脑缺血再灌注后大鼠血脑屏障通透性的免疫组织化学研究

目的:研究脑缺血再灌注后大鼠血脑屏障(BBB)通透性改变。方法:采用线栓法大鼠大脑中动脉闭塞的局灶性脑缺血模型,缺血1h后再灌注,分别于再灌注后0h、3h、5h、12h、及24h,采用免疫组织化学SABC法,观察内源性免疫球蛋白G(IgG)在脑组织中的表达。结果:再灌注0h、3h时,脑组织中未见IgG的表达。再灌注5h时,缺血侧大脑半球纹状体有局灶性的IgG表达。再灌注12h时,缺血侧纹状体及新皮层可见有广泛的IgG的表达。再灌注24h时,表达更加明显。结论:缺血1h再灌注3~5h时,BBB开始受损开放,通透性增加。纹状体的BBB较新皮层更易受损。再灌注12h、24h,外渗的血清蛋白累积增加。     

高压氧对脑缺血再灌注小鼠明胶酶、一氧化氮合酶及血脑屏障的影响

目的:通过检测脑缺血再灌注小鼠明胶酶A(MMP－2)、明胶酶B(MMP－9)及一氧化氮合酶(NOS)活性,探讨高压氧(HBO)对脑缺血再灌注小鼠明胶酶、NOS及血脑屏障(BBB)通透性的影响。方法:复制脑缺血再灌注模型,并于再灌注期间经0.25MPaHBO治疗5次,采用明胶酶谱分析法及比色法,检测脑组织海马区明胶酶及NOS的活性,同时经尾静脉注射2%伊文思兰(EB),检测脑组织EB含量。结果:脑缺血再灌注后明胶酶(A、B)活性增加,HBO+脑缺血再灌注组明胶酶B(MMP－9)活性明显低于脑缺血再灌注组;而HBO对明胶酶A(MMP－2)作用不显著。脑缺血再灌注后NOS活性增加,HBO+脑缺血再灌注组NOS含量显著低于脑缺血再灌注组(P＜0.01)。脑组织EB含量以再灌注4h最高,11h、23h、48h、72h脑组织EB含量逐渐减少。高压氧+脑缺血再灌注组脑组织EB含量明显低于脑缺血再灌注组(P＜0.05,P＜0.01)。结论:高压氧具有降低脑缺血再灌注小鼠明胶酶B及NOS活性,降低血脑屏障的通透性的作用。     

醒脑静注射液对全脑缺血再灌注大鼠血脑屏障ZO-1表达的影响

目的:探讨醒脑静(XNJ)注射液对大鼠全脑缺血再灌注后血脑屏障通透性及紧密连接蛋白1(ZO-1)表达的影响。方法:采用改良Pulsinelli四血管闭塞法建立大鼠全脑缺血再灌注模型。将雄性Wistar大鼠随机分为4组,即假手术组、全脑缺血再灌注模型组、溶剂对照组和XNJ组。每组均在缺血再灌注后24 h、48 h和72 h处理。用干湿重法测定脑组织中水含量,分光光度计法检测脑组织伊文思蓝(EB)含量,Western blot检测大脑皮层的ZO-1蛋白含量。结果:缺血再灌注后24 h,模型组、溶剂对照组和XNJ组的脑组织含水量均显著高于假手术组(P0.05),但在缺血再灌注后48 h和72 h,模型组和溶剂对照组脑组织含水量显著高于XNJ组和假手术组(P0.05)。缺血再灌注后24 h,模型组、溶剂对照组和XNJ组大鼠脑组织内EB含量均高于假手术组(P0.05),缺血再灌注后48 h和72 h,假手术组和XNJ组的EB含量显著低于模型组和溶剂对照组(P0.05)。缺血再灌注后24h,模型组、溶剂对照组和XNJ组大鼠脑皮层中的ZO-1蛋白表达水平显著低于假手术组(P0.05),同样缺血再灌注后48 h和72 h,假手术组和XNJ组皮层中ZO-1蛋白含量显著高于模型组和溶剂对照组(P0.05)。结论:在缺血再灌注后的48 h和72 h,醒脑静注射液对血脑屏障具有保护作用,可能与醒脑静注射液上调ZO-1蛋白的表达有关。     

