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. Mikroglial Hücre Kültürü Bakım <ol><li> Sıcak hücre kültürü ortamı (Dulbecco Modifiye Eagle Ortamı DMEM) 37 ° C&#39;de% 10 fetal sığır serumu (FBS) ve% 1 penisilin / streptomisin / neomisin (PSN) ile takviye edilmiştir. </li><li> -80 ° C&#39;de depolamadan 25 - geçit 18 BV2 mikroglial hücreler dondurulmuş stok elde edilir. Hızla 37 ° C su banyosu içinde hücreleri eritin. </li><li> Yavaşça 10 ml hücre kültür ortamı içeren 75 cm 2 Bacalı şişeye hücreleri aktarmak. </li><li> 37 ° C&#39;de% 5 CO2 içinde balon inkübe edin. Medya aspire 24 saat sonra ve taze medya ile değiştirin. % 80 confluency - yaklaşık 70 kadar hücreleri büyür. </li></ol> 2. Sayma ve Kaplama Hücreler <ol><li> 37 ° C su banyosu, sıcak DMEM ve 1 x-tripsin-EDTA çözeltisi içinde. </li><li> hücrelerden aspire medya ve tripsin 500 ul ekleyin. 5 dakika - 2 37 ° C&#39;de inkübe edin. </li><li> kazıyıcı kullanarak şişeye hücreleri çıkarmak ve 5 ml askıyaDMEM. 50 ml&#39;lik bir tüp içine üç kez 70 mikron süzgeç vasıtasıyla hücrelere geçerler. </li><li> Bir hemasitometre kullanarak hücreleri sayın ve% 10 FBS ve% 1 PSN ile desteklenmiş 200 ul DMEM nihai hacimde net bir alt plaka duvarlı / göz, bir siyah 8.000 hücre tohum. 24 saat boyunca 37 ° C&#39;de% 5 CO2 içinde plaka inkübe edin. </li><li> Eski DMEM çıkarın ve mikroglia açlıktan serum% 1 PSN ile takviye ısıttı DMEM 200 ul ile değiştirin. 24 saat boyunca 37 ° C&#39;de% 5 CO2 içinde plaka inkübe edin. </li></ol> 3. etkinleştirilmesi Mikroglia <ol><li> Ayrı tüplerde, AGNPS seyreltilmiş (0.01, 0.05 ya da 0.1 ug / ml) ve araç / nötr kontrolü son işleme konsantrasyonları% 1 PSN ile takviye edilmiş serumsuz DMEM (sodyum sitrat, 0.04 mM). </li><li> her bir ortamın 100 ul çıkarın ve uygun tedavi, bileşiğin 100 ul ile değiştirin. </li><li> 24 saat boyunca 37 ° C&#39;de% 5 CO2 içinde plaka inkübe edin. </li></ol> 4. FiKoşullu ortam edilmiş filtre edilmiş <ol><li> Her ortam süpernatan 200 ul iyi toplama toplama tüpünün (1,5-2 mi kapasite) sahip bir filtre (10 kDa moleküler ağırlık cutoff) aktarmak. </li><li> 15 dakika boyunca, oda sıcaklığında 14,000 x g&#39;de santrifüjleyin. </li><li> flow-through (parçacıkları ile süpernatant) atın ve yeni bir toplama tüpüne filtre ters yerleştirin. </li><li> 2 dakika boyunca oda sıcaklığında 1000 x g&#39;de santrifüjleyin. Not: Elde edilen filtre ortamı (salgılanmış sitokinler ihtiva eden) altı kat konsantre edilir. </li><li> Buz üzerinde,% 10 FBS ve% 1 PSN ve mağaza ile desteklenmiş taze DMEM ile 400 ul, konsantratın