Name: autoradiographic measurements of [14c]-iodoantipyrine in rat brain following central post-stroke pain
Uploaded: 2016-07-18T15:00:00.000+00:00
Duration: 7 min 27 s
Description: Watch this Scientific Journal Video about Autoradiographic Measurements of [14C]-Iodoantipyrine in Rat Brain Following Central Post-Stroke Pain at JoVE.com

본 연구는 박사 바이 - 추앙 Shyu (NSC 99-2320-B-001-016-MY3, NSC 100-2311-B-001-003-MY3 및 NSC 102-2320-에 국가 과학위원회의 보조금 지원 B-001-026-MY3). 이 작품은 중앙 연구원에서 자금을받은 생명 과학 연구소에서 실시 하였다.

본 연구의 프로토콜은 대만의 중앙 연구원 기관 동물 관리의 승인 및 이용위원회를 받았다. 1. 동물 준비 <ol><li> (- 400g 약 300) 수컷 흰쥐를 가져옵니다. 음식과 물을 무료로 이용할 수로 (오전 8:00에 불이) 12 시간 / 12 시간 빛 / 어둠 사이클에서 - (22 °의 C, 습도 50 % 21) 에어컨 방에 ​​쥐를 유지한다. </li></ol> 2. 실험 절차 <ol><li> 쥐의 모든 실험 전에 1 주간 자신의 홈 케이지의 환경에 적응하도록 허용합니다. 적응하는 동안, 기준을 설정하는 데 프레이와 발바닥 테스트를 수행합니다. </li><li> 프레이 테스트 <ol><li> 거주 30 분 동안 아크릴 케이스 (30 cm × 30 cm × 80 cm)에 쥐를 놓습니다. </li><li> (. 아니 필라멘트 11-20) 본 프레이 필라멘트를 구하는 동일한 길이를 가지고 있지만, 직경이 변화하는 범위를 제공하도록100g - 2의 힘. </li><li> 쥐의 뒷발의 발 움츠림 역치를 평가 5 분 intertrial 간격 아크릴 판에 그물 형 포트를 통해 hindpaws의 중심을 자극 프레이 필라멘트를 사용한다. 최대인가 된 압력이 기록 될 때까지 로우에서 하이로 오름차순으로 필라멘트를 사용하여, 최대인가 된 압력을 기록한다. (10) </li><li> 쥐 자극에 대한 발 움츠림 반응을 나타낼 때, 필라멘트 수를 기록한다. 가장 낮은 자극에 따라이 재판에서 철수 응답을 확인합니다. </li><li> 동일한 래트에 연속 3 회, 총 즉시 동일한 절차를 반복한다. (g)에 대응하는 힘에 필라멘트 번호를 변환하고 값을 평균. </li></ol></li><li> 발바닥 테스트 <ol><li> 거주 30 분 (4 개의 프레임 80 cm × 30 cm × 15 센티미터로 구분) 투명한 플렉시 글라스 상자에 쥐를 놓습니다. </li><li> INF를 사용하여rared 광선은 유리판을 통해 뒷발의 중심을 자극한다. 약 10 초의 평균 발 움츠림 응답 대기 시간을 얻기 위해 적외선 강도를 조정한다. 적외선 광원을 켜고 디지털 솔리드 스테이트 타이머를 시작하는 키를 누름으로써 시험을 실시한다. 적외선 광선의 지속 시간을 조작 할 수 있습니다. </li><li> 래트가 발 움츠림 반응을 나타낼 때에 ​​외광의 지속 시간을 기록한다. 최장 기간은 조직의 손상을 방지하기 위해 각 시험에서 20 초를 초과 할 수 없습니다. 연속적인 자극을 피하기 위해 적어도 5 분의 intertrial 간격을 사용합니다. </li><li> 좌우 hindpaws 세 실험 테스트를 반복하고, 각 래트 뒷발에 대한 각각의 평균을 계산한다. </li></ol></li><li> 콜라게나 병변 수술 <ol><li> 수술 자극이 발생에 발가락 - 핀치 응답의 손실과 체세포 응답 할 때까지 4 % 이소 플루 란과 쥐를 마취. 2 % isoflur - 1.5 마취 유지수술 시간 동안 메탄. </li><li> 37.5 °의 C - 36.5에서 체온을 유지하기 위해 간단한 가열 패드와 정위 장치에서 쥐를 놓습니다. 마취 동안 건조를 방지하기 위해 눈에 아이 크림을 적용합니다. </li><li> 멸균 전기 면도기로 털을 면도하고 두피의 정중선을 따라 메스와 부드러운 절개 (약 2-2.5 cm)를 확인합니다. 섭리의 요오드 및 소독 75 % 알코올 용액을 교대로 피부와 두개골을 청소합니다. 수술 단계 동안 모든 기기, 도구 살균 75 %의 알코올을 사용하고, 작업대는 무균 상태를 유지하기. 