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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cells/Reagents</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&#160;</td>
			<td>Cellutions Biosystems Inc.</td>
			<td>CLU172</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium</td>
			<td>Invitrogen</td>
			<td>10313-039</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum&#160;</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 (20nm)</td>
			<td>Sigma-Aldrich</td>
			<td>730793</td>
			<td></td>
		</tr>
		<tr>
			<td> 
			PrestoBlue Cell Viability Reagent</td>
			<td>Invitrogen</td>
			<td>A13262</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse TNF-&#945; 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></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96W 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 50mL Conical Centrifuge Tubes</td>
			<td>Corning, Inc</td>
			<td> 
			14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Cell Strainers 70 &#956;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

Nerve Excitability Assessment in Chemotherapy-induced Neurotoxicity

Chemotherapy-induced neurotoxicity is a serious consequence of cancer treatment, which occurs with some of the most commonly used chemotherapies1,2. Chemotherapy-induced peripheral neuropathy produces symptoms of numbness and paraesthesia in the limbs and may progress to difficulties with fine motor skills and walking, leading to functional impairment. In addition to producing troubling symptoms, chemotherapy-induced neuropathy may limit treatment success leading to dose reduction or early cessation of treatment. Neuropathic symptoms may persist long-term, leaving permanent nerve damage in patients with an otherwise good prognosis3. As chemotherapy is utilised more often as a preventative measure, and survival rates increase, the importance of long-lasting and significant neurotoxicity will increase.
There are no established neuroprotective or treatment options and a lack of sensitive assessment methods. Appropriate assessment of neurotoxicity will be critical as a prognostic factor and as suitable endpoints for future trials of neuroprotective agents. Current methods to assess the severity of chemotherapy-induced neuropathy utilise clinician-based grading scales which have been demonstrated to lack sensitivity to change and inter-observer objectivity4. Conventional nerve conduction studies provide information about compound action potential amplitude and conduction velocity, which are relatively non-specific measures and do not provide insight into ion channel function or resting membrane potential. Accordingly, prior studies have demonstrated that conventional nerve conduction studies are not sensitive to early change in chemotherapy-induced neurotoxicity4-6. In comparison, nerve excitability studies utilize threshold tracking techniques which have been developed to enable assessment of ion channels, pumps and exchangers in vivo in large myelinated human axons7-9.
Nerve excitability techniques have been established as a tool to examine the development and severity of chemotherapy-induced neurotoxicity10-13. Comprising a number of excitability parameters, nerve excitability studies can be used to assess acute neurotoxicity arising immediately following infusion and the development of chronic, cumulative neurotoxicity. Nerve excitability techniques are feasible in the clinical setting, with each test requiring only 5 -10 minutes to complete. Nerve excitability equipment is readily commercially available, and a portable system has been devised so that patients can be tested in situ in the infusion centre setting. In addition, these techniques can be adapted for use in multiple chemotherapies.
In patients treated with the chemotherapy oxaliplatin, primarily utilised for colorectal cancer, nerve excitability techniques provide a method to identify patients at-risk for neurotoxicity prior to the onset of chronic neuropathy. Nerve excitability studies have revealed the development of an acute Na+ channelopathy in motor and sensory axons10-13. Importantly, patients who demonstrated changes in excitability in early treatment were subsequently more likely to develop moderate to severe neurotoxicity11. However, across treatment, striking longitudinal changes were identified only in sensory axons which were able to predict clinical neurological outcome in 80% of patients10. These changes demonstrated a different pattern to those seen acutely following oxaliplatin infusion, and most likely reflect the development of significant axonal damage and membrane potential change in sensory nerves which develops longitudinally during oxaliplatin treatment10. Significant abnormalities developed during early treatment, prior to any reduction in conventional measures of nerve function, suggesting that excitability parameters may provide a sensitive biomarker.

This abstract describes a novel method to assess the development of neurotoxicity in patients receiving chemotherapy treatment. While conventional assessment methods are limited in their ability to detect early changes in nerve function, nerve excitability techniques provide early identification of patients at risk of severe neurotoxicity and insight into pathophysiology.

화학 요법 - 유도 Neurotoxicity의 신경 흥분 평가

Chemotherapy-induced neurotoxicity is a serious consequence of cancer treatment, which occurs with some of the most commonly used chemotherapies1,2. ...

