Name: microglia as a surrogate biosensor to determine nanoparticle neurotoxicity
Uploaded: 2016-10-25T14:00:00.000+00:00
Duration: 8 min 37 s
Description: Watch this Scientific Journal Video about Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity at JoVE.com

This work was funded by the US Department of Veterans Affairs BLR&amp;D IK2 BX001686 (to TAB), and grants from the University of Minnesota Healthy Foods, Healthy Lives Institute (to CMD, JPN, and TAB) and the Minnesota Veterans Medical Research &amp; Education Foundation (to TAB). We thank Drs. Philippe Marambaud (Feinstein Institute for Medical Research, Manhasset, NY) and Weihua Zhao (Methodist Hospital, Houston, TX) for providing the BV2 cell line.

1. 소교 세포 배양 유지 보수 <ol><li> 따뜻한 세포 배양 배지 (둘 베코 변형 이글 중간; DMEM) 37 ° C로 10 % 소 태아 혈청 (FBS) 및 1 % 페니실린 / 스트렙토 마이신 / 네오 마이신 (PSN)로 보충. </li><li> -80 ° C에서 저장 25 - 통로 (18)에 BV2 소교 세포의 냉동 주식을 얻습니다. 빠르게 37 ° C의 물을 욕조에 세포를 해동. </li><li> 조심스럽게 10 ㎖ 세포 배양 배지를 포함하는 75cm 2 배기 플라스크에 세포를 옮긴다. </li><li> 37 ° C에서 5 % CO 2에서 플라스크를 품어. 기음 미디어 24 시간 후 신선한 용지로 교체합니다. 80 % 컨 플루 - 약 70 때까지 세포를 성장. </li></ol> 2. 계수 및 도금 세포 <ol><li> 37 ° C의 물을 욕조, 따뜻한 DMEM과 1 배 - 트립신 EDTA 용액합니다. </li><li> 세포에서 대기음 매체와 트립신의 500 μl를 추가합니다. 5 분 - 2 37 ° C에서 품어. </li><li> 스크레이퍼를 사용하여 플라스크로부터 세포를 제거하고, 5 ml의 정지DMEM의. 50 ML 튜브에 세 번 70 μm의 스트레이너를 통해 세포를 전달합니다. </li><li> 혈구 세포를 사용하여 계산하고, 10 % FBS, 1 %의 PSN이 보충 된 DMEM을 200㎕의 최종 부피에서 선명한 바닥 판 성벽 / 웰 블랙 8000 세포를 시드. 24 시간 동안 37 ° C에서 5 % CO 2 판을 품어. </li><li> 오래 된 DMEM를 제거하고 미세 아교 세포를 굶어 혈청 1 %의 PSN 보충 따뜻하게 DMEM 200 μL로 교체합니다. 24 시간 동안 37 ° C에서 5 % CO 2 판을 품어. </li></ol> 3. 활성화 미세 아교 세포 <ol><li> 별도의 튜브에 AGNPS 희석 (0.01, 0.05, 0.1 μg의 / mL) 및 비히클 / 중립 제어 최종 작업 농도 1 % PSN이 보충 된 무 혈청 DMEM에서 (구연산 나트륨, 0.04 mm)이다. </li><li> 각 우물에서 미디어 100 μl를 제거하고 적절한 치료 화합물 100 ㎕로 교체합니다. </li><li> 24 시간 동안 37 ° C에서 5 % CO 2 판을 품어. </li></ol> 4. 