In vitro engineered models of neurodegenerative diseases

Neurodegeneration is a catastrophic process that develops progressive damage leading to functional andstructural loss of the cells of the nervous system and is among the biggest unavoidable problems of our age.Animalmodels do not reflect the pathophysiology observed in humans due to distinct differences between the neuralpathways,gene expression patterns,neuronal plasticity,and other disease-related mechanisms in animals andhumans.Classical in vitro cell culture models are also not sufficient for pre-clinical drug testing in reflecting thecomplex pathophysiology of neurodegenerative diseases.Today,modern,engineered techniques are applied to developmulticellular,intricate in vitro models and to create the closest microenvironment simulating biological,biochemical,and mechanical characteristics of the in vivo degenerating tissue.In THIS review,the capabilities and shortcomings ofscaffold-based and scaffold-free techniques,organoids,and microfluidic models that best reflect neurodegeneration invitro in the biomimetic framework are discussed.

Animal models in the study of Alzheimer's disease and Parkinson's disease:A historical perspective

Alzheimer's disease and Parkinson's disease are two of the most prevalent and disabling neurodegenerative diseases globally.Both are proteinopathic conditions and while occasionally inherited,are largely sporadic in nature.Although the advances in our understanding of the two have been significant,they are far from complete and neither diagnosis nor the current practices in treatment and rehabilitation is adequately helpful.Animal models have historically found application as testing beds for novel therapeutics and continue to be valuable aids in pharmacological research.This review chronicles the development of those models in the context of Alzheimer's and Parkinson's disease,and highlights the shifting paradigms in studying two humanspecific conditions in non-human organisms.

Effects of natural-cerebrolysin-containing serum on neurotoxicity and synaptogenesis in amyloid-beta 1-40-induced Alzheimer's disease in vitro models

BACKGROUND： Neuronal loss, synapse mutilation, and increasing malnourished axons are pathologically related to Alzheimer＇s disease. Microtubule-associated protein 2 （MAP2） is of importance for neuronal, axonal, and dendritic generation, extension, and stabilization, as well as for the regulation of synaptic plasticity. OBJECTIVE： To investigate the antagonistic effects of natural-cerebrolysin-containing serum on beta amyloid protein 1-40 （Aβ1-40）-induced neurotoxicity from the standpoints of cell proliferation, synaptogenesis, and cytoskeleton formation （MAP2 expression）. DESIGN, TIME AND SETTING： A paralleled, controlled, neural cell, and molecular biology experiment was performed at the Institute of Integrated Chinese and Western Medicine, Shenzhen Hospital, Southern Medical University between February 2006 and April 2008. MATERIALS： PC12 cells, derived from the rat central nervous system, were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences, China. A β1-40 was provided by Sigma, USA. Natural-cerebrolysin was provided by Shenzhen Institute of Integrated Chinese and Western Medicine, China. The natural-cerebrolysin was predominantly composed of Renshen （Radix Ginseng）, Tianma （Rhizoma Gastrodiae）, and Yixingye （Ginkgo Leaf） in a proportion of 1：2：2. Following conventional water extraction technology, an extract （1：20） was prepared. Each gram of extract equaled 20 grams of crude drug. In a total of 12 adult male New Zealand rabbits, six underwent intragastric administration of natural-cerebrolysin extract for 1 month to prepare natural-cerebrolysin-containing serum, and the remaining six rabbits received intragastric administration of physiological saline to prepare normal blank serum. METHODS： An AIzheimer＇s disease in vitro model was induced in PC12 cells using Aβ1-40. The cells were incubated with varying doses of natural-cerebrolysin-containing serum （2.5%, 5%, and 10%）. Normal blank serum-treated PC12 cells served as a blank control group. MAIN OUTCOME MEASURES： Through the use of inverted phase contrast microscope, cell morphology and neurite growth were observed, neurite length was measured, and the percentage of neurite-positive cells was calculated. Cell proliferation rate was determined by MTT assay, and MAP 2 expression was detected by fluorescent immunocytochemistry. RESULTS： Following Aβ1-40 treatments, some PC12 cells were apoptotic/dying, and only a few short neurites were observed. Following interventions with natural-cerebrolysin-containing serum, the PC12 cells proliferated, there was an increased number of neurites, and neurite length was enhanced. After middle- and high-dose natural-cerebrolysin treatments, the percentage of neurite-positive cells, as well as the average length of neurites, was significantly greater than the normal blank serum-treated PC12 cells （P 〈 0.05 or P 〈 0.01）. Compared with the blank control group, MAP2 expression in the Aβ1-40-treated PC12 cells was significantly inhibited, and the cell proliferation rate was significantly decreased （P 〈 0.01）. Following incubations with natural-cerebrolysin-containing serum, MAP2 expression and cell proliferation rate in the PC12 cells were significantly increased in a dose-dependent manner, compared with treatments with blank control serum （P 〈 0.05 or P 〈 0.01 ）. CONCLUSION： Natural-cerebrolysin exhibited antagonistic effects on neurotoxicity in Aβ1-40 induced Alzheimer＇s disease in vitro models. These effects were likely related to cell proliferation and the upregulation of intracellular MAP2 expression.