大鼠脑缺血/再灌注损伤后梗死灶周围水通道蛋白4与血脑屏障通透性的动态变化

目的： 研究大鼠局灶性脑缺血/再灌注损伤对梗死灶周围脑组织水通道蛋白4（AQP4）的表达及血脑屏障通透性的影响。〖HJ1.7mm〗方法: 健康雄性Sprague-Dawley大鼠随机分为脑缺血/再灌注6 h、24 h、48 h、7 d组及相应时点的假手术组，每组各6只。采用线栓法阻塞大脑中动脉（MCAo）建立局灶性脑缺血/再灌注模型，于相应时点进行神经症状评分后断头取脑，采用免疫组织化学方法检测大鼠脑缺血/再灌注后不同时点梗死灶周围AQP4的表达及IgG的渗出以评价血脑屏障通透性的改变。结果: (1)大鼠脑缺血/再灌注损伤后神经功能缺失症状较假手术组明显（P<0.01），6-48 h呈逐渐加重趋势，72 h后有所缓解，7 d恢复正常；(2)大鼠脑缺血/再灌注6 h时AQP4表达增加不明显，至再灌注24 h后表达明显增加（P<0.05），7 d仍处于表达高峰；(3)再灌注6 h时IgG渗出不明显，再灌注24 h后开始增加（P<0.05），于再灌注48 h IgG渗出达高峰后开始下降; (4)大鼠脑缺血/再灌注损伤后血脑屏障通透性的增强与AQP4表达的增加呈显著相关(P<0.01)。结论: 大鼠脑缺血/再灌注损伤后AQP4表达增加与血脑屏障通透性增强密切相关，两者是脑缺血/再灌注损伤后脑水肿发生的重要因素。     

姜黄素对脑缺血再灌注模型大鼠的神经保护作用及其机制

目的:观察姜黄素对脑缺血再灌注大鼠模型的神经保护作用及其机制。方法:雄性SD大鼠随机分为3组:模型组、姜黄素组和假手术组,模型组采用血管内线栓阻断法制备大鼠局灶性脑缺血再灌注损伤模型,姜黄素组在缺血前1h腹腔注射姜黄素200mg/kg,随后每12h腹腔注射姜黄素60mg/kg(共5次)。再灌注后3h、24h和72h测定脑组织含水量、伊文斯蓝(EB)含量(反映血脑屏障破坏情况),免疫印迹法测定脑组织基质金属蛋白酶-9(MMP-9)表达水平。结果:脑缺血再灌注损伤大鼠脑组织含水量及EB含量增加,MMP-9高表达。姜黄素治疗能够减轻神经功能损伤,降低脑组织含水量和EB含量,并降低MMP-9表达水平(P<0.01)。结论:姜黄素通过降低脑缺血再灌注大鼠MMP-9表达水平及血脑屏障通透性,对脑缺血再灌注损伤起保护作用。     

大鼠局灶性脑缺血-再灌注时ε型蛋白激酶与热休克蛋白70表达变化及相关性

目的探讨大鼠脑缺血—再灌注时ε型蛋白激酶C（PKCε）与热休克蛋白70（HSP70）表达间关系。方法60只大鼠，随机分成缺血-再灌注组及假手术组，根据再灌注时间的不同，分别分为再灌注1、6、12、24、48、72h亚组，应用蛋白印迹法观察缺血皮层、基底节区各时间点胞浆内PKCε及HSP70及胞膜PKCε的表达。结果脑缺血后再灌注1h胞浆内PKCε含量开始下降，至再灌注24h达最低点，持续至再灌注72h。而膜内PKCε变化趋势与此相反，含量逐渐上升，于再灌注24h达到最高。再灌注1hHSP70有少量表达，再灌注6h即增高，至再灌注48h达最高，再灌注72h稍有减弱。假手术组各时间点均低含量表达。脑缺血-再灌注时HSP70与胞膜PKCε表达呈正相关，但无差异性，（r=0．55，P〉0．05）；HSPT0与胞浆内PKCε表达间呈显著负相关（r=-0．672，P〈0．05）。结论脑缺血再灌注时缺血皮层、基底节区PKCε发生了膜转位激活现象，同时有HSP70的表达，两者间呈一定的相关性。     