인터넷금연실 미디어 ltering <ol><li> 각에서 미디어 상층 액 200 μl를 잘 수집하고 수집 관 (1.5-2 ml의 용량)와 필터 (10 kDa의 분자량 차단)로 전송합니다. </li><li> 실온에서 15 분 동안 14,000 × g으로 원심 분리기. </li><li> 플로우를 통해 (입자 상층 액)을 버리고 새 컬렉션 튜브에 필터 거꾸로 놓습니다. </li><li> 2 분 동안 실온에서 1,000 XG에 원심 분리기. 참고 : 결과 필터링 미디어는 (분비되는 사이토 카인을 포함) 6 배 농축한다. </li><li> 얼음에 10 % FBS와 1 % PSN 저장 보충 된 신선한 DMEM 400 ㎕의 농축액의 양을 준비한다. 주 : - 420 내지 약 10 nm (390)에서 특징적인 피크에 근거 AgNP 농도, 잔류 AGNPS 여과 매체로부터 제거되도록 생성한다. (3.1 단계에 설명 된대로 0, 0.01, 0.025, 0.05, 0.1, 0.2 ㎍ / ㎖) 및 여과 요약하면, 필터링되지 않은 AGNPS의 분취 량을 준비차량 제어 무 혈청 DMEM에서 (구연산 나트륨, 0.04 mm)이다. 나누어지는 검은 색으로 필터링 된 시료와 표준의 50 μl를 명확 바닥 판을 벽으로 둘러싸인. 분광 광도계를 사용하여 390에서의 흡광도를 측정 - 410 nm의 상기 AgNP 농도 곡선 판독을 비교한다. 필터링 된 샘플은 차량 제어 샘플과 동일한 흡광도 값을 갖는 경우, 잔여 AGNPS 제거한 것으로 가정 할 수있다. </li><li> 시중에서 판매하는 키트에서 TNF-α 분비 다음 지시 사항을 결정하기 위해 여과 매체의 절반을 사용합니다. </li></ol> 5. 시상 하부 세포 배양 유지 보수 <ol><li> 따뜻한 DMEM 세포 배양 배지는 37 ° C로 10 % FBS와 1 % PSN 보충. </li><li> 80 ° C에서 보관 25 - 시상 하부의 냉동 재고 (mHypo-A1 / 2) 통로 (18)에 세포를 얻습니다. 빠르게 37 ° C의 물을 욕조에 세포를 해동. </li><li> 조심스럽게 10 ㎖ 세포 배양 배지를 포함하는 75cm 2 배기 플라스크에 세포를 옮긴다. </li><li> CO 2 5 %에 플라스크를 품어 </s37 ° C에서 UB&gt;. 기음 미디어 24 시간 후 신선한 용지로 교체합니다. 80 % 컨 플루 - 약 70 때까지 세포를 성장. </li></ol> 6. 확인 시상 하부의 세포 독성 <ol><li> 따뜻한 DMEM과 37 ° C의 물을 욕조에 1 배 - 트립신 EDTA 솔루션입니다. </li><li> 시상 하부 세포에서 대기음 매체와 트립신의 500 μl를 추가합니다. 5 분 - 2 37 ° C에서 품어. 2 단계에서 상술 한 바와 같이 스크레이퍼를 사용하여 플라스크에서 세포를 제거합니다. </li><li> 혈구를 이용하여 세포 수를 계산하고, 10 % FBS, 1 % PSN 보충을 200㎕ DMEM 최종 부피에 클리어 바닥 판 성벽 / 웰 블랙 5000 세포에서 시상 세포 플레이트 및 37 ℃에서 5 % CO 2에서 하룻밤 부화 . </li><li> 멀티 채널 피펫을 사용하여 이전 미디어 100 ㎕를 제거하고 1 배의 최종 농도로 여과하고, 농축 된 배지 100 ㎕를 추가한다. </li><li> 24 시간 동안 37 ° C에서 5 % CO 2 판을 품어. </li><li> 22 추가1; 레사 주린 시약 L 및 37 ℃에서 5 % CO 2에서 20 분 동안 플레이트를 배양한다. </li><li> 멀티 모드 분광 광도계, 기록 형광 (560 nm의 EX / 590 나노 EM)을 사용하여 세포 생존율을 측정합니다. 상대 형광 단위 (RFU) 등의 결과를보고합니다. </li></ol>

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The authors have no conflicts to disclose.