Toward three-dimensional in vitro models to study neurovascular unit functions in health and disease

The high metabolic demands of the brain require an efficient vascular system to be coupled with neural activity to supply adequate nutrients and oxygen.This supply is coordinated by the action of neurons,glial and vascular cells,known collectively as the neurovascular unit,which temporally and spatially regulate local cerebral blood flow through a process known as neurovascular coupling.In many neurodegenerative diseases,changes in functions of the neurovascular unit not only impair neurovascular coupling but also permeability of the blood-brain barrier,cerebral blood flow and clearance of waste from the brain.In order to study disease mechanisms,we need improved physiologicallyrelevant human models of the neurovascular unit.Advances towards modeling the cellular complexity of the neurovascular unit in vitro have been made using stem-cell derived organoids and more recently,vascularized organoids,enabling intricate studies of non-cell autonomous processes.Engineering and design innovations in microfluidic devices and tissue engineering are progressing our ability to interrogate the cerebrovasculature.These advanced models are being used to gain a better understanding of neurodegenerative disease processes and potential therapeutics.Continued innovation is required to build more physiologically-relevant models of the neurovascular unit encompassing both the cellular complexity and designed features to interrogate neurovascular unit functionality.

Lipopolysaccharide mouse models for Parkinson's disease research:a critical appraisal

Parkinson's disease,the most common movement disorder,has a strong neuroinflammatory aspect.This is evident by increased pro-inflammatory cytokines in the serum,and the presence of activated microglial cells,and inflammatory cytokines in the substantia nigra of post-mortem brains as well as cerebrospinal fluid of Parkinson's disease patients.The central and peripheral neuroinflammatory aspects of Parkinson's disease can be investigated in vivo via administration of the inflammagen lipopolysaccharide,a component of the cell wall of gram-negative bacteria.In this mini-review,we will critically evaluate different routes of lipopolysaccharide administration(including intranasal systemic and ste reotasic),their relevance to clinical Parkinson's disease as well as the recent findings in lipopolysaccharide mouse models.We will also share our own expe riences with systemic and intrastriatal lipopolysaccharide models in C57BL/6 mice and will discuss the usefulness of lipopolysaccharide mouse models for future research in the field.

Fabrication of paper-based devices for in vitro tissue modeling

Paper devices have recently attracted considerable attention as a class of cost-effective cell culture substrates for various biomedical applications.The paper biomaterial can be used to partially mimic the in vivo cell microenvironments mainly due to its natural three-dimensional characteristic.The paper-based devices provide precise control over their structures as well as cell distributions,allowing recapitulation of certain interactions between the cells and the extracellular matrix.These features have shown great potential for the development of normal and diseased human tissue models.In this review,we discuss the fabrication of paper-based devices for in vitro tissue modeling,as well as the applications of these devices toward drug screening and personalized medicine.It is believed that paper as a biomaterial will play an essential role in the field of tissue model engineering due to its unique performances,such as good biocompatibility,eco-friendliness,cost-effectiveness,and amenability to various biodesign and manufacturing needs.