그 전에, 모든 수술 재료는 증기 오토 클레이브 멸균해야합니다. </li><li> (시상의 (복부 후 내측 시상 핵 [VPM]와 복부 외측 시상 핵 [VPL] 포함) VB를 통해 전기 드릴로 두개골에 3.0 작은 구멍 (3 mm 직경)를 멸균 장갑과 악기를 사용하여 드릴 -3.5 mm 후방, 3.0-3.5 mm 측면) 정수리, 두개골 표면 5.5-5.8 mm 깊이합니다. </li><li> Microinject 생리 식염수 0.5 μL 또는 각각 제어 및 실험 그룹에서 4 형 콜라겐 용액 0.125 U. </li><li> 약물의 확산을 허용하도록 추가로 5 분 동안 곳에 주사 바늘을 유지. </li><li> 두개골에 구멍을 채우기 위해 치과 시멘트를 사용하고 절개를 봉합. 봉합 절개 후 상처 로컬 진통 마취제 (리도카인 연고)를 적용하고, 자신의 홈 케이지로 돌아갑니다. </li><li> 수술 후 단독으로 그들은 흉골 드러 누움을 유지할 때까지 플라스틱 케이지의 쥐를 수용하고, 마취에서 회복 될 때까지 따뜻하게 유지합니다. </li></ol></li><li> 수술 회복 후 행동 테스트 <ol><li> 수술에서 회복 7 일 후, 섹션 테스트 단계 2.2과 2.3의 절차를 반복합니다. 건강과 동물의 발달 상태를 모니터링하면서 사주를 통해 행동 테스트를 수행합니다. </li><li&gt; 이후 수술 단계의 제어 및 실험군의 동물의 건강 상태 (예를 들어, 체중, 먹이 양, 그리고 자유로운 움직임)을 모니터한다. </li></ol></li><li> PE-50 튜브와 함께 정맥 주입을 수행 <ol><li> 4 % 이소 플루 란 마취 래트 및 36.5에서 체온을 유지 - 단순한 가열 패드와 37.5 ° C. </li><li> 메스로 각각 왼쪽 어깨와 흉강의 복부 부분의 지느러미 앞발의 일부 교차로의 중간 선에 두 개의 구멍 (2cm 직경 각)을 잘라. </li><li> 두 구멍 사이 가위로 피부와 근육을 떼어 놓다. </li><li> 복부 구멍을 통해 외부 경정맥에 PE-50 튜브 (20cm 길이)의 한쪽 끝을 연결합니다. 지느러미 구멍 같은 PE-50 튜브의 말단을 연결하고, 피부에 부착합니다. </li><li> 확인하기 위해 경정맥에 식염수를 주입하는 주사기를 사용하여 그 PE-50 튜브방해하지 않습니다. </li><li> 절개 부위를 봉합하고, 6 ㎎ / ㎏ 겐타 마이신 (복강 내)로 쥐를 주입. </li><li> 20 U / ㎖ 헤파린과 생리 식염수 0.1 ml의 다음에, 0.3 ml의 0.9 %의 식염수로 튜브를 수술 후 매일을 플래시합니다. </li></ol></li><li> 최종 행동 테스트 <ol><li> 반복이 그 동작을 확인 2.2-2.3 단계 단계 2.6 이후 1 주일 수술 복구 후 단계 2.5에 비해 안정적이다. </li></ol></li><li> 추적자 주입 <ol><li> 적응을위한 10 분 - 5 휴식 케이지에 쥐를 놓습니다. </li><li> 스플리터를 사용하여, 두 1ml를 주사기에 PE-50 튜브를 연결합니다. 생리 식염수 하나의 주사기를 입력하고 (- 0.5 ml의 0.3의 볼륨에 125 μCi를 / kg) [14 C] -IAP 솔루션을 다른 입력합니다. </li><li> 외부 경정맥에 추적자를 주입하고, 3 M 염화칼륨 가득 다른 주사기 주사기를 교체합니다. </li><li> 텐 초 추적자의 INJ 후ection, 표준 안락사 방법에 따라 동물을 이소 플루 란의 과다 복용에서 3 M 염화칼륨을 주입하고, 희생 (즉, 50 분 동안 기화기에서 이소 플루 란) 대만하여 중앙 연구원 기관 동물 관리의 지침과 이용위원회를 기반으로. </li><li> 1 분 후, 두개골을 노출하고, 나머지 근육 잘라내. rongeurs를 사용하여 뇌에서 두개골의 지느러미 표면을 벗겨. rongeurs을 이용하여 두개골의 측면을 얻어 낸다. 다음에, 주걱을 이용하여, 뇌의 복부 표면을 따라 후각 신경 전구 연결을 잘라 뇌를 제거한다. </li><li> 드라이 아이스와 메틸 부탄의 두뇌를 동결 최적의 절삭 온도 OCT () 화합물을 사용하여 (약 -55 ° C). 냉동실에 뇌 조직을 저장합니다. 11, 12 </li></ol></li><li> 뇌 슬라이스 <ol><li> 후뇌이 아래를 향하도록하여, 마이크로톰에서 조직의 방향. 20로 뇌를 슬라이스 그라 이오 스탯을 사용하여# 181; m 두께의 섹션. </li><li> 각 조각 사이에 240 μm의 간격으로, -20 ° C에서 저온 유지 장치에 현미경 슬라이드에 뇌 조각을 놓습니다. </li><li> 현미경 슬라이드와 -20 ° C에서 3 일간 노출 카세트에 등급 방사능 다섯 표준 필터 종이를 놓습니다. 