Recent studies support that environmental exposure contributes to obesity and other neurodegenerative diseases 11,12. However, techniques used in previous studies are time consuming and expensive. Economic considerations, physiologically relevant delivery systems, ethical issues with extensive use of in vivo animal models, and difficulty translating findings into meaningful health advisories are a few of the major challenges that have impeded advancements in studying NP-induced neurotoxicity 13</...

환경 오염은 특히 나노 입자 (NP)의 범위들 (1-20 nm의 직경), 비만 및 혈액 뇌 장벽 1-3을 통과 할 수있는 능력으로 인해 다른 신경 퇴행성 질환과 관련이있다. 오염에 대한 높은 노출은 시상 하나를 포함하는 중추 신경계의 염증을 유발할 수있다. 이 미세 아교 세포의 나노 입자 유도 활성화 (뇌 면역 세포) (4)를 통해 할 수 발생하는 하나의 잠재적 인 메커니즘. 선행 연구는 시간 소모, 고가 뇌의 건강에 NP에의 영향을 연구하기 위해 생체 내 모델에서 사용한 직접 NPS에서 미세 아교 세포에 영향을 미치는 방법에 대한 질문에 응답하지 않는다. 미세 아교 세포는 뇌의 미세 환경의 유지를 포함하여 분비 인자 및 사이토 카인의 방출을 통해 신경 주변 통신, 중추 신경계 다각적 역할을한다. 자극에 따라, 미세 아교 세포는 M1 홍보에 활성화 될 수있다오 염증 또는 M2 항 염증 상태. M2는 인터루킨 -4 (IL-4)를 포함하여 미세 아교 세포 분리 항 염증성 사이토 카인으로 활성화 예를 들어, M1은, 미세 아교 세포는 종양 괴사 인자 알파 (TNF-α)와 같은 염증성 사이토 카인 방출 활성화. 대기 오염 물질의 신경 독성을 결정하기위한 시험 관내 바이오 센서 우리의 대리를 확인하기 위해, 우리는 20 nm의 나노 입자 (AGNPS)에 소교 반응을 측정했다. 이 문서의 목적은 시험 관내 소교 세포주가 NP에에 쥐 소교 반응을 테스트하는 방법과 미세 아교 활성화가 시상 하부 세포에 영향을 미치는위한 대리 바이오 마커로 사용하는 방법을 설명하는 것입니다. 장기는이 검증 된 모델의 응용 프로그램이 뇌 건강과 신경 퇴행성 질환에 대한 실제 오염 물질의 효과를 테스트하는 것입니다 예정. 우리는 마이크의 노출 후 미세 아교 활성화 및 시상 세포 생존율을 측정하기위한 시험 관내 96 웰 형식 분석의 상세한 설명을 제공한다<html> 에어컨 미디어를 roglial. 소교 활성화는 (ELISA)을 면역 측정법 링크 된 TNF-α의 효소를 사용하여 노광 AgNP 따라 결정 하였다. 시상 세포에서 활성화 된 미세 아교 세포의 효과를 결정하기 위해 AGNPS는 여과 장치를 이용하여 상층 액 소교 (조정 배지)에서 제거 하였다. 크기에 따라 AGNPS을 배제하면서 여과 장치 사이토킨을 유지한다. 간단히, AGNPS 또는 처리없이 미세 아교 세포의 상등액을 수거 필터에 첨가하고, 15 분 동안 14,000 × g으로 원심 분리 하였다. 그런 다음 시상 세포 생존에 소교 분비 된 사이토 카인의 영향을 확인할 수 있었다. 이전 5,6 설명 된대로 에어컨 미디어 (포함하는 사이토 카인)에 노출 된 후 세포 독성은 레사 주린 기반 분석을 통해 측정 하였다. 대사 적 활성 세포 레사 주린을 줄이고 생세포 (7)의 수에 비례하여 형광 신호를 생성한다. NT &quot;&gt; (예, 또는 생체 내 실험에서 공동 배양 트랜스 웰 설정 등) 등을 통해이 기술을 사용하여 여러 가지 이점이있다. 우리의 모델은 직접적으로 미세 아교 세포 활성화 및 분비 인자 뉴런 8 독성 여부를 결정하는 능력을 제공한다 . 현재의 프로토콜은 불후의 BV2의 미세 아교 세포는 쥐의 시상 하부 세포를이 프로토콜은 이러한 특정 조건에 최적화되었지만 (지정하는 mHypo-A1 / 2) 이후의 반응의 결정에 대한 9., 방법이 될 수 있습니다 20 나노 미터 직경의 나노 입자로 자극하고, 불멸 사용합니다 변경된은 소교에 의한 세포 사멸의 다른 모델에 사용, 또는 기본 미세 아교 세포와 신경 세포를 포함한 다른 세포 유형으로합니다.