雌激素对大鼠脑缺血再灌注后血脑屏障作用研究

目的观察雌激素对大鼠脑缺血再灌注后血脑屏障(blood-brain barrier,BBB)的通透性、Occludin表达的影响,探讨雌激素在脑缺血中的作用。方法随机将去势雌性大鼠分为假手术组、模型组、雌激素预处理组,选取缺血再灌后4 h,24 h,3 d3个时间点作为观察点,对各个时间点脑水肿情况、Occludin蛋白表达、血脑屏障通透性改变进行考察分析,并选取24 h和3 d作BBB超微结构电镜观察,脑水肿改变情况采用脑含水量百分数测定;蛋白质表达采用western blot方法;血脑屏障通透性改变采用伊文思蓝(EB)比色法。结果与假手术组比较,局灶性脑缺血再灌4 h模型组大鼠脑组织含水量及EB含量均增加(P0.05),随缺血再灌时间延长,脑组织含水量、脑组织EB含量持续增加,至24 h,达到高峰(P0.01),与同时间点模型组比较,各治疗组大鼠脑组织含水量、EB含量均有不同程度降低(P0.05或P0.01),以24 h组降低最为显著(P0.01)。电镜观察雌激素预处理组较模型组同时间点比较BBB TJ开放减轻,星形胶质细胞足突及毛细血管管周水肿较轻,以3 d组显著。Western blot检测Occludin蛋白表达,发现模型组4 h时Occludin蛋白表达较假手术组减弱,但无显著性差异(P0.05),24 h组Occludin蛋白表达较假手术组减弱,有显著性差异(P0.05),3 d组Occludin蛋白表达进一步减弱,有明显差异(P0.01),雌激素组4 h时Occludin蛋白表达较同时间点模型组比较无显著性差异(P0.05),雌激素24 h及3 d组Occludin蛋白表达较同时间点模型组比较均升高,有显著性差异(P0.05)。结论局灶性脑缺血大鼠动物血脑屏障超微结构可见紧密连接发生断裂,内皮细胞内小泡数量增加及星形胶质细胞足突肿胀,可能是大鼠局脑缺血时血管源性脑水肿的重要因素。大鼠局灶性脑缺血,随缺血再灌时间延长,紧密连接相关蛋白occludin的表达明显下降,提示occludin在调节紧密连接通透性变化的过程中有重要作用。雌激素上调紧密连接Occludin蛋白的表达,有可能是其维护血脑屏障完整性减轻脑水肿的机制之一。     

高血脂对大鼠脑缺血再灌注损伤后大脑皮质基质金属蛋白酶9表达的影响

目的:研究高血脂对脑缺血再灌注损伤后大脑皮质基质金属蛋白酶9(MMP-9)表达的影响,探讨高血脂加重脑缺血再灌注损伤的机制。方法:高脂饲料喂养和线栓法分别建立高血脂动物模型和局灶性脑缺血再灌注模型,采用蛋白印迹、TTC染色、EB染色以及神经行为学评分,检测MMP-9的表达和脑梗死体积、血脑屏障通透性及神经行为的变化。结果:与假手术组比较,单纯脑缺血再灌注组和高血脂合并脑缺血再灌注组大脑皮质MMP-9表达增加,再灌注2 h时开始增加,24 h时达高峰,48、72 h时降低。相同再灌注时间点,与单纯脑缺血再灌注组比较,高血脂合并脑缺血再灌注组大脑皮质MMP-9表达明显增加,且脑梗死体积、血脑屏障通透性及神经行为评分均明显升高。结论:高血脂可通过上调大脑皮质MMP-9的表达而加重脑缺血再灌注损伤。     