뇌 조각의 순서에 따라 위에서 아래로 현미경 슬라이드를 준비. 마지막으로, 카세트의 저부에 여과지를 배치했다. 12 </li><li> 노광 카세트로부터 형광면을 제거하고 뇌 슬라이스 [14 C] -IAP 흡수를 보여주기 위해 이미지를 형광체 스크린을 읽고 생성하는 가변 모드 이미 저를 사용한다. </li></ol></li></ol> 3. 데이터 분석 <ol><li> 단계 2.9.4 후에 통계적 매개 변수 사상 (SPM) 및 ImageJ에 소프트웨어를 사용하여 이미지를 조정한다. 직렬 관상 섹션을 사용하여 모든 이미지를 재구성. 부드럽고 기준 쥐 뇌 모델에 따라 이미지를 정규화.12 13 </li><li> 정량적 평가를 위해, 픽셀 신호 강도를 판정하고, 통계 해석 소프트웨어를 사용 ImageJ에 소프트웨어를 사용하여 관심 영역 뇌 영상 (ROI)을 측정한다. (12, 13) </li><li> 다른 뇌 핵 사이의 연결을 조사하기 위해, 지역간 상관 행렬의 방사능 비율을 표시하는 MATLAB 상관 분석 소프트웨어를 사용하여 컬러 맵 행렬로 시각화. 마지막으로, 네트워크 분석 Pajek 소프트웨어를 사용한다. (12) (13) </li><li> 베이스 라인에서 가짜 및 CPSP 그룹의 프레이 시험에서 발바닥 시험 및 기계적 힘에 열 관용의 지속 기간을 비교, 2 × 편차의 5 양방향 혼합 분석 (ANOVA), 그룹 및 주 요인으로 사용하고 주 1 - 5. ANOVA 그룹 뇌 영역에있어서 방사능 비율을 측정하는 2 × 31 양방향. 적절한 경우, Tukey에의 정직 유의 한 차이 (HSD) 사후 검사를 실시하고 있습니다. 칼culate 피어슨 상관 관계는 가짜와 CPSP 그룹에서 선택된 뇌 영역의 모든 사이에 상관 관계를 평가하는 계수. </li></ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+[14C]iodoantipyrine+study+of+inter-regional+correlations+of+neural+substrates+following+central+post-stroke+pain+in+rats.">A [14C]iodoantipyrine study of inter-regional correlations of neural substrates following central post-stroke pain in rats.</a> Mol Pain. 11 (1), (2015).</li><li>Wang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+brain+activation+during+retrieval+of+visceral+pain-conditioned+passive+avoidance+in+the+rat.">Functional brain activation during retrieval of visceral pain-conditioned passive avoidance in the rat.</a> Pain. 152, 2746-2756 (2011).</li><li>Wasserman, J. K., Koeberle, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+a+hemorrhagic+rat+model+of+central+post-stroke+pain.">Development and characterization of a hemorrhagic rat model of central post-stroke pain.</a> Neuroscience. 161 (1), 173-183 (2009).</li></ol>

저자는 공개 아무것도 없어.

행동 검사에서 CPSP 그룹 기준의 본 프레이 시험 열 통증 검사 기계적 힘의 발 움츠림 역치의 감소 및 일주 전시 -. 5. 결과는 이전의 연구와 일치 하였다 14 에서 [14 C] -IAP있어서 다른 뇌 조각의 정량적 분석을 위해 뇌 영상의 화소 강도에 의존한다. 뇌 영상의 데이터를 평가하기 위해, 픽셀 신호 강도를 정의 하였다. 46.​​979 CPM - 본 연구에서, 환경 배경 신호는...