<table><tbody><tr><td>&lt;strong&gt;Cells/Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Mouse microglial cell line (BV2)</td><td>Interlab Cell Line Collection (Genoa, Italy)</td><td>ATL03001</td><td></td></tr><tr><td>Adult Mouse Hypothalamus Cell Line mHypoA-1/2&amp;nbsp;</td><td>Cellutions Biosystems Inc.</td><td>CLU172</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium</td><td>Invitrogen</td><td>10313-039</td><td></td></tr><tr><td>Fetal bovine serum&amp;nbsp;</td><td>PAA Labs</td><td>A15-751</td><td></td></tr><tr><td>Penicillin/Streptomycin/Neomycin</td><td>Thermo Fisher Scientific</td><td>15640-055</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Thermo Fisher Scientific</td><td>25200056</td><td></td></tr><tr><td>Silver nanoparticles (20 nm)</td><td>Sigma-Aldrich</td><td>730793</td><td></td></tr><tr><td>&lt;br/&gt; PrestoBlue Cell Viability Reagent</td><td>Invitrogen</td><td>A13262</td><td></td></tr><tr><td>Mouse TNF-&amp;alpha; ELISA Max Delux</td><td>Biolegend</td><td>430904</td><td></td></tr><tr><td>Lipopolysaccharide</td><td>Sigma-Aldrich</td><td>L4391</td><td></td></tr><tr><td>Sodium Citrate</td><td>Sigma-Aldrich</td><td>S4641</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>96 W Optical Bottom Plate, Black Polystyrene, Cell Culture Treated, with lid, Sterile</td><td>Thermo Fisher Scientific</td><td>165305</td><td></td></tr><tr><td>Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-10 membrane</td><td>EMD Millipore</td><td>UFC501008</td><td></td></tr><tr><td>SpectraMax M5 Multi-Mode Microplate</td><td>Molecular Devices</td><td>M5</td><td></td></tr><tr><td>Falcon 50 ml Conical Centrifuge Tubes</td><td>Corning, Inc</td><td>&lt;br/&gt; 14-432-22</td><td></td></tr><tr><td>Falcon Cell Strainers 70 &amp;mu;m</td><td>Corning, Inc</td><td>08-771-2</td><td></td></tr><tr><td>Tabletop centrifuge 5430</td><td>Eppendorf</td><td>22620560</td><td></td></tr></tbody></table>

microglia as a surrogate biosensor to determine nanoparticle neurotoxicity

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay described here will aid in elucidating the role of microglia in neuroinflammation and neurodegenerative diseases. The use of microglia, resident immune cells of the brain, as a surrogate biosensor provides novel insight into how inflammatory responses mediate neuronal insults. Here, we utilize an immortalized murine microglial cell line, designated BV2, and describe a method for nanoparticle exposure using silver nanoparticles (AgNPs) as a standard. We describe how to expose microglia to nanoparticles, how to remove nanoparticles from supernatant, and how to use supernatant from activated microglia to determine toxicity, using hypothalamic cell survival as a measure. Following AgNP exposure, BV2 microglial activation was validated using a tumor necrosis factor alpha (TNF-&#945;) enzyme linked immunosorbent assay (ELISA). The supernatant was filtered to remove the AgNP and to allow cytokines and other secreted factors to remain in the conditioned media. Hypothalamic cells were then exposed to supernatant from AgNP activated microglia and survival of neurons was determined using a resazurin-based fluorescent assay. This technique is useful for utilizing microglia as a surrogate biomarker of neuroinflammation and determining the effect of neuroinflammation on other cell types.

