经皮腹主动脉球囊扩张成形后炎性因子的变化

背景：炎性反应在动脉粥样硬化疾病的发生与发展，以及冠状动脉介入治疗后再狭窄的过程中起重要作用，但具体机制尚未明确。 目的：观察兔腹主动脉球囊扩张后炎性因子白细胞介素8、白细胞介素6、肿瘤坏死因子α以及C-反应蛋白不同时间点的水平变化。 设计、时间及地点：随机对照动物实验，于2008-01/08在河北省人民医院心脏中心导管室及临床研究中心完成。 材料：选取8~12月龄新西兰大白兔24只，雌雄不拘，体质量2.50~3.25 kg。 方法：24只新西兰大白兔，随机抽签法分为实验组和对照组，每组12只。实验组穿刺股动脉后留置鞘管，经鞘管送入球囊行球囊扩张，拉伤腹主动脉。对照组仅穿刺留鞘，不进行球囊拉伤。分别于实验前、实验后4 h，1，3 d，1，2周，耳缘静脉采血4 mL，3 000 r/min离心20 min后，取上清液得血清标本，-80 ℃冰箱统一保存。 主要观察指标：①酶联免疫吸附法分别测定实验前后不同时间点白细胞介素8、白细胞介素6、肿瘤坏死因子α和C-反应蛋白水平。②白细胞介素8水平与其他3种因子的相关性分析。③造影观察实验动物腹主动脉球囊损伤前后的形态学改变。 结果：24只新西兰大白兔均进入结果分析。①兔腹主动脉球囊损伤后炎性因子白细胞介素8、白细胞介素6、肿瘤坏死因子α以及C-反应蛋白水平明显升高，与术前及对照组相比，差异具有显著性意义(P < 0.01)。②各种炎性因子中，白细胞介素8水平最先升高，达峰值时间最短，相关分析显示，白细胞介素8水平表达与炎性反应标志物C-反应蛋白以及白细胞介素6、肿瘤坏死因子α呈正相关，相关系数r分别为0.569，0.673，0.597(P均< 0.05)。③球囊扩张拉伤后2周，实验组动物腹主动脉拉伤处较对照组管腔明显狭窄。 结论：兔腹主动脉球囊扩张后引起炎性因子白细胞介素8、白细胞介素6、肿瘤坏死因子α以及C-反应蛋白水平升高，导致动脉管腔狭窄，其中白细胞介素8在此过程中发挥了重要作用。     

瑞舒伐他汀对Medtronic球囊导管损伤大鼠颈动脉平滑肌细胞增殖及凋亡的影响

背景：球囊扩张及支架置入后再狭窄、管腔丢失等问题制约了支架置入治疗的进一步发展,血管内膜增殖和凋亡在再狭窄中的作用及干预方法尚在积极探索中。 目的：采用Medtronic球囊建立大鼠颈动脉球囊损伤模型,观察瑞舒伐他汀对球囊损伤大鼠颈动脉平滑肌细胞增殖及凋亡的影响。 方法：将雄性SD大鼠随机数字表法分为球囊损伤组和治疗组。均采用Medtronic球囊建立大鼠颈动脉球囊损伤模型,并取球囊损伤组大鼠的右侧颈总动脉(未行球囊损伤)作为正常对照。治疗组于球囊损伤前3 d始连续给予瑞舒伐他汀5 mg/(kg•d)灌胃,球囊损伤组给予9 g/L氯化钠溶液灌胃。术后7,14 d取颈总动脉进行苏木精-伊红染色,免疫组织化学检测SM α-actin、增殖细胞核抗原,采用TUNEL法检测平滑肌细胞凋亡情况。 结果与结论：与球囊损伤组比较,治疗组造模后14 d,损伤血管新生内膜面积、新生内膜/中膜面积比值明显减少(P < 0.05),管腔面积增加26%;增殖细胞核抗原阳性细胞率降低,凋亡阳性细胞率增高(P < 0.05)。提示支架置入球囊损伤前给予瑞舒伐他汀可抑制大鼠颈动脉球囊损伤后新生内膜增生及新生内膜细胞的增殖,促进平滑肌细胞的凋亡,从而减少动脉新生内膜形成,抑制再狭窄。     