hacimle getirin. NOT: - 420 nm 10 yaklaşık 390 karakteristik tepe dayalı bir AgNP konsantrasyonu, artık AGNPS süzülmüş ortamdan kaldırılır sağlamak oluşturmak için. (Adım 3.1 de tarif edildiği gibi, 0, 0.01, 0.025, 0.05, 0.1, ve 0.2 ug / ml) ve süzüldü Kısaca, süzülmemiş AGNPS hacimde hazırlanmasıAraç kontrol serumsuz DMEM (sodyum sitrat, 0.04 mM). Kısım Bir siyah süzülür örnekler ve standartlar 50 ul açık alt plaka duvarlı. bir spektrofotometre kullanılarak, 390 absorbans ölçmek - 410 nm ve AgNP konsantrasyon eğrisine okumaları karşılaştırın. süzüldü örnekler araç kontrol numunesi ile aynı emilme değeri varsa, kalıntı AGNPS kaldırılır varsayılabilir. </li><li> Bir ticari olarak temin edilebilir kit TNF-α salgılanmasını talimatlarını belirlemek için, filtre ortamının yarısı kullanın. </li></ol> 5. Hipotalamik hücre kültürü Bakım <ol><li> Sıcak DMEM hücre kültür aracı 37 ° C,% 10 FBS ve% 1 PSN ile takviye edilmiştir. </li><li> 80 ° C de muhafaza 25 - hipotalo dondurulmuş stok (mHypo-A1 / 2) geçit 18 hücreleri elde etmek. Hızla 37 ° C su banyosu içinde hücreleri eritin. </li><li> Yavaşça 10 ml hücre kültür ortamı içeren 75 cm 2 Bacalı şişeye hücreleri aktarmak. </li><li> CO2% 5 balon inkübe </s37 ° C&#39;de UB&gt;. Medya aspire 24 saat sonra ve taze medya ile değiştirin. % 80 confluency - Yaklaşık 70 kadar hücreleri büyür. </li></ol> 6. belirlenmesi Hipotalamus Hücre zehirlilik <ol><li> Sıcak DMEM ve 37 ° C su banyosu içinde 1x-tripsin-EDTA çözeltisi. </li><li> hipotalamik hücrelerden aspire medya ve tripsin 500 ul ekleyin. 5 dakika - 2 37 ° C&#39;de inkübe edin. 2. adımda, yukarıda tarif edildiği gibi, bir kazıyıcı kullanılarak şişeden hücreler çıkarın. </li><li> Bir hemasitometre kullanarak hücreleri sayın ve% 10 FBS ve% 1 PSN ile desteklenmiş 200 ul DMEM nihai hacimde net bir alt plaka duvarlı / göz, bir siyah 5000 hücre olacak şekilde hipotalamus hücreleri plaka ve 37 ° C&#39;de% 5 CO2 içinde gece boyunca inkübe . </li><li> Çok kanallı bir pipet kullanılarak, eski ortamın 100 ul çıkarın ve 1x bir son konsantrasyon elde etmek üzere filtre konsantre hale koşullu ortamın 100 ul, ilave edin. </li><li> 24 saat boyunca 37 ° C&#39;de% 5 CO2 içinde plaka inkübe edin. </li><li> 22 ekle1 &#39;dir; resazurin ayıracı ve 37 ° C&#39;de% 5 CO2 içinde 20 dakika süreyle plaka inkübe edin. </li><li> Bir modlu spektrofotometre, kayıt floresan (560 nm EX / 590 nm EM) kullanarak hücre canlılığını ölçmek için. bağıl floresan üniteleri (RFU) olarak sonuçları rapor. </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</...