우리는 위의 프로토콜을 사용하여 나노 입자에 대한 뇌의 반응에 대한 대리 바이오 센서 등이 미세 아교 세포의 기능을 보여줍니다. 우리의 결과는 다운 스트림 신경 세포 사멸에 소교 활성화의 독성 효과의 측정을 포함한다. (1) 미세 아교 세포를 활성화하고 분비되는 사이토 카인은 시상 하부 신경 세포의 생존을 줄일 수 있는지를 확인하는 프로토콜의 워크...

Watch this Scientific Journal Video about Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity at JoVE.com

Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity

Microglia (immune cells of the brain), are used as a surrogate biosensor to determine how nanoparticles influence neurotoxicity. We describe a series of experiments designed to assay microglial response to nanoparticles and exposure of hypothalamic neurons to supernatant from activated microglia to determine neurotoxicity.

나노 입자 신경 독성을 결정하는 대리 바이오 센서로 미세 아교 세포

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay ...

microglia-as-surrogate-biosensor-to-determine-nanoparticle

Research

JoVE Journal

Neuroscience

6.9K 조회수.  Minneapolis Veterans Affairs Health Care System. Microglia (immune cells of the brain), are used as a surrogate biosensor to determine how nanoparticles influence neurotoxicity. We describe a series of experiments designed to assay microglial response to nanoparticles and exposure of hypothalamic neurons to supernatant from activated microglia to determine neurotoxicity.

비디오: Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring 

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end organs. The latter include the lung, RES, and brain and are operative during human immunodeficiency virus type one (HIV-1) infection. Macrophage entry into tissues is notable in areas of active HIV-1 replication and sites of inflammation. In order to assess the potential of macrophages as nanocarriers, superparamagnetic iron-oxide and/or drug laden particles coated with surfactants were parenterally injected into HIV-1 encephalitic mice. This was done to quantitatively assess particle and drug biodistribution. Magnetic resonance imaging (MRI) test results were validated by histological coregistration and enhanced image processing. End organ disease as typified by altered brain histology were assessed by MRI. The demonstration of robust migration of nanoformulations into areas of focal encephalitis provides '"proof of concept" for the use of advanced bioimaging techniques to monitor macrophage migration. Importantly, histopathological aberrations in brain correlate with bioimaging parameters making the general utility of MRI in studies of cell distribution in disease feasible. We posit that using such methods can provide a real time index of disease burden and therapeutic efficacy with translational potential to humans.

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Bioimaging methods used to assess cell biodistribution of nanoparticles are applicable for therapeutic and diagnostic monitoring of nanoformulated compounds. The methods described herein are sensitive and specific when assessed by histological coregistration. The methodologies provide a translational pathway from rodent to human applications.

진단 및 치료 모니터링을위한 Nanomaterials 등록 Bioimaging

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end ...

연구

생체공학

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

일반 CNS의 실시간 PCR에 의한과 Neuroinflammation 동안 Microglia에 MicroRNAs의 탐지

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

면역학 및 감염병학

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure for isolation of microglia from neonatal murine cortices by mechanical agitation with a rotary shaker. This microglia isolation method yields highly pure cortical microglia that exhibit morphological and functional characteristics indicative of quiescent microglia in normal, nonpathological conditions in vivo. This procedure also preserves the microglial immunophenotype and biochemical functionality as demonstrated by the induction of morphological changes, nuclear translocation of the p65 subunit of NF-&#954;B (p65), and secretion of the hallmark proinflammatory cytokine, tumor necrosis factor-&#945; (TNF-&#945;), upon lipopolysaccharide (LPS) and Pam3CSK4 (Pam) challenges. Therefore, the present isolation procedure preserves the immunophenotype of both quiescent and activated microglia, providing an experimental method of investigating microglia biology in ex vivo conditions.