스트로크 출혈이 신경 병증 성 통증으로 고통 환자의 8 % 이상에서 발생하는 것으로 나타났다과 같은 중앙 뇌졸중 후 통증 (CPSP)라고합니다. 1-3 CPSP가 체 감각 장애, 이에 과민과 이질통을 유도 발생할 수 있습니다. (4) 그러나 , CPSP의 체성 감각 기능 장애의 병태 생리 학적 메커니즘은 불확실 남아있다. 예를 들어, 체성 감각의 손실은 출혈성 뇌 영역에서 신경 deafferentation 기인. 통각 과민은 중앙 통각 신경 세포 또는 중앙 탈 억제, 5, 6하지만 CPSP 증상에 관여하는 신경 기판을 알 수없는 남아의 hyperexcitability에 의해 발생할 수 있습니다. 일부 연구에서는 전전두엽 피질 (dPFC), 주동이의 앞쪽에 cingulate 피질 (ACC), 편도체, 해마, periaqueductal 그레이 (PAG), 주동이의 ventromedial 수질, 서로와의 연결이 침해 처리를 중재 할 것을 제안했다. 추가 7LY, 내측 전두엽 피질 (mPFC) -amygdala 회로는 CPSP의 병태 생리 학적 메커니즘에 8 데이터가 다양하다. 통증 관련 인식에 관여하는 것으로 도시 및 CPSP에 신경 기판의 활성화는 더 조사가 필요했다. [14 C] -Iodoantipyrine (IAP) 흡수는 뇌 활동 CBF의 관계를 가정하여, 간접적으로 지방 뇌 혈류 (rCBF)을 관찰하는 데 사용된다. 비록 [14 C] -IAP 이러한 기능 자기 공명 영상 (fMRI)와 같이, 실시간으로 뇌 활동을 평가할 수 없으며, 이는 여러 가지 장점을 갖는다. 예를 들어, [14 C] -IAP는 자발적으로 측정 병적 인 상태 동안 뇌 이벤트를 발생하기에 적합하다. (9) 또한, [14 C] -IAP 흡수가 마취없이 측정한다. 또한, 자기 공명과 양전자 방출 단층 촬영 (PET)를 포함하는 다른 이미징 방법보다 적은 비용이 든다. 에서 [14 C] -IAP 방법은 measurin에 적합한 것으로 제안되었다시상의 복부 기저 핵 (VB)의 병변에 의해 유도되는 g 자발 통증 (예를 들어, CPSP). (9) 본 프로토콜은 동물 모델에서 시상의 VB의 병변에 의해 유도되는 CPSP의 신경 기판의 참여를 평가하는 [14 C] -IAP 방법을 수행하는 방법에 대해 설명합니다. 이 기술은 행동과의 연결 수준 CPSP 증상을 기초 병리 생리 학적 메카니즘을 결정하는 방법을 제공한다.

<table><tbody><tr><td>&lt;strong&gt;Anesthetic:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Halocarbon Products Corporation&amp;nbsp;</td><td>NDC 12164-002-25</td><td>4%</td></tr><tr><td>&lt;strong&gt;Surgery&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>homeothermic blanket system</td><td>Harvard Apparatus</td><td>Model 50&amp;ndash;7079</td><td>body temperature were maintained at 36.5 - 37.5 &amp;deg;C.</td></tr><tr><td>10 &amp;micro;l micro syringe</td><td>Hamilton</td><td>80008, Model 1701SN</td><td>injected with collagenase</td></tr><tr><td>polyethylene-50 tubing</td><td>Becton, Dickinson and Company</td><td>427411</td><td>catheterized into external jugular vein</td></tr><tr><td>1 c.c syringe</td><td>Terumo Medical Products</td><td>SS-01T</td><td>injected with &lt;sup&gt;14&lt;/sup&gt;C-IAP and saline.</td></tr><tr><td>saline (Sodium Chloride 0.9 gm)</td><td>Taiwan Biotech Co., LTD.</td><td>100-130-0201</td><td>To flush the tube</td></tr><tr><td>&lt;strong&gt;Drugs&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>type 4 collagenase</td><td>Sigma</td><td>C5138-500MG</td><td>0.125 U</td></tr><tr><td>Gentamicin</td><td>Sigma</td><td>G1264-250MG</td><td>6 mg/kg</td></tr><tr><td>Heparin</td><td>Sigma</td><td>H9399</td><td>20 U/ml; 