Three-dimensional cell culture systems as an in vitro platform for cancer and stem cell modeling

Three-dimensional(3D)culture systems are becoming increasingly popular due to their ability to mimic tissue-like structures more effectively than the monolayer cultures.In cancer and stem cell research,the natural cell characteristics and architectures are closely mimicked by the 3D cell models.Thus,the 3D cell cultures are promising and suitable systems for various proposes,ranging from disease modeling to drug target identification as well as potential therapeutic substances that may transform our lives.This review provides a comprehensive compendium of recent advancements in culturing cells,in particular cancer and stem cells,using 3D culture techniques.The major approaches highlighted here include cell spheroids,hydrogel embedding,bioreactors,scaffolds,and bioprinting.In addition,the progress of employing 3D cell culture systems as a platform for cancer and stem cell research was addressed,and the prominent studies of 3D cell culture systems were discussed.

Lipid accumulation in hepatocytes induces fibrogenic activation of hepatic stellate cells

Despite the initial belief that non-alcoholic fatty liver disease is a benign disorder, it is now recognized that fibrosis progression occurs in a significant number of patients. Furthermore, hepatic steatosis has been identified as a risk factor for the progression of hepatic fibrosis in a wide range of other liver diseases. Here, we established an in vitro model to study the effect of hepatic lipid accumulation on hepatic stellate cells （HSCs）, the central mediators of liver fibrogenesis. Primary human hepatocytes were incubated with the saturated fatty acid palmitate to induce intracellular lipid accumulation. Subsequently, human HSCs were incubated with conditioned media （CM） from steatotic or control hepatocytes. Lipid accumulation in hepatocytes induced the release of factors that accelerated the activation and proliferation of HSC, and enhanced their resistance to apoptosis, largely mediated via activation of the PI-3-kinase pathway. Furthermore, CM from steatotic hepatocytes induced the expression of the profibrogenic genes TGF-β, tissue inhibitor of metallo-proteinase-1 （TIMP-1）, TIMP-2 and matrix-metallo-proteinase-2, as well as nuclear-factor κB-dependent MCP-1 expression in HSC. In summary, our in vitro data indicate a potential mechanism for the pathophysiological link between hepatic steatosis and fibrogenesis in vivo. Herewith, this study provides an attractive in vitro model to study the molecular mechanisms of steatosis-induced fibrogenesis, and to identify and test novel targets for antifibrotic therapies in fatty liver disease.

Hydrophobic surface induced pro-metastatic cancer cells for in vitro extravasation models

In vitro vascularized cancer models utilizing microfluidics have emerged as a promising tool for mechanism study and drug screening.However,the lack of consideration and preparation methods for cancer cellular sources that are capable of adequately replicating the metastatic features of circulating tumor cells contributed to low relevancy with in vivo experimental results.Here,we show that the properties of cancer cellular sources have a considerable impact on the validity of the in vitro metastasis model.Notably,with a hydrophobic surface,we can create highly metastatic spheroids equipped with aggressive invasion,endothelium adhesion capabilities,and activated metabolic features.Combining these metastatic spheroids with the well-constructed microfluidic-based extravasation model,we validate that these metastatic spheroids exhibited a distinct extravasation response to epidermal growth factor(EGF)and normal human lung fibroblasts compared to the 2D cultured cancer cells,which is consistent with the previously reported results of in vivo experiments.Furthermore,the applicability of the developed model as a therapeutic screening platform for cancer extravasation is validated through profiling and inhibition of cytokines.We believe this model incorporating hydrophobic surface-cultured 3D cancer cells provides reliable experimental data in a clear and concise manner,bridging the gap between the conventional in vitro models and in vivo experiments.