One key to successful investigation of microglial biology is the preservation of microglial immunofunction ex vivo during isolation from CNS tissue. Isolating microglia via rotary shaking results in highly pure and immunofunctional cell cultures as assessed by fluorescent imaging, immunocytochemistry, and ELISA following microglia activation with the proinflammatory stimuli lipopolysaccharide (LPS) and Pam3CSK4 (Pam).

쥐과 신생아에서 보존 면역 표현형과 기능으로 두피 소교 세포의 분리

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure ...

An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and subsequently digests this material, commonly through lysosomal degradation. Microglia are the resident immune cells of the central nervous system (CNS) whose phagocytic function has been described in a broad range of conditions from neurodegenerative disease (e.g., beta-amyloid clearance in Alzheimer&#8217;s disease) to development of the healthy brain (e.g., synaptic pruning)1-6. The following protocol is an engulfment assay developed to visualize and quantify microglia-mediated engulfment of presynaptic inputs in the developing mouse retinogeniculate system7. While this assay was used to assess microglia function in this particular context, a similar approach may be used to assess other phagocytes throughout the brain (e.g., astrocytes) and the rest of the body (e.g., peripheral macrophages) as well as other contexts in which synaptic remodeling occurs (e.g. ,brain injury/disease).

Microglia are the resident immune cells of the central nervous system (CNS) with a high capacity to phagocytose or engulf material in their extracellular environment. Here, a broadly applicable, reliable, and highly quantitative assay for visualizing and measuring microglia-mediated engulfment of synaptic components is described.

말림 분석 : CNS 식세포와 신경 세포 사이의 상호 작용을 평가하기위한 프로토콜

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and ...

신경과학

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

망막 미세 아교 식세포의 기능을 평가<em&gt; 생체</em&gt;은 유동 세포 계측법 기반 분석을 사용하여

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders.
This protocol outlines methods for long-term, in vivo imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1GFP/+ reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200-300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in microglial activation and microgliosis during early stages of retinal neurodegeneration in a mouse model of chronic glaucoma. This approach should be useful to investigate the contributions of microglia to neuronal and axonal decline in chronic CNS disorders that affect the retina and optic nerve.

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia activation and microgliosis are key responses to chronic neurodegeneration. Here, we present methods for in vivo, long-term visualization of retinal CX3CR1-GFP+ microglial cells by confocal ophthalmoscopy, and for threshold and morphometric analyses to identify and quantify their activation. We monitor microglial changes during early stages of age-related glaucoma.

마우스 녹내장에 공 촛점 안저 이미징 및 세포 형태 계측 : 신경 퇴행 중 망막 미세 아교 활성화의 생체 역학

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with ...

의학

Rapid and Refined CD11b Magnetic Isolation of Primary Microglia with Enhanced Purity and Versatility

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. Microglia are mesodermal cells that function similarly to macrophages in surveying inflammatory stress. The classical (M1-type) and alternative (M2-type) activations of macrophages have also been extended to microglia in an effort to better understand the underlying interplay these phenotypes have in neuroinflammatory conditions such as Parkinson&#39;s, Alzheimer&#39;s, and Huntington&#39;s Diseases. In vitro experimentation utilizing primary microglia offers rapid and reliable results that may be extended to the in vivo environment. Although this is a clear advantage over in vivo experimentation, isolating microglia while achieving adequate yields of optimal purity has been a challenge. Common methods currently in use either suffer from low recovery, low purity, or both. Herein, we demonstrate a refinement of the column-free CD11b magnetic separation method that achieves a high cell recovery and enhanced purity in half the amount of time. We propose this optimized method as a highly useful model of primary microglial isolation for the purposes of studying neuroinflammation and neurodegeneration.