연구

신경과학

Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis

Antibodies provide the ability to gain novel insight into various events taking place in living systems. The ability to produce highly specific antibodies to target proteins has allowed for very precise biological questions to be addressed. Importantly, antibodies have been implicated in the pathogenesis of a number of human diseases including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), paraneoplastic syndromes, multiple sclerosis (MS) and human T-lymphotropic virus type 1 (HTLV-1) associated myelopathy/tropical spastic paraparesis (HAM/TSP) 1-9. How antibodies cause disease is an area of ongoing investigation, and data suggests that interactions between antibodies and various intracellular molecules results in inflammation, altered cellular messaging, and apoptosis 10. It has been shown that patients with MS and HAM/TSP produce autoantibodies to the intracellular RNA binding protein heterogeneous ribonuclear protein A1 (hnRNP A1) 3, 5-7, 9, 11. Recent data indicate that antibodies to both intra-neuronal and surface antigens are pathogenic 3, 5-9, 11. Thus, a procedure that allows for the study of intracellular antibody:protein interactions would lend great insight into disease pathogenesis.
Genes are commonly transfected into primary cells and cell lines in culture, however transfection of antibodies into cells has been hindered by alteration of antibody structure or poor transfection efficiency 12. Other methods of transfection include antibody transfection based on cationic liposomes (consisting of DOTAP/DOPE) and polyethylenimines (PEI); both of which resulted in a ten-fold decrease in antibody transfection compared to controls 12. The method performed in our study is similar to cationic lipid-mediated methods and uses a lipid-based mechanism to form non-covalent complexes with the antibodies through electrostatic and hydrophobic interactions 13. We utilized Ab-DeliverIN reagent, which is a lipid formulation capable of capturing antibodies through non-covalent electrostatic and hydrophobic interactions and delivering them inside cells. Thus chemical and genetic couplings are not necessary for delivery of functional antibodies into living cells. This method has enabled us to perform various antibody tracing and protein localization experiments, as well as the analyses of the molecular consequences of intracellular antibody:protein interactions 9.
In this protocol, we will show how to transfect antibodies into neurons rapidly, reproducibly and with a high degree of transfection efficiency. As an example, we will use anti-hnRNP A1 and anti-IgG antibodies. For easy quantification of transfection efficiency we used anti-hnRNP A1 antibodies labelled with Atto-550-NHS and FITC-labeled IgG. Atto550 NHS is a new label with high molecular absorbtion and quantum yield. Excitation source and fluorescent filters for Atto550 are similar to Cy3 (Ex. 556 Em. 578). In addition, Atto550 has high photostability. FITC-labeled IgG were used as a control to show that this method is versatile and not dye dependent. This approach and the data that is generated will assist in understanding of the role that antibodies to intracellular target antigens might play in the pathogenesis of human diseases.

A rapid approach to investigate interactions and effects on molecular mechanisms related to the presence of antibodies in an intracellular environment is described. The method involves transfection of antibodies into live cells using a non-covalent complex formation based on a lipid formulation. The technique is adaptable to immortalized cell lines and primary cells.

질병 Pathogenesis를 공부하는 도구로 뉴런에 항체 Transfection

Antibodies provide the ability to gain novel insight into various events taking place in living systems.  The ability to produce highly specific ...

Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and decreases waste in costs and reagents. Here we describe optimization of a multiplex assay to assess apoptosis following a palmitic acid (PA) challenge in an in vitro hypothalamic model, using both fluorescent and luminescent based assays to measure viable cell counts and caspase-3/7 activity in a 96-well microtiter plate format. Following PA challenge, viable cells were determined by a resazurin-based fluorescent assay. Caspase-3/7 activity was then determined using a luminogenic substrate, DEVD, and normalized to cell number. This multiplexing assay is a useful technique for determining change in caspase activity following an apoptotic stimulus, such as saturated fatty acid challenge. The saturated fatty acid PA can increase hypothalamic oxidative stress and apoptosis, indicating the potential importance of assays such as that described here in studying the relationship between saturated fatty acids and neuronal function.