Enhancing Parkinson's disease severity assessment through voice-based wavelet scattering,optimized model selection,and weighted majority voting

Parkinson's disease(PD)is a neurodegenerative disorder characterized by motor and non-motor symptoms that significantly impact an individual's quality of life.Voice changes have shown promise as early indicators of PD,making voice analysis a valuable tool for early detection and intervention.This study aims to assess and detect the severity of PD through voice analysis using the mobile device voice recordings dataset.The dataset consisted of recordings from PD patients at different stages of the disease and healthy control subjects.A novel approach was employed,incorporating a voice activity detection algorithm for speech segmentation and the wavelet scattering transform for feature extraction.A Bayesian optimization technique is used to fine-tune the hyperparameters of seven commonly used classifiers and optimize the performance of machine learning classifiers for PD severity detection.AdaBoost and K-nearest neighbor consistently demonstrated superior performance across various evaluation metrics among the classifiers.Furthermore,a weighted majority voting(WMV)technique is implemented,leveraging the predictions of multiple models to achieve a near-perfect accuracy of 98.62%,improving classification accuracy.The results highlight the promising potential of voice analysis in PD diagnosis and monitoring.Integrating advanced signal processing techniques and machine learning models provides reliable and accessible tools for PD assessment,facilitating early intervention and improving patient outcomes.This study contributes to the field by demonstrating the effectiveness of the proposed methodology and the significant role of WMV in enhancing classification accuracy for PD severity detection.

阿尔茨海默病“肾虚酿毒伤髓”病机演变模型构建及其应用

目的 根据肾-骨髓-血细胞-脑髓相互影响的中西医理论,建立一种阿尔茨海默病(Alzheimer′s disease, AD)“肾虚酿毒伤髓”模型,筛查AD有效中药,并初步探讨该模型的临床应用。方法 由于AD患者B淋巴细胞可以促进神经细胞产生Aβ毒素。将肾虚型AD患者外周血提取B淋巴细胞永生化,建立淋巴母细胞系(lymphoblastoid cell lines, LCL),并与人神经上皮瘤细胞(SKNMC)共培养,产生Aβ毒素,制成阿尔茨海默病“肾虚酿毒伤髓”模型。通过免疫荧光法检测Aβ_(1-42)的表达量,筛查有抑制Aβ_(1-42)生成的中药。并初步探讨该模型的临床应用。结果 与正常对照组相比,肾虚型AD模型中神经细胞Aβ_(1-42)的生成明显增多,具有统计学意义(P<0.01)。与肾虚模型组相比,白术、人参、黄芪、生地黄、梓醇减少了神经细胞Aβ_(1-42)的生成,差异具有统计学意义(P<0.01)。结论 白术、人参、黄芪、生地黄、梓醇可以减少AD中Aβ_(1-42)的生成。该模型除了筛查有效中药外,可以作为AD患者“肾虚酿毒”严重程度的生物标记物,及评价中药干预的疗效指标。

Functional Connectivity-Based Modelling Simulates SubjectSpecific Network Spreading Effects of Focal Brain Stimulation

Neurostimulation remarkably alleviates the symptoms in a variety of brain disorders by modulating the brain-wide network. However, how brain-wide effects on the direct and indirect pathways evoked by focal neurostimulation elicit therapeutic effects in an individual patient is unknown. Understanding this remains crucial for advancing neural circuit-based guidance to optimize candidate patient screening, pre-surgical target selection, and post-surgical parameter tuning. To address this issue, we propose a functional brain connectome-based modeling approach that simulates the spreading effects of stimulating different brain regions and quantifies the rectification of abnormal network topology in silico. We validated these analyses by pinpointing nuclei in the basal ganglia circuits as top-ranked targets for 43 local patients with Parkinson’s disease and 90 patients from a public database. Individual connectome-based analysis demonstrated that the globus pallidus was the best choice for 21.1% and the subthalamic nucleus for 19.5% of patients. Down-regulation of functional connectivity(up to 12%) at these prioritized targets optimally maximized the therapeutic effects. Notably, the priority rank of the subthalamic nucleus significantly correlated with motor symptom severity(Unified Parkinson’s Disease Rating Scale III) in the local cohort. These findings underscore the potential of neural network modeling for advancing personalized brain stimulation therapy,and warrant future experimental investigation to validate its clinical utility.