Çevre kirliliği, özellikle nanopartikül (NP) aralığı olan (1-20 nm çapında), obezite ve kan-beyin bariyerinden 1-3 geçebilmekte bağlı başka nörodejeneratif hastalıklar ile bağlantılı olmuştur. Kirliliğine yükseltilmiş maruz hipotalamus 1 de dahil olmak üzere, merkezi sinir sisteminde enflamasyonun neden olabilir. Bu mikroglia nanoparçacık kaynaklı aktivasyonu (beyin bağışıklık hücreleri) 4 kanalıyla olabilir meydana geldiği bir potansiyel mekanizma. Önceki çalışmalar zaman alıcı, pahalı beyin sağlığı üzerinde NPS etkilerini incelemek için in vivo modellerinde kullanmış ve doğrudan NPS mikroglia nasıl etkilediği sorusuna cevap yok. Mikroglia beyin mikro bakım dahil olmak üzere ve salgılanan faktörler ve sitokinlerin açığa ile nöronları çevreleyen iletişim merkezi sinir sisteminde, çok yönlü bir rol oynamaktadır. uyaranlara bağlı olarak, mikroglia bir M1 pr aktif hale getirilebiliro-inflamatuar veya M2 anti-inflamatuar durum. M2, interlökin-4 (IL-4) dahil olmak üzere, mikroglia salma anti-enflamatuar sitokinler aktive Örneğin, M1, mikroglia tümör nekroz faktörü alfa (TNF-α) gibi pro-enflamatuar sitokinleri serbest aktif. Hava kirleticilerin nörotoksik belirlenmesi için in vitro biyosensörle bizim vekil doğrulamak için, biz 20 nm gümüş nanopartikülleri (AGNPS) için mikroglial tepki ölçülür. Bu makalenin amacı bir in vitro mikroglial hücre hattı NP için fare mikroglial tepki test ve nasıl mikroglial aktivasyon hipotalamus hücrelerini etkiler için bir vekil biyosensör belirteci olarak nasıl kullanılabileceğini açıklamaktır. Uzun vadeli bu valide modelin uygulama, beyin sağlığı ve nörodejeneratif hastalık üzerinde gerçek dünya kirleticilerin etkilerini test etmek amaçlanmıştır. Bu mikrofon maruz kaldıktan sonra mikroglial aktivasyonu ve hipotalamik hücre yaşamını ölçmek için bir in vitro 96 oyuklu bir format ayrıntılı bir açıklama temin<html> koşullu ortam roglial. Mikroglia aktivasyonu testi (ELISA) bağlı immünosorban bir TNF-α enzim kullanılarak AgNP maruz kalındıktan sonra belirlendi. hipotalamik hücrelerde aktif mikroglia etkisini belirlemek için, AGNPS bir süzme cihazı kullanılarak mikroglial süpernatan (koşullu ortam) çıkarıldı. boyutuna göre AGNPS hariç ise filtrasyon cihazı sitokinler korur. Kısaca, AGNPS ile veya tedavi mikroglia alınan üst faz, toplanmış, filtrelere eklenmiş ve 15 dakika için 14,000 x g&#39;de santrifüjlenmiştir. Daha sonra hipotalamik hücre canlılığı üzerine mikroglial salgılanan sitokinlerin etkisini belirlemek başardık. Daha önce tarif edildiği gibi 5,6 koşullu ortam (ihtiva eden sitokinler) maruz kaldıktan sonra hücre toksisitesi resazurin tabanlı tahlil ile belirlenmiştir. Metabolik olarak aktif hücrelerin resazurin azaltmak ve canlı hücreler 7 sayısı ile orantılı bir flüoresan sinyal üretir. nt &quot;&gt; (örneğin, ya da in vivo deneyler ko-kültür, trans-kuyu kurulumları gibi) diğerleri üzerinde bu tekniği kullanarak birçok avantajı vardır. Bizim modeli doğrudan mikroglia etkinleştirmek ve salgılanan faktörler nöronlar 8 için toksik olup olmadığını belirlemek için yeteneği sağlar . geçerli protokol ölümsüzleştirilmiş BV2 mikroglia sıçangil hipotalamik hücreleri bu protokol bu spesifik koşullar için optimize edilmiş olsa da (belirlenmiş mHypo-A1 / 2) daha sonra karşılık belirlenmesi için 9., yöntemler olabilir 20 nm çapında nano ile uyarılmış ve ölümsüzleştirilmiş kullanır değiştirilmiş mikroglial kaynaklı hücre ölümünün diğer modellerde kullanılan veya birincil mikroglia ve nöronlar gibi diğer hücre tipleri ile edilmesi.