川芎嗪对颈动脉球囊损伤后基质金属蛋白酶组织抑制物-1基因表达的影响

目的观察川芎嗪对兔颈动脉球囊损伤后新生内膜增生、管腔狭窄度和管壁基质金属蛋白酶组织抑制物-1(TIMP-1)表达的影响,探讨其预防再狭窄的可能机制和作用。方法应用直径为3 mm的球囊导管制作家兔左颈动脉去内皮损伤模型,术后14 d测量对照组和川芎嗪治疗组动物新生内膜及中膜厚度、新生内膜面积和管腔狭窄度,并应用Brdu和α-actin免疫组化方法观察新生内膜细胞的增殖指数,同时应用半定量逆转录聚合酶链式反应(RT-PCR)方法比较对照组和治疗组间管壁TIMP-1的基因表达。结果治疗组新生内膜及中膜厚度、新生内膜面积、管腔狭窄度和细胞增殖指数均低于对照组,而川芎嗪治疗组动脉壁TIMP-1mRNA的表达明显高于对照组。结论川芎嗪具有明显增加动脉损伤后管壁TIMP-1mRNA形成的作用,从而抑制新生内膜的形成和降低管腔狭窄度。     

小鼠胚胎干细胞来源平滑肌细胞的鉴定及其功能分析

背景：胚胎干细胞来源的平滑肌细胞是血管组织工程潜在的细胞来源之一,但这些细胞是否具有成熟平滑肌细胞的表型和功能仍不清楚。 目的：对小鼠胚胎干细胞分化的平滑肌细胞进行表型鉴定及功能分析。 设计、时间及地点：细胞学体外观察,于2007-05/2008-12在解放军沈阳军区总医院全军心血管病研究所完成。 材料：清洁级孕12.5 d昆明小鼠1只用于制备饲养层细胞,SD大鼠1只用于制备主动脉平滑肌细胞。小鼠胚胎干细胞系R1、小鼠微血管内皮细胞系由美国ATCC公司提供,转染pSPIE载体的胚胎干细胞R1由本实验室制备。 方法：①诱导分化及筛选：用转染pSPIE载体的胚胎干细胞R1制备拟胚体,拟胚体悬浮培养5 d后贴壁培养1 d,第7天加入全反维甲酸诱导分化4 d,第 11天用10 mg/L嘌呤霉素对诱导分化后的细胞筛选2 d。②表型鉴定：对筛选到的平滑肌细胞进行SM α-actin、SM22α、SM-MHC免疫荧光染色,以大鼠原代主动脉平滑肌细胞、未经筛选的第10天分化细胞作为对照,进行Western Blot分析。③收缩功能：10-5 mol/L卡巴可处理筛选到的平滑肌细胞 30 min。④体外成血管功能：将等量的内皮细胞和筛选到的平滑肌细胞混合,在Matrigel上培养。 主要观察指标：平滑肌细胞标志物的表达,平滑肌细胞的收缩,平滑肌细胞向内皮管腔样结构的募集。 结果：分化的平滑肌细胞主要分布于贴壁生长的拟胚体的外周部位,胚胎干细胞来源的平滑肌细胞3种标志物SM α-actin、SM22α、SM-MHC免疫荧光染色均呈阳性表达,Western Blot结果显示其SM α-actin、SM22α表达水平与大鼠原代主动脉平滑肌细胞相似,高于未经筛选的第10天分化细胞。筛选到的平滑肌细胞在10-5 mol/L卡巴可刺激下出现明显收缩,且能够募集到内皮管腔样结构周围。 结论：小鼠胚胎干细胞来源的平滑肌细胞具有与成熟平滑肌细胞相似的表型和功能。     

川芎嗪对兔颈动脉球囊损伤后内膜增生的影响

目的研究川芎嗪对兔颈动脉球囊损伤后新生内膜及管腔狭窄度的影响，探讨其预防再狭窄的可能性。方法应用直径为3mm的球囊导管造成家兔左颈动脉去内皮损伤模型。术后14d和28d分别测量对照组和川芎嗪治疗组动脉新生内膜及中膜厚度、新生内膜面积和管腔狭窄度并应用Brdu和α-actin免疫组化方法观察新生内膜细胞的增殖指数。结果治疗组新生内膜及中膜厚度、新生内膜面积．管腔狭窄度和细胞增殖指数均明显低于对照组。结论川芎嗪具有明显抑制动脉损伤后新内膜的形成及降低管腔狭窄度的作用。     