Multiplex assays can provide beneficial information for basic cellular mechanisms&#160;and eliminate waste of reagents and unnecessary repetitive experiments. We describe here a multiplex caspase-3/7 activity assay, using fluorescent- and luminescent-based methods, to determine cell viability in an in vitro hypothalamic model following oxidative challenge with palmitic acid.

시상 하부 세포 모델에서 세포 사멸을 결정하는 카스파 다중 분석의 사용

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and ...

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic effects of drugs and downstream pathways involved would help gain insight into the molecular mechanisms of neuroprotection/neurotoxicity and could potentially facilitate drug development. However, many existing in vitro toxicity assays have major limitations &#8211; most assess neurotoxicity and neuroprotection at a single time point, not allowing to observe the time-course and kinetics of the effect. Furthermore, the opportunity to collect information about downstream signaling pathways involved in neuroprotection in real-time would be of great importance. In the current protocol we describe the use of a real-time impedance-based cell analyzer to determine neuroprotective effects of serotonin 2A (5-HT2A) receptor agonists in a neuronal cell line under label-free and real-time conditions using impedance measurements. Furthermore, we demonstrate that inhibitors of second messenger pathways can be used to delineate downstream molecules involved in the neuroprotective effect. We also describe the utility of this technique to determine whether an effect on cell proliferation contributes to an observed neuroprotective effect. The system utilizes special microelectronic plates referred to as E-Plates which contain alternating gold microelectrode arrays on the bottom surface of the wells, serving as cell sensors. The impedance readout is modified by the number of adherent cells, cell viability, morphology, and adhesion. A dimensionless parameter called Cell Index is derived from the electrical impedance measurements and is used to represent the cell status. Overall, the real-time impedance-based cell analyzer allows for real-time, label-free assessment of neuroprotection and neurotoxicity, and the evaluation of second messenger pathways involvement, contributing to more detailed and high-throughput assessment of potential neuroprotective compounds in vitro, for selecting therapeutic candidates.

Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

신경 세포주에 신경 독성과 신경에 관여하는 분자 진학의 윤곽을하는 도구로 실시간 임피던스 기반의 세포 분석기

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic ...

Intravital Imaging of Axonal Interactions with Microglia and Macrophages in a Mouse Dorsal Column Crush Injury

Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. Intra-vital two-photon microscopy enables the study of macrophages and microglia in the spinal cord lesion in the living animal. This can be performed in adult animals with a traumatic injury to the dorsal column. Here, we describe methods for distinguishing macrophages from microglia in the CNS using an irradiation bone marrow chimera to obtain animals in which only macrophages or microglia are labeled with a genetically encoded green fluorescent protein. We also describe a injury model that crushes the dorsal column of the spinal cord, thereby producing a simple, easily accessible, rectangular lesion that is easily visualized in an animal through a laminectomy. Furthermore, we will outline procedures to sequentially image the animals at the anatomical site of injury for the study of cellular interactions during the first few days to weeks after injury.

Two-photon intravital imaging can be used to investigate interactions among different cell types in the spinal cord in their native tissue environment in a bone marrow chimeric animal with a dorsal column traumatic spinal cord crush injury.

마우스 지느러미 열 압박 손상에 마이크로 글 리아와 대 식세포와 축삭의 상호 작용의 생체 내에 이미징

Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

Organotypic Hippocampal 슬라이스 문화 연구 Neuroprotection 및 종양 세포의 침입을 모델로

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium

Microglia represent 5 - 10% of all central nervous system (CNS) cells and are increasingly drawing attention due to their contributions during development, homeostasis, and disease. Although macrophages have been studied in detail for decades, specialized features of microglia, the tissue-resident macrophages of the CNS, have remained largely mysterious, in part due to limitations in the ability to recapitulate mature microglial properties in culture. Here, we illustrate a straightforward procedure for the rapid isolation of pure microglia from the mature rodent brain. We also describe serum-free culture conditions that support high levels of microglial viability over time. Microglia cultured under these defined-medium conditions exhibit elaborate ramified processes and dynamic surveillance behavior. We illustrate some effects of serum exposure on cultured microglia and discuss how these serum-free cultures compare to both serum-exposed cultures as well as microglia in vivo.

Efforts to understand microglial function in detail have been hindered by the lack of microglial culture models that recapitulate the properties of mature in vivo microglia. This protocol describes an isolation and culture approach designed to maintain robust survival of highly ramified mature rat microglia under defined-medium conditions.