局灶性脑缺血再灌注致血脑屏障破坏与zileuton的保护作用

目的： 探讨局灶性脑缺血再灌注损伤（CIRI）致血脑屏障(BBB) 通透性破坏的影响因素,观察高选择性5-脂氧合酶（5-LO）抑制剂zileuton的保护作用并分析其机制。方法: 线栓法制备大鼠大脑中动脉闭塞2 h/再灌注24 h模型（MCAO2 h/R24 h）,于缺血前2 h、再灌注0、5、10 h,分别灌胃给予zileuton10、50 mg·kg-1。伊文氏蓝示踪剂检测BBB;AutoCAD图像分析软件计算脑水肿程度;RT-PCR法检测脑组织白三烯受体1mRNA（CysLTR1 mRNA）;ELISA法检测血清白三烯B4（LTB4）;免疫组化法检测脑组织水通道蛋白4（AQP4）、基质金属蛋白酶9（MMP-9）蛋白。结果: MCAO2 h/R24 h后,BBB通透性明显增高,出现脑水肿,血清LTB4含量升高,梗塞侧脑组织CysLTR1mRNA表达增强,缺血区与梗塞灶周边缺血半暗带（IP）区AQP4与MMP-9蛋白表达上调; zileuton10、50 mg·kg-1均能有效控制CIRI所致的BBB破坏,改善脑水肿,降低血清LTB4含量,下调脑组织CysLTR1mRNA、AQP4与MMP-9表达。结论: CIRI后BBB通透性明显破坏,zileuton的保护作用与抑制脑组织和循环血液中的5-LO通路活化、下调脑组织缺血区与IP区的AQP4与MMP-9蛋白表达有关。     

人工麝香对大鼠局灶性脑缺血再灌注后大脑MMP-9 mRNA及其蛋白表达的影响

目的观察人工麝香对大鼠脑缺血再灌注后脑组织基质金属蛋白酶-9（MMP-9）mRNA及其蛋白表达的影响。方法采用SD大鼠制作大脑中动脉局灶性脑缺血再灌注模型,用高、中、低浓度人工麝香治疗缺血2h再灌注24、48h大鼠。应用实时荧光定量PCR法检测缺血再灌注脑组织MMP-9 mRNA的表达水平,免疫组化方法检测脑组织MMP-9蛋白表达量。结果与假手术组相比,各组在缺血再灌注不同时间段脑组织MMP-9 mRNA及MMP-9蛋白表达水平均升高（P〈0.05）,其中,以模型组MMP-9 mRNA及MMP-9蛋白表达水平最高（P〈0.01）,高、中浓度人工麝香治疗组MMP-9 mRNA及MMP-9蛋白表达水平最低（P〈0.05）;与模型组相比,高、中浓度人工麝香治疗组脑组织MMP-9 mRNA及MMP-9蛋白表达水平降低最为显著（P〈0.01）。结论人工麝香能降低大鼠局灶性脑缺血再灌注后脑组织MMP-9 mRNA及MMP-9蛋白的表达水平。人工麝香可能通过抑制缺血再灌注后大鼠脑组织MMP-9的表达,降低脑组织血脑屏障基底膜细胞外基质的降解和血脑屏障（BBB）的通透性,减轻脑缺血后脑水肿。     

Renal dysplasia and asplenia in two sibs

A family is reported in which two sibs, one male and the other female, both died within 24 hours of birth with enlarged polycystic kidneys. Postmortem histology in the second child showed gross renal dysplasia. In both children the pancreas was enlarged, nodular and cystic but the liver appeared macroscopically normal. In the second child, histological examination confirmed pancreatic fibrosis with cystic dilation of ducts, but showed portal fibrosis with bile duct proliferation in the liver.
This combination of findings is very reminiscent of those in a girl and her brother reported by Ivemark et al. (1959). The children reported here also showed absence or hypoplasia of the spleen, cardiac anomalies and other features of the Ivemark syndrome (Ivemark 1955), a quite different, usually sporadic, congenital disorder. It is suggested that the children described here have a distinct lethal congenital disorder, probably inherited in an autosomal recessive manner.     