0.1 ml/day</td></tr><tr><td>&lt;sup&gt;14&lt;/sup&gt;C-iodoantipyrine (IAP)</td><td>PerkinElmer</td><td>NEC712</td><td>125 mCi/kg in 300 ml of 0.9% saline</td></tr><tr><td>Potassium chloride</td><td>Merck</td><td>1.04936.1000</td><td>3 M</td></tr><tr><td>&lt;strong&gt;Behavior system:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>von Frey esthesiometer</td><td>Fabrication Enterprises, Inc.</td><td>Baseline Tactile Monofilaments 12-1666</td><td>mechanical hyperalgesia was assessed by measuring the withdrawal response to a mechanical stimulus</td></tr><tr><td>plantar test apparatus</td><td>IITC Life Science</td><td>IITC 390G Plantar Test</td><td>Thermal hyperalgesia was assessed by measuring the hind paw withdrawal latency in response to radiant heat.</td></tr><tr><td>&lt;strong&gt;Brain slice:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Optimal Cutting Temperature compound</td><td>Sakura Fintek Inc</td><td>4583</td><td>embedded the brain</td></tr><tr><td>dry ice</td><td></td><td></td><td>frozen in dry ice/methylbutane (approximately &amp;minus;55 &amp;deg;C)</td></tr><tr><td>methylbutane</td><td>Sigma</td><td>M32631-1L</td><td>frozen in dry ice/methylbutane (approximately &amp;minus;55 &amp;deg;C)</td></tr><tr><td>Cryostat</td><td>&amp;nbsp;Leica Biosystems Nussloch GmbH, Germany</td><td>Leica CM1850</td><td>Coronal brain slice were sectioned on this machine.</td></tr><tr><td>&lt;strong&gt;Data analyze&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>exposure cassettes with a phosphor screen</td><td>Amersham Biosciences&amp;nbsp;</td><td>20 cm x 25 cm</td><td>The slices were dried on glass slides and placed alongside five standard filter papers with graded radioactivity. All of the slides were exposed to the cassettes at &amp;minus;20 &amp;deg;C.</td></tr><tr><td>&amp;gamma;-counter</td><td>Beckman Coulter</td><td>Beckman LS 6500 Liquid Scintillation Counter</td><td>To measure the radioactivity count of the filter papers.</td></tr><tr><td>Typhoon 9410 Variable Mode Imager</td><td>&amp;nbsp;GMI, Inc.