早期高压氧治疗对急性脑梗死患者血清hs-CRP、IL-6、IL-8、ET-1的影响及预后的临床分析

目的 探讨早期高压氧治疗后急性脑梗死(ACI)患者血清超敏反应蛋白(hs-CRP)、IL-6、IL-8和ET-1水平变化及临床意义.方法 将120例急性脑梗死患者随机分为2组,应用放射免疫法测定血清hs-CRP水平,用ELISA法检测血清IL-6、IL-8和ET-1水平以及用NIHSS对患者的神经功能进行评估.结果 高压氧治疗组1周后血清 hs-CRP、IL-6、IL-8、ET-1水平较对照组明显下降(P均＜0.05),并与神经功能恢复有关.结论 急性脑梗死患者血清hs-CRP、IL-6、IL-8、ET-1水平与神经功能缺损程度评分有关.     

颈动脉支架置入术后内皮功能的变化及与再狭窄的相关研究

目的 探讨颈动脉支架置入术后假性血友病因子(vWF)、内皮素1(ET-1)的水平变化及与再狭窄的关系.方法 检测67例短暂性脑缺血发作患者行颈动脉支架置人术前,术后1 h、2周、1个月、6个月、1年血浆vWF、ET-1水平.患者术后1年常规进行头颈血管CTA检查,根据结果分为再狭窄组和无狭窄组,并对两组vWF、ET-1水平进行比较.结果 随访中1例死亡,4例失访.14例患者存在不同程度的再狭窄,其中3例再狭窄≥50%.与术前比较,术后1 h、6个月vWF 水平升高,术后1 h、2周、6个月ET-1水平升高,差异均有统计学意义(P<0.05).与无狭窄组比较,再狭窄组术后1 h、2周、6个月vWF水平升高,术后1 h、2周、1个月、6个月ET-1水平升高,差异均有统计学意义(P<0.05).结论 颈动脉支架置入术后短期(2周内)vWF、ET-1水平持续升高,术后6个月再次升高者发生再狭窄的危险性增加,监测vWF、ET-1水平对判断患者的远期预后有一定价值.     

颈动脉支架置入术后内皮功能的变化及与再狭窄的相关研究

目的 探讨颈动脉支架置入术后假性血友病因子(vWF)、内皮素1(ET-1)的水平变化及与再狭窄的关系.方法 检测67例短暂性脑缺血发作患者行颈动脉支架置人术前,术后1 h、2周、1个月、6个月、1年血浆vWF、ET-1水平.患者术后1年常规进行头颈血管CTA检查,根据结果分为再狭窄组和无狭窄组,并对两组vWF、ET-1水平进行比较.结果 随访中1例死亡,4例失访.14例患者存在不同程度的再狭窄,其中3例再狭窄≥50%.与术前比较,术后1 h、6个月vWF 水平升高,术后1 h、2周、6个月ET-1水平升高,差异均有统计学意义(P<0.05).与无狭窄组比较,再狭窄组术后1 h、2周、6个月vWF水平升高,术后1 h、2周、1个月、6个月ET-1水平升高,差异均有统计学意义(P<0.05).结论 颈动脉支架置入术后短期(2周内)vWF、ET-1水平持续升高,术后6个月再次升高者发生再狭窄的危险性增加,监测vWF、ET-1水平对判断患者的远期预后有一定价值.     