<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.

Yukarıdaki protokolünü kullanarak nanopartiküller beyin yanıtı için bir vekil biyosensör olarak bu mikroglia işlevini göstermektedir. Bizim sonuçlarımız aşağı nöronal hücre ölümü üzerindeki mikroglial aktivasyon toksik etkileri ölçülmesini içerir. 1 mikroglia etkinleştirmek ve salgılanan sitokinler hipotalamik nöronların canlılığını azaltır olmadığını belirlemek için protokol iş akışı gösterir Şekil. TNF-α ...

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.

Nanoparçacık Neurotoksite Belirleyen Bir Vekil Biosensor olarak mikroglia

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

6.9K Görüntüleme.  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.

Video: 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.

Teşhis ve Tedavi İzleme Nanomalzemelerin Kayıtlı bio

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

Biyomühendislik

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.

Normal MSS Real-time PCR ve Nöro sırasında mikroglia içinde microRNA tespiti

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 ...

İmmünoloji ve Enfeksiyon

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).

Kemirgen Yenidoğan itibaren Korunan immunfenotipine ve İşlevselliği ile Kortikal mikrogliya İzolasyonu

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.

Bir yutulma Deneyi: CNS Fagositler ve nöronlar arasındaki etkileşimi değerlendirmek için bir protokol

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

Nörobilim

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.

Retina Mikroglial Fagositik Fonksiyonu değerlendirilmesi<em&gt; İn Vivo</em&gt; A Akış Sitometri bazlı analiz kullamlarak

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.

Fare Glokomda Konfokal Oftalmoskopik Görüntüleme ve Hücre morfometrisi: Nörodejenerasyonda sırasında Retina Mikroglial Aktivasyonu Vivo Dynamics

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

Tıp

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.

Geliştirilmiş Saflık ve Çok Yönlü İlköğretim mikroglia hızlı ve Rafine CD11 Manyetik İzolasyon

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.

Karakterizasyonu ve yoğunluk Gradient Santrifüjü tarafından fare birincil Microglia izolasyonu

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

Sinaptik Materyallerin Mikroglial Yutulması için Akış Sitometrisi Tabanlı Testler

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.

Yazar Spotlight: Psikonöroimmünolojide Teknolojik Yenilikler Yoluyla Anlayışı Geliştirme

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

Akış Sitometrisi Kullanılarak Sinaptik Materyalin Mikroglial Yutulmasının Miktar Tayini

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

Nanoparçacık Neurotoksite Belirleyen Bir Vekil Biosensor olarak mikroglia

Özet

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Bu videodaki bölümler

Teşhis ve Tedavi İzleme Nanomalzemelerin Kayıtlı bio

Normal MSS Real-time PCR ve Nöro sırasında mikroglia içinde microRNA tespiti

Kemirgen Yenidoğan itibaren Korunan immunfenotipine ve İşlevselliği ile Kortikal mikrogliya İzolasyonu

Bir yutulma Deneyi: CNS Fagositler ve nöronlar arasındaki etkileşimi değerlendirmek için bir protokol

Retina Mikroglial Fagositik Fonksiyonu değerlendirilmesi

Fare Glokomda Konfokal Oftalmoskopik Görüntüleme ve Hücre morfometrisi: Nörodejenerasyonda sırasında Retina Mikroglial Aktivasyonu Vivo Dynamics

Geliştirilmiş Saflık ve Çok Yönlü İlköğretim mikroglia hızlı ve Rafine CD11 Manyetik İzolasyon

Karakterizasyonu ve yoğunluk Gradient Santrifüjü tarafından fare birincil Microglia izolasyonu

Akış Sitometrisi Kullanılarak Sinaptik Materyalin Mikroglial Yutulmasının Miktar Tayini

Akış Sitometrisi Kullanılarak Sinaptik Materyalin Mikroglial Yutulmasının Miktar Tayini