Schizophrenia in a North Swedish geographical isolate, 1900–1977. Epidemiology, genetics and biochemistry

Over 200 schizophrenic patients belonging to three major and interrelated pedigree complexes have been investigated over the past 30 years in a North Swedish geographically isolated population, presently numbering about 6,000. An intensive investigation of a number of biochemical correlates and genetic markers in a few selected families belonging to one of the major pedigrees has indicated new strategies for the current research program.
Schizophrenia, as defined operationally, is significantly associated with decreased activities of two enzymes (1) blood platelet monoamine oxidase, (2) plasma dopamine-β-hydroxylase, and (3) with the genetic marker Gc² (group specific antigen). Both enzymes are subject to genetic variation. A positive score for linkage between schizophrenia and low plasma DBH activity has been calculated, but, so far, available data are insufficient for discrimination between linkage and partial contribution of genetically controlled low plasma DBH to the pathogenesis of the disease. Alternatively, both mechanisms could be involved.
As a model for continued research, schizophrenia is explained as based on a double dominant-recessive genotype (Aabb), representing a vulnerability which in about 50 % of cases develops into clinical schizophrenia. It is suggested that the dominant mutation (A) operates on or affects MAO activity, and that the recessive genotype (bb) is instrumental in low variates of DBH activity and very likely such variates within the normal range of physiological variation. Moreover, it is suggested that the combined effects of MAO- and DBH-reduced efficiency on the metabolism of e.g. dopamine could be an essential pathogenic mechanism for the schizophrenic illness which is segregating in this population.     

The dominant diseases of modernized societies as omega-3 essential fatty acid deficiency syndrome: Substrate beriberi

About 1900, modern food selection and processing caused widespread $\text{epidemics}$ of the B vitamin deficiency diseases of beriberi and pellagra which, for genetic reasons, often expressed as different diseases ranging from bowel and heart disease to dermatoses and psychoses. But the B vitamins merely help convert essential fatty acids (EFA) into the prostaglandin (PG) tissue regulators and it now turns out that, through hydrogenation, milling and selection of w3-poor southern foods, we have also been systematically depleting, by as much as 90%, a newly discovered trace Nordic EFA (w3) of special importance to primates and sole precursor of the PG3(4) series, even as a concurrent fiber deficiency increases body demand for EFA. Since substrate EFA is processed by many B vitamin catalysts, an EFA deficiency will mimic a $\text{panh}$ypovitaminosis B, i.e., a mixture of $\text{substrate}$ beriberi and $\text{substrate}$ pellagra resembling vitamin beriberi and pellagra but exhibiting as even more diverse $\text{endemic}$ disease. This would consitute a second stage of the Modern Malnutrition and explain why some workers now hold the dominant diseases of modermized societies to be new, nutritionally based, pellagraform yet lipid-related and to range, once again, from heart disease to psychosis. It is an assumption that our dominant diseases are unrelated to each other or are merely revealed by our diagnostic acumen and therapeutic success; and that hydrogenating millions of tons of food oils annually, to destroy the rancidity producing w3-EFA, is safe for $\text{primates}$. Extensive beriberiform disease is reported here in 32 typical cases taken from medical practice which responds strikingly to linseed oil supplements (60% w3-EFA) in confirmation of identical results in Capuchins.     

What will studies of Fulani individuals naturally exposed to malaria teach us about protective immunity to malaria?

There are an estimated over 200 million yearly cases of malaria worldwide. Despite concerted international effort to combat the disease, it still causes approximately half a million deaths every year, the majority of which are young children with Plasmodium falciparum infection in sub-Saharan Africa. Successes are largely attributed to malaria prevention strategies, such as insecticide-treated mosquito nets and indoor spraying, as well as improved access to existing treatments. One important hurdle to new approaches for the treatment and prevention of malaria is our limited understanding of the biology of Plasmodium infection and its complex interaction with the immune system of its human host. Therefore, the elimination of malaria in Africa not only relies on existing tools to reduce malaria burden, but also requires fundamental research to develop innovative approaches. Here, we summarize our discoveries from investigations of ethnic groups of West Africa who have different susceptibility to malaria.     

'Very sore nights and days': the child's experience of illness in early modern England, c.1580-1720

Sick children were ubiquitous in early modern England, and yet they have received very little attention from historians. Taking the elusive perspective of the child, this article explores the physical, emotional, and spiritual experience of illness in England between approximately 1580 and 1720. What was it like being ill and suffering pain? How did the young respond emotionally to the anticipation of death? It is argued that children’s experiences were characterised by profound ambivalence: illness could be terrifying and distressing, but also a source of emotional and spiritual fulfilment and joy. This interpretation challenges the common assumption amongst medical historians that the experiences of early modern patients were utterly miserable. It also sheds light on children’s emotional feelings for their parents, a subject often overlooked in the historiography of childhood. The primary sources used in this article include diaries, autobiographies, letters, the biographies of pious children, printed possession cases, doctors’ casebooks, and theological treatises concerning the afterlife.     