加味补阳还五汤对兔髂动脉球囊损伤血管狭窄和氧化应激的影响

背景：近年研究证明,补阳还五汤具有扩张血管、改善微循环、抗炎、抗氧化应激和保护血管内皮细胞的功能,但其具体机制尚不清楚,尤其是对经皮腔内冠状动脉成形后再狭窄形成过程有何影响,目前很少见报道。 目的：观察加味补阳还五汤对兔髂动脉球囊损伤后血管狭窄及氧化应激的影响。 方法：将新西兰兔以随机抽签法分为对照组、模型组和药物组。对照组给予普通饲料,模型组、药物组给予高脂饮食。饲养2周后,模型组、药物组行髂动脉内膜剥脱术,对照组兔行假手术对照,药物组术后饲料中添加加味补阳还五汤药颗粒 2 mL/(kg•d),对照组及模型组喂食同前。4周后光镜观察兔髂动脉内膜的损伤情况,并检测血脂水平、血清超氧化物歧化酶活性和丙二醛水平的变化。 结果与结论：对照组髂动脉内膜薄且结构完整,无动脉硬化斑块;模型组内膜增厚,管腔明显狭窄,可见明显动脉粥样硬化斑块;药物组动脉粥样硬化斑块厚度减小,管腔狭窄程度较轻。药物组兔血清总胆固醇、三酰甘油和低密度脂蛋白胆固醇、丙二醛水平明显低于模型组,而高密度脂蛋白胆固醇、血清超氧化物歧化酶水平明显高于模型组(P < 0.05)。结果说明加味补阳还五汤具有较好的防止家兔球囊扩张损伤髂动脉所致的管腔狭窄及抗实验性动脉粥样硬化作用,其机制可能与其清除氧自由基抗氧化应激、调节脂质代谢等作用有关。     

一种新型聚合物携带雷帕霉素洗脱血管支架抑制内膜增生的效应

背景：课题组前期已进行了系列体内生物相容性实验，证实了聚砜-聚氧化乙烯共聚物材料具有良好的细胞相容性、血液相容性、组织相容性和使用安全性。 目的：在前期实验基础上进一步评价聚砜-聚氧化乙烯共聚物作为携带雷帕霉素涂层的新型血管支架抑制血管内膜增生防治再狭窄的安全性及有效性。 设计、时间及地点：随机对照动物实验，于2007-01在东南大学附属中大医院心内科完成。 材料：采用超声雾化的方法将含聚氧化乙烯30%的聚砜-聚氧化乙烯共聚物均匀喷涂于316 L不锈钢金属裸支架上，制成含雷帕霉素130 μg的涂层支架，由江苏省微纳生物医疗器械重点实验室制作；金属裸支架采用相同材质和结构制作。 方法：通过球囊扩张小型猪冠状动脉形成冠状动脉狭窄模型。6只小型猪随机抽签法分为2组，实验组置入雷帕霉素洗脱支架；对照组置入金属裸支架，每组3只。置入后第28天行冠状动脉造影和血管内超声检查。 主要观察指标：支架置入后冠状动脉管腔丢失情况、新生内膜面积和厚度、内皮生长情况；免疫组织化学分析测定增殖细胞核抗原；TUNEL法检测血管平滑肌细胞凋亡数；扫描电镜观察内皮细胞的覆盖情况。 结果：血管支架置入后第28天冠状动脉造影检查显示，实验组血管腔直径丢失及直径狭窄率较对照组明显减少(P < 0.05)。病理学证实，实验组较对照组管腔面积增加、支架内新生内膜面积和面积再狭窄率减小(P < 0.05)。对照组增殖细胞核抗原细胞阳性率较实验组明显减少(P < 0.05)，单位面积内血管平滑肌细胞凋亡数明显增加(P < 0.05)。扫描电镜显示对照组支架置入28 d血管内皮细胞完全覆盖，实验组基本覆盖。 结论：在小型猪/冠状动脉支架置入模型中，聚砜-聚氧化乙烯共聚物携带的雷帕霉素洗脱血管支架显示出良好的安全性和降低冠状动脉再狭窄的有效性。     

视频脑电图在小儿癫痫诊断中的应用

目的评价视频脑电图(video-EEG)在小儿癫诊断中的应用价值。方法对126例具有发作性症状的患儿进行连续8h的包括清醒、睡眠、诱发试验及必要的认知测验的视频脑电图监测。结果经发作期视频脑电图证实,39例初诊为癫性发作的患儿中14例(35%)为非癫性发作;15例其他症状发作中13例(86%)为非癫性发作。64例样放电患儿中51例(80%)确定发作类型,22例(34%)确定癫类型。视频脑电图可发现短暂轻微的癫发作及样放电引起的一过性认知损伤。结论视频脑电图在排除非癫性发作、确定癫性发作的类型、评价脑电-临床关系方面可提供准确可靠的证据,进一步提高癫的临床诊断水平。     

Neuroprotective properties of memantine in different in vitro and in vivo models of excitotoxicity