阿尔茨海默病发病机制及体内外模型研究进展

阿尔茨海默病(AD)是一种以认知功能障碍为主要特征的神经退行性疾病,具体发病机制目前尚不清晰。随着我国人口老龄化越来越严重,患有AD人群的比例越来越高,亟需深入探究其发病机制并建立合适的疾病模型。本文从当前AD的发病假说和体内外模型进行了综述,详细介绍了神经细胞β-淀粉样前体蛋白(APP)转基因模型、D-半乳糖(D-gal)模型、D-半乳糖联合β-淀粉样蛋白(Aβ)模型等10种体内模型和3种体外模型(HT22细胞模型、PC12细胞模型和SH-SY5Y细胞模型),以期为AD疾病的基础研究和新药开发提供模型参考和研究思路。

体外功能性肺组织模型构建、调控及在肺纤维化和COVID-19中的应用

肺纤维化是很多肺部疾病发生发展过程中出现的病理现象。近年出现的2019冠状病毒病(coronavirus disease 2019,COVID-19)引发的呼吸系统综合征也会出现弥漫性肺泡损伤,并诱发肺纤维化。因此,开展肺纤维化和COVID-19体外模型构建与调控研究在肺部疾病治疗和药物筛选方面意义重大。目前,现有研究已建立了多种体外肺组织二维、三维细胞培养模型,本文将全面概述这些模型的构建方法,并结合这些模型在肺纤维化及COVID-19中的应用研究,对体外肺组织模型在药物传递、高通量药物筛选及发病机制研究等生物医学领域中的应用前景进行综述,为其进一步研究提供参考。

An Efficient WRF Framework for Discovering Risk Genes and Abnormal Brain Regions in Parkinson's Disease Based on Imaging Genetics Data

As an emerging research field of brain science,multimodal data fusion analysis has attracted broader attention in the study of complex brain diseases such as Parkinson's disease(PD).However,current studies primarily lie with detecting the association among different modal data and reducing data attributes.The data mining method after fusion and the overall analysis framework are neglected.In this study,we propose a weighted random forest(WRF)model as the feature screening classifier.The interactions between genes and brain regions are detected as input multimodal fusion features by the correlation analysis method.We implement sample classification and optimal feature selection based on WRF,and construct a multimodal analysis framework for exploring the pathogenic factors of PD.The experimental results in Parkinson's Progression Markers Initiative(PPMI)database show that WRF performs better compared with some advanced methods,and the brain regions and genes related to PD are detected.The fusion of multi-modal data can improve the classification of PD patients and detect the pathogenic factors more comprehensively,which provides a novel perspective for the diagnosis and research of PD.We also show the great potential of WRF to perform the multimodal data fusion analysis of other brain diseases.

雷公藤红素醇质体的制备及体外透皮性能研究

【目的】制备雷公藤红素(Cel)醇质体,并考察醇质体作为雷公藤红素经皮给药载体的渗透特性。【方法】采用乙醇注入法制备雷公藤红素醇质体,并对其包封率、粒径、多分散指数(PDI)及Zeta电位进行分析;采用TP2A型智能透皮试验仪进行体外透皮试验,比较雷公藤红素醇质体、空白醇质体/Cel溶液和Cel溶液的透皮行为。【结果】此方法制备的雷公藤红素醇质体为类球形结构,平均粒径为(401.3±5.5)nm,PDI为(0.21±0.02),Zeta电位为(-2.75±0.1)m V,平均包封率为(80.6±0.7)%;醇质体48 h的累积透过量76.86μg·cm-2,渗透速率为1.640 9μg·cm-2·h-1,与空白醇质体/Cel溶液和Cel溶液比较,均有显著提高。【结论】雷公藤红素醇质体的包封率较高,粒径分布均匀,质量稳定,且可显著促进雷公藤红素的透皮吸收。

牛血清白蛋白与血管性疾病

牛血清白蛋白是一种球蛋白,目前已成功运用于血管性疾病实验动物模型的诱导及体外实验中。本文就牛血清白蛋白在血管性疾病中应用的相关机理进行综述。

吸烟相关疾病模型的研究进展

吸烟对呼吸系统、消化系统、生殖系统、感官系统等多系统造成危害,可导致各种疾病的发生、发展。为了研究烟草与人类疾病的相关性,吸烟相关疾病模型的建立及完善越来越受到人们的关注,目前吸烟相关疾病模型主要包括体外吸烟细胞模型、动物吸烟模型和临床样本吸烟模型3类。本文就吸烟疾病模型的发展过程和研究进展作一综述。