Guidelines for the Validation and Use of Immunoassays for Determination of Introduced Proteins in Biotechnology Enhanced Crops and Derived Food Ingredients

Recent advancements in agricultural biotechnology have created a need for analytical techniques to determine introduced proteins in crops enhanced through modern biotechnology techniques. These proteins are expressed in plant tissues and may be present in food ingredients. Immunoassays are ideally suited for protein detection and may be used as both quantitative and threshold methods. Microplate ELISA and lateral flow devices are two of the most commonly used immunoassay formats for agricultural biotechnology applications. This paper provides general background information and a discussion of criteria for the validation and application of immunochemical methods to the analysis of proteins introduced into plants and food ingredients using biotechnology methods. It is the result of a collaborative effort of members of the Analytical Environmental Immunochemical Consortium. This collaborative effort represents the combined expertise of several organizations to reach consensus on establishing guidelines for the validation and use of immunoassays. Further, the paper offers developers and users a consistent approach to adopting the technology as well as aid in producing accurate and meaningful results.     

HLA in North Colombia Chimila Amerindians

HLA-A,-B,-C,-DRB1 and -DQB1 alleles have been studied in Chimila Amerindians from Sabana de San Angel (North Colombian Coast) by using high resolution molecular typing. A frequent extended haplotype was found:HLA-A*24:02-B*51:10-C*15:02-BRB1*04:07-DQB1*03:02 (28.7%) which has also been described in Amerinndian Mayos Mexican population (Mexico, California Gulf, Pacific Ocean). Other haplotypes had already been found in Amerindians from Mexico (Pacific and Atlantic Coast), Peru (highlands and Amazon Basin), Bolivia and North USA. A geographic pattern according to HLA allele or haplotype frequencies is lacking in Amerindians, as already known. Also, five new extended haplotypes were found in Chimila Amerindians. Their HLA-A*24:02 high frequencies characteristic is shared with aboriginal populations of Taiwan; also, HLA-C*01:02 high frequencies are found in New Zealand Maoris, New Caledonians and Kimberly Aborigines from Australia. Finally, this study may show a model of evolutionary factors acting and rising one HLA allele frequency (-A*24:02), but not in others that belong to the same or different HLA loci.     

Ultracryotomy: development and application (with examples from the yeast cytology) (author's transl)

The preparation steps usually necessary for obtaining ultrathin frozen sections of biological material (chemical prefixation, enclosing, cryoprotective treatment, freezing, sectioning, and post-staining the sections for transmission electron microscopy) are submitted to a critical analysis. The application of cryo-ultramicrotomy, in particularly for cytochemical purposes, is reviewed. Fundamental considerations of chemical prefixation and poststaining are supported by examples from yeast cytology. Furthermore, the efficiency of the cryo-ultramicrotomy (electron optical resolution of ultrastructural details) is demonstrated on yeast cells and protoplasts.     

The universal muscular chamber. A basic unit of the genitourinary, cardiovascular, gastro-intestinal, skeletal, cutaneous, respiratory and other systems, endocameral hypertension; and spasm, the key to their idiopathic diseases and arthropathies

Starting with the integument, we see many organs are contractile sacs or multiples thereof, which tubes or bags constitute the major part of the entire body. Recognition of this basic unit and its characteristics sheds new light, individually and collectively, on many disorders previously considered unrelated. Muscular tears and perforations develop in the walls of these chambers, being no way peculiar to those organs, wherein, hydrochloric acid occurs. So, it is not necessary to explain the absence of excessive acid from patients who exhibit holes in the gastric, uterine, aortic, duodenal, rectal, pulmonary, retina, and other walls. Muscle, not acid is the great common factor relating idiopathic disorders in the gastrointestinal tract to each other and to similar diseases in other systems. When the units are linked together, the lesions tend to appear as arthropathies, i.e. at the joints. Rephrasing common-place observations, frees us from conventional, conceptual cul-de-sacs. An observation is only as good as its interpretation, so all possibilities must be considered, otherwise, we will remain blinded by our misconceptions.