절연 및 동적 홍보 설치류 Microglia의 문화 Ramified 혈 청 자유로운 매체에 있는 외관

Microglia represent 5 - 10% of all central nervous system (CNS) cells and are increasingly drawing attention due to their contributions during ...

Adaptation of Microelectrode Array Technology for the Study of Anesthesia-induced Neurotoxicity in the Intact Piglet Brain

Every year, millions of children undergo anesthesia for a multitude of procedures. However, studies in both animals and humans have called into question the safety of anesthesia in children, implicating anesthetics as potentially toxic to the brain in development. To date, no studies have successfully elucidated the mechanism(s) by which anesthesia may be neurotoxic. Animal studies allow investigation of such mechanisms, and neonatal piglets represent an excellent model to study these effects due to their striking developmental similarities to the human brain.
This protocol adapts the use of enzyme-based microelectrode array (MEA) technology as a novel way to study the mechanism(s) of anesthesia-induced neurotoxicity (AIN). MEAs enable real-time monitoring of in vivo neurotransmitter activity and offer exceptional temporal and spatial resolution. It is hypothesized that anesthetic neurotoxicity is caused in part by glutamate dysregulation and MEAs offer a method to measure glutamate. The novel implementation of MEA technology in a piglet model presents a unique opportunity for the study of AIN.

This study explores the novel use of enzyme-based microelectrode array (MEA) technology to monitor in vivo neurotransmitter activity in piglets. The hypothesis was that glutamate dysregulation contributes to the mechanism of anesthetic neurotoxicity. Here, we present a protocol to adapt MEA technology to study the mechanism of anesthesia-induced neurotoxicity.

마 취 유도 Neurotoxicity 그대로 돼지 뇌에서의 연구에 대 한 Microelectrode 배열 기술의 적응

Every year, millions of children undergo anesthesia for a multitude of procedures. However, studies in both animals and humans have called into question ...

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which enables them to seek out their preferred thermal landscape. To define the temperature preferences of larvae over a defined linear range, we developed an assay using a temperature gradient. To establish a single-directional gradient, two aluminum blocks are connected to independent water baths, each of which controls the temperature of individual blocks. The two blocks set the lower and upper limits of the gradient. The temperature gradient is established by placing an agarose-coated aluminum plate over the two water-controlled blocks so that the plate spans the distance between them. The ends of the aluminum plate that is set on the top of the water blocks defines the minimum and maximum temperatures, and the regions in-between the two blocks form a linear temperature gradient. The gradient assay can be applied to&#160;larvae of different ages and can be used to identify mutants that exhibit phenotypes, such as those with mutations affecting genes encoding TRP channels and opsins, which are required for temperature discrimination.

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Here, we present a protocol to determine the preferred environmental temperature of Drosophila larvae using a continuous thermal gradient.

초파리 애벌레의 열 환경 설정을 결정 하는 온도 분석 결과

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which ...

Cell-based Assay to Study Antibody-mediated Tau Clearance by Microglia

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing neuronal dysfunction which eventually leads to cognitive decline. Hyperphosphorylated tau aggregates in the neuron are believed to cause most of the pathology associated with AD. These aggregates are assumed to be released into the extracellular compartment and taken up by adjacent healthy neurons where they induce further tau aggregation. This &#34;prion-like&#34; spreading can be interrupted by antibodies capable of binding and &#34;neutralizing&#34; extracellular tau aggregates as shown in preclinical mouse models of AD. One of the proposed mechanisms by which therapeutic antibodies reduce pathology is antibody-mediated uptake and clearance of pathological aggregated forms of tau by microglia. Here, we describe a quantitative cell-based assay to assess tau uptake by microglia. This assay uses the mouse microglial cell line BV-2, allows for high specificity, low variability and medium throughput. Data generated with this assay can contribute to a better characterization of anti-tau antibody effector functions.

Here we describe a cell-based assay to quantitatively assess tau uptake by microglia with the aim of creating an investigational tool to better characterize the mechanisms of action of anti-tau antibodies.

Microglia에 의해 항 체 중재 타우 정리 공부 세포 기반 분석 결과

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing ...