD8 阳性细胞数明显高于出血后24 h组。结论:①脑出血期以中性粒细胞反应为主,以后以淋巴细胞反应为主,胶质细胞反应在出血后24 h已发生,持续一周以上;②CD3 、CD8 阳性细胞的出现提示它们参与了出血后脑损伤的病理过程,在出血7天内, CD3 和CD8-阳性细胞反应呈增强趋势。     

慢性局灶脑缺血区血管周细胞的形态学观察

为了深入研究血管周细胞的生物学特性和在脑缺血病理过程中的形态学变化规律 ,对不同缺血时间的大鼠大脑皮质缺血区和尾壳核区的血管周细胞的变化进行了免疫组织化学探查。结果显示 :(1)在脑缺血早期 (3 d,1周 ) ,ED2阳性反应的血管周细胞呈圆形或卵圆形 ,无明显的突起。数量增加的阳性细胞主要位于大脑皮质缺血灶外围的血管周围 ,并随血管走行而分布 ,缺血灶中央区的阳性细胞数量少。在缺血侧尾壳核 ,阳性细胞的数量明显增加 ,呈卵圆形或杆状 ,阳性细胞大多数位于尾壳核的实质内 ,少数位于血管壁内 ;(2 )在脑缺血后期 (4周 ,6周 ) ,大脑皮质缺血灶外周的 ED2阳性细胞的数量、形态和分布形式与脑缺血早期相比 ,也无明显变化 ;但在脑缺血灶的中央区有大量的 ED2阳性反应细胞 ,呈圆形 ,无明显突起 ;脑缺血后期 ED2阳性细胞主要位于尾壳核外侧区 ,其数量与缺血早期无明显的变化 ,但细胞形态变化显著 ,大多数细胞胞体肥大并有 2～ 3个明显的突起。脑缺血后期 ,在侧脑室脉络丛出现大量 ED2阳性反应的细胞 ,其胞体肥大 ,并有多个突起。结果表明 :慢性局灶脑缺血导致大脑皮质缺血区和尾壳核区的血管周细胞发生特征性的形态学改变。提示 ,血管周细胞与慢性脑缺血病理变化有联系     

Discrimination between different types of neuroglial cells in rat central nervous system using combined immuno- and enzyme-histochemical methods

The central nervous system (CNS) contains several types of neuroglial cells. In the present study, we characterized different types of glial cells in rat CNS by using single and combined immuno- and enzyme-histochemical methods, and immunofluorescence techniques. Two recently developed monoclonal antibodies (mAbs) against rat macrophages-associated antigens appeared to recognize a subpopulation of glial cells in the CNS of normal adult rats. These ED4- and ED8-positive glial cells were predominantly located in the white matter of adult rat CNS and shared morphological features with microglia. ED4 and ED8 were applied in a double staining combined with mAbs and an antiserum raised against galactocerebroside (GalC) to identify oligodendrocytes, or with anti-glial fibrillary acidic protein antiserum (GFA) to identify astrocytes. We also used a mAb against myelin basic protein (MBP) to identify oligodendrocytes. It appeared that ED4 and ED8 recognized a subpopulation of oligodendrocytes. MAbs against GalC and MBP recognized cells in an immunoperoxidase staining with a morphology identical to that of the ED8-positive cells and part of the ED4-positive cells. Frozen sections of Lewis rats CNS with acute experimental allergic encephalomyelitis (EAE) were investigated, where infiltrating brain macrophages could be found which stained positively with ED4 and ED8 as well as with the monocyte/macrophage mAbs ED1 and ED2. These brain macrophages did not stain when GalC, MBP and GFA markers were applied. Furthermore, ED4+GalC+ and ED8+GalC+ oligodendrocytes were present in the CNS white matter of EAE animals with similar appearance as in normal adult rats. With the currently used markers, we could not detect a third type of neuroglial cell, besides the astrocytes and oligodendrocytes. Thus, none of our anti-macrophage monoclonals recognized the presumptive microglia. Only under pathological conditions, e.g., in inflammatory infiltrates in the course of EAE, could brain macrophages be detected in the CNS parenchyma and only in the direct vicinity of blood vessels, indicating their hematogenous origin.     

Postischemic brain infiltration of leukocyte subpopulations differs among murine permanent and transient focal cerebral ischemia models

Cellular and humoral inflammations play important roles in ischemic brain injury. The effectiveness of immunomodulatory therapies may critically depend on the chosen experimental model. Our purpose was to compare the post‐ischemic neuroinflammation among murine permanent and transient middle cerebral artery occlusion (MCAO) models. Permanent MCAO was induced by transtemporal electrocoagulation and 30 minutes or 90 minutes transient MCAO was induced by intraluminal filament in C57BL/6 mice. Infiltration of leukocyte subpopulations was quantified by immunohistochemistry and fluorescence‐activated cell sorting. Cerebral cytokine and adhesion molecule expression was measured by real‐time polymerase chain reaction (RT‐PCR). Neutrophil infiltration was noted at 24 h after transient MCAO, but did not further increase until 5 days in the permanent MCAO model. Few T cells were observed in both MCAO models at 24 h, but permanent MCAO demonstrated much more infiltrating T cells at 5 days. Pronounced microglial activation was evident at 24 h and 5 days after permanent but not after transient MCAO. The number of invading NK cells and expression of MHCII on CD11b+ cells did not differ among the three groups. Five days after MCAO, the expression of IL‐1, TNF‐α and IFN‐γ and of the adhesion molecules ICAM‐1 and VCAM‐1 was significantly higher in the permanent than in the transient MCAO groups. Cellular and humoral inflammation differs substantially among commonly used MCAO models. Neuroinflammation is more pronounced after permanent electrocoagulatory MCAO compared with 30 minutes and 90 minutes filament‐MCAO.