Here, we present a protocol to isolate microglia from postnatal mouse pups (day 1) for in vitro experimentation. This improvised method of isolation generates both high yield and purity, a significant advantage over alternate methods that allows broad range experimentation for the purposes of elucidating microglial biology.

향상된 순도와 다양성에 차 미세 아교 세포의 신속하고 세련된 CD11b를 자기 절연

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. ...

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has revealed microglia to be dynamic, capable of assuming both pro-inflammatory and anti-inflammatory phenotypes. Both M1 (pro-inflammatory) and M2 (pro-reparative) phenotypes play an important role in neuroinflammatory conditions such as perinatal brain injury, and exhibit differing functions in response to certain environmental stimuli. The modulation of microglial activation has been noted to confer neuroprotection thus suggesting microglia may have therapeutic potential in brain injury. However, more research is required to better understand the role of microglia in disease, and this protocol facilitates that. The protocol described below combines a density gradient centrifugation process to reduce cellular debris, with magnetic separation, producing a highly pure sample of primary microglial cells that can be used for in vitro experimentation, without the need for 2-3 weeks culturing. Additionally, the characterization steps yield robust functional data about microglia, aiding studies to better our understanding of the polarization and priming of these cells, which has strong implications in the field of regenerative medicine.

A protocol for the isolation of primary microglia from murine brains is presented. This technique aids in furthering the current understanding of neurological conditions. Density gradient centrifugation and magnetic separation are combined to produce sufficient yield of a highly pure sample. Furthermore, we outline the steps for characterization of microglia.

특성화 및 조밀도 기온 변화도 원심 분리 하 여 마우스 기본 Microglia의 절연

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has ...

시냅스 물질의 미세아교세포 삼킴을 위한 유세포분석 기반 분석

Quantification of Microglial Engulfment of Synaptic Material Using Flow Cytometry

Microglia play a pivotal role in synaptic refinement in the brain. Analysis of microglial engulfment of synapses is essential for comprehending this process; however, currently available methods for identifying microglial engulfment of synapses, such as immunohistochemistry (IHC) and imaging, are laborious and time-intensive. To address this challenge, herein we present in vitro and in vivo* assays that allow fast and high-throughput quantification of microglial engulfment of synapses using flow cytometry.
In the in vivo* approach, we performed intracellular vGLUT1 staining following fresh cell isolation from adult mouse brains to quantify engulfment of vGLUT1+ synapses by microglia. In the in vitro synaptosome engulfment assay, we used freshly isolated cells from the adult mouse brain to quantify the engulfment of pHrodo&#160;Red-labeled synaptosomes by microglia. These protocols together provide a time-efficient approach to quantifying microglial engulfment of synapses and represent promising alternatives to labor-intensive image analysis-based methods. By streamlining the analysis, these assays can contribute to a better understanding of the role of microglia in synaptic refinement in different disease models.

저자 스포트라이트: 정신신경면역학의 기술 혁신을 통한 이해 증진

Here we present two protocols to quantify microglial engulfment of vGLUT1-positive synapses and pHRodo&#160;Red-labeled crude synaptosomes using flow cytometry.

유세포분석을 사용한 시냅스 물질의 미세아교세포 삼킴 정량화

Microglia play a pivotal role in synaptic refinement in the brain. Analysis of microglial engulfment of synapses is essential for comprehending this ...

나노 입자 신경 독성을 결정하는 대리 바이오 센서로 미세 아교 세포

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특성화 및 조밀도 기온 변화도 원심 분리 하 여 마우스 기본 Microglia의 절연

유세포분석을 사용한 시냅스 물질의 미세아교세포 삼킴 정량화

유세포분석을 사용한 시냅스 물질의 미세아교세포 삼킴 정량화