The pathogenesis of stroke, trauma and chronic degenerative diseases, such as Alzheimer's disease (AD), has been linked to excitotoxic processes due to inappropriate stimulation of the N-methyl-D-aspartate receptor (NMDA-R). Attempts to use potent competitive NMDA-R antagonists as neuroprotectants have shown serious side-effects in patients. As an alternative approach, we were interested in the anti-excitotoxic properties of memantine, a well-tolerated low affinity uncompetitive NMDA-R antagonist presently used as an anti-dementia agent. We explored in a series of models of increasing complexity, whether this voltage-dependent channel blocker had neuroprotective properties at clinically relevant concentrations. As expected, memantine protected neurons in organotypic hippocampal slices or dissociated cultures from direct NMDA-induced excitotoxicity. However, low concentrations of memantine were also effective in neuronal (cortical neurons and cerebellar granule cells) stress models dependent on endogenous glutamate stimulation and mitochondrial stress, i.e. exposure to hypoxia, the mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP+) or a nitric oxide (NO) donor. Furthermore, memantine reduced lethality and brain damage in vivo in a model of neonatal hypoxia-ischemia (HI). Finally, we investigated functional rescue (neuronal capacity to migrate along radial glia) by memantine in cerebellar microexplant cultures exposed to the indirect excitotoxin 3-nitropropionic acid (3-NP). Potent NMDA-R antagonists, such as (+)MK-801, are known to block neuronal migration in microexplant cultures. Interestingly, memantine significantly restored the number of neurons able to migrate out of the stressed microexplants. These findings suggest that inhibition of the NMDA-R by memantine is sufficient to block excitotoxicity, while still allowing some degree of signalling.     

Storage of lipofuscin in neurons in mucopolysaccharidosis

Summary A histochemical and ultrastructural study was made on the brain of a 23-year-old man with Sanfilippo's syndrome. In accordance with previous reports the cortical nerve cells contained a PAS-positive lipid storage substance. This showed intense autofluorescence in UV-light and was positive with various stains for lipofuscin. The storage material appeared ultrastructurally as inclusion bodies composed of short lamellated membranes, granular material, and vacuoles. In addition, concentrically and transversely lamellated membranous cytoplasmic bodies were observed in the nerve cells. It is concluded that the PAS-positive lipid storage material in the neurons was composed partly of lipofuscin in addition to other lipids presumably glycosphingolipids.Supported by a grant from the Expressen Prenatal Research Foundation     

Role of vincristine in the inhibition of angiogenesis in glioblastoma

Objective: Vincristine, a microtubule-destabilizing drug, was found to exhibit anti-angiogenic effects and anti-tumoral activity. However, the precise mechanism by which vincristine inhibits angiogenesis in glioblastomas is not well understood. Our aim was to investigate whether vincristine affects vascular endothelial growth factor (VEGF) expression in glioblastoma cells and determine whether it is mediated by the downregulation of hypoxia-inducible factor-1α (HIF-1α).

Methods: We investigated the expression of HIF-1α in glioblastoma tissues resected from patients and in human glioblastoma cell lines using immunohistochemistry, Western blot analysis, and immunocytochemistry. In addition to an MTT assay assessing the effect of vincristine on cell proliferation and viability, the effects of vincristine on VEGF mRNA expression and HIF-1α protein were examined using real-time RT-PCR and Western blot analysis under 1% O₂ (hypoxia).

Results: HIF-1α was expressed in the majority of glioblastoma tissues and was detected mainly in the nucleus. Strong immunoreactivity for HIF- 1 α was found often in the hypercellular zones. Under hypoxic conditions, HIF-1α protein levels in the glioblastoma cell lines increased, primarily localizing into the nucleus similar to glioblastoma tissues. Exposure of glioblastoma cells to vincristine resulted in enrichment of the G₂-M fraction of the cell cycle, which suggests that vincristine-mediated growth inhibition of glioblastoma is correlated with mitotic inhibition. Using doses lower than those found to reduce the viability and proliferation of cells by 50% (IC₅₀), vincristine decreased both the expression of VEGF mRNA and the level of HIF-1α protein in hypoxic glioblastoma cells. In addition, following exposure to vincristine, the expression of VEGF mRNA was correlated with HIF-1α protein levels.

Conclusions: Our results suggest that the mechanism by which vincristine elicits an anti-angiogenic effect in glioblastomas under hypoxic conditions might be mediated, in part, by HIF-1α inhibition.     