影响再生育继发不孕妇女体外受精——胚胎移植临床妊娠结局的多因素分析

目的：比较再生育继发不孕妇女行体外受精—胚胎移植（IVF－ET）后不同临床妊娠结局的影响因素，评估各影响因素对临床妊娠结局的相对重要性，为妇女再生育指导提供科学依据。方法：回顾性分析2012年7月至2014年7月浙江大学医学院附属妇产科医院因再生育继发不孕行IVF－ET的1099例妇女1129个IVF－ET治疗周期的临床、实验室及随访资料。按是否临床妊娠分为两组，通过单因素分析和二元logistic回归分析研究再生育继发不孕妇女行IVF－ET治疗临床妊娠结局的影响因素；比较各年龄阶段不同胚胎移植数的临床妊娠结局；并分析比较促性腺激素释放激素（ GnRH ）激动剂长方案、GnRH激动剂短方案、GnRH拮抗剂方案等3种常用控制性超促排卵（ COH ）方案治疗≥40岁高龄再生育继发不孕妇女的助孕效果。结果：1129个治疗周期中临床妊娠376例次（33．30％），未临床妊娠753例次（66．70％），这两组女方年龄、女方体质量指数、基础卵泡刺激素、窦卵泡数、移植胚胎数和男方年龄差异有统计学意义（均P＜0．05）。二元logistic回归分析发现年龄（ OR ＝0．900，95％CI：0．873-0．928， P ＜0．001）、移植胚胎数（ OR＝2．248，95％CI：1．906-2．652， P＜0．001）是再生育继发不孕妇女行IVF－ET治疗临床妊娠是否成功的独立影响因素。30-40岁移植双胚胎与＜30岁移植多胚胎妇女的临床妊娠率差异无统计学意义（ P＞0．05）。年龄≥40岁再生育继发不孕妇女应用GnRH激动剂长方案、GnRH激动剂短方案和GnRH拮抗剂方案三组妇女临床妊娠率差异无统计学意义（ P＞0．05）。结论：年龄和胚胎移植数是预测再生育继发不孕妇女行IVF－ET治疗临床妊娠结局的独立影响因素。30-40岁妇女建议移植胚胎数不超过2个。年龄≥40岁再生育继发不孕妇女的临床妊娠率显著降低，应用GnRH激动剂长方案、GnRH激动剂短方案、GnRH拮抗剂方案对其临床妊娠率无明显影响。

游离脂肪酸诱导小鼠肝实质细胞体外脂肪化并加速细胞凋亡

目的建立小鼠非酒精性脂肪肝病(Non-alcoholic fatty liver disease,NAFLD)体外细胞模型,为进一步体外药物高通量的筛选以及深入探讨NAFLD发生发展的分子机制奠定基础。方法胶原酶灌注分离并培养小鼠原代肝实质细胞,随后给予不同浓度的游离脂肪酸(free fatty acids,FFA)刺激,进行油红染色以及三酰甘油测定;通过MTS法以及流式细胞术分析游离脂肪酸对肝实质细胞增殖以及细胞凋亡的影响,使用Western印迹法检测肝实质细胞内的MAPK相关信号通路的变化,实时定量PCR检测细胞中bim与fas的表达。结果肝实质细胞内的脂质累积程度随加入的FFA浓度上升而呈明显增加(P<0.05);肝实质细胞的细胞增殖受到明显抑制,细胞凋亡明显增加,肝实质细胞内JNK信号通路随刺激时间延长强度增加,ERK信号通路随刺激时间延长强度减弱,细胞内bim和fas的表达量上升。结论成功建立了非酒精性脂肪肝病的体外细胞模型,能够很好地模拟体内肝实质细胞内脂肪累积以及细胞凋亡现象。