自噬参与帕金森病发病的研究进展

近年来,蛋白质的降解障碍被认为是帕金森病（Parkinson’Sdisease,PD）发病过程中的重要因素,人们已经公认泛素一蛋白酶体系统（ubiquitin--pro—teasomesystem,UPS）功能异常或衰竭能够导致细胞内异常蛋白蓄积、细胞功能障碍,甚至细胞凋亡。与此同时,蛋白降解的另一条途径——自噬-溶酶体途径（autophagy—lysosomepathway,ALP）也已成为了生命科学领域的研究热点,自噬与神经变性疾病,尤其是PD的关系日益受到人们的重视。     

脑电图预测痫性发作研究进展

癫痫(epilepsy)是由脑部神经元高度同步化异常放电所致的临床综合征,系神经系统的常见病,困扰着全世界约1%的人群.每次神经元的阵发性放电或短暂的脑功能异常称为痫性发作(seizures).     

Midazolam in treatment of various types of seizures in children

Midazolam is a recently developed water-soluble benzodiazepine that shares anxiolytic, muscle relaxant, hypnotic and anticonvulsant actions with other members of this class. There are limited studies that midazolam can be used successfully to treat seizures in adults and children. In this study, 0.2 mg/kg intramuscular (IM) midazolam was administered to 11 children (eight boys and three girls), aged 3 days to 4 years (mean age 1.8±1.4 years), with seizures of various types. In all but one child, seizures stopped in 15 s–5 min after injection. No side effects were observed. These results suggest that IM administration of midazolam may be useful in a variety of seizures during childhood, especially in case of intravenous (IV) line problem.     

Developmental effects of in vivo and in vitro inhibition of nitric oxide synthase in neurons

The diffusible chemical messenger nitric oxide (NO) is involved in neuronal plasticity and it is, therefore, supposed to play a role in brain development. A shortage of NO during the critical period of brain maturation may theoretically have long-lasting consequences on the organization of the adult brain. We have performed in neonatal rats a chronic inhibition of the enzyme responsible for NO production, nitric oxide synthase (NOS), from postnatal day 3 to postnatal day 23, through administration of the competitive antagonist N-nitro-L-arginine methylester (L-NAME). The calcium-dependent catalytic activity resulted almost completely inhibited throughout the period of treatment and it took more than 4 days after its suspension to get a full recovery. The expression of the neuronal isoform of the enzyme (nNOS), revealed by immunoblotting, was unchanged during the treatment and after it. The histochemical reaction for NADPH diaphorase was reduced at the end of the treatment and recovered in concomitance with the recovery of the catalytic NOS activity. No gross structural alterations were detected in brain morphology. The levels of three neurotransmitter-related and one astrocytic marker were unchanged in the cerebellum, hippocampus and cortex of 60-day-old rats which had been neonatally treated. A similar lack of significant effects on neurochemical brain maturation was also noticed in a parallel series of experiments, in which a short pulse of NOS inhibition was performed at a critical prenatal time of brain development, from gestational day 14 to gestational day 19. In vitro, chronic exposure of cerebellar granule cells to L-NAME (500 microM) resulted in slight decrease of surviving neurons after 8 days in culture and in better resistance to the challenge of stressful culture conditions. The present results suggest that the basic plan of brain organization can be achieved despite an almost complete NOS inhibition during the maturation period. In vitro, NOS inhibition may bring to more pronounced consequences on neuronal viability and function.     

Issues in evaluation of cognition in the elderly in developing countries

Background:

Developing regions of the world host the majority of elderly subjects who are at risk for dementia. Reliable epidemiological data from these countries is invaluable in tackling this global problem. Scarcity of such data in literature is largely attributable to problems that are unique to developing communities worldwide.

Objective:

To classify and describe the problems that interfere with the collection of reliable epidemiological data on cognitive impairment in the elderly in developing communities, and to suggest practical solutions for some of them.

Methods:

Inferring from the experiences of a large, ongoing, population-based study on the cognitive impairments in the elderly in South India and from the review of literature.

Conclusion:

A fatalistic attitude regarding aging in the communities, significant heterogeneity in educational abilities and activities of daily living, high illiteracy among rural subjects, and lack of an organized health care system and updated demographic figures are some of the major factors that contribute to technical, namely, methodology-related problems and practical, namely, subject-related problems in such epidemiological studies.