</td><td>WS-S9410</td><td>To read&amp;nbsp; phosphor screen which was exposed by brain slice</td></tr><tr><td>Statistical Parametric Mapping (SPM)</td><td>Wellcome Centre for Neuroimaging</td><td>version 8</td><td>all of the brains were averaged to create the final brain template. To determine significant differences between the images in these two groups, the images were derived by subtracting the sham group from the CPSP group.</td></tr><tr><td>ImageJ</td><td>http://imagej.nih.gov/ij</td><td>version 1.46</td><td>Adjacent sections were aligned both manually and using Stack- Reg, an automated pixel-based registration algorithm in ImageJ software. All of the original three-dimensionally reconstructed brains were smoothed and normalized to the reference rat brain model.</td></tr><tr><td>Matlab</td><td>MathWorks</td><td>version 2009b</td><td>used Pearson correlation coefficients to examine the relationships between the CPSP and sham groups. An inter-regional correlation matrix was calculated across animals from each group.</td></tr><tr><td>Pajek</td><td>http://Pajek.imfm.si/</td><td>version 3.06</td><td>Graphical theoretical analysis was performed on networks defined by the above correlation matrices using Pajek software.</td></tr></tbody></table>

autoradiographic measurements of [14c]-iodoantipyrine in rat brain following central post-stroke pain

Approximately 8% of stroke patients present symptoms of central post-stroke pain (CPSP). CPSP is associated with allodynia and hypersensitivity to nociceptive stimuli. Although some studies have shown that neuropathic pain may involve the dorsolateral prefrontal cortex, rostral anterior cingulate cortex, amygdala, hippocampus, periaqueductal gray, rostral ventromedial medulla, and medial thalamus, the neural substrates and their connections that mediate CPSP remain unclear. [14C]-Iodoantipyrine (IAP) uptake can be measured to evaluate spontaneously active pain. It can be used to assess the activation of neural substrates that may be involved in CPSP in an animal model. The [14C]-IAP method in rats is less expensive to perform compared with other brain mapping techniques. The present [14C]-IAP protocol is used to measure the activation of neural substrates that are involved in CPSP that is induced by lesions of the ventral basal nucleus (VB) of the thalamus in a rodent model.

도 1a는 실험적인 타임 라인을 도시한다. 쥐가 행동 검사에 대한 사기 및 CPSP 그룹 (즉, 본 프레이 시험 및 발바닥 시험)에 할당되었다. 실험의 첫 날 기준으로 제공하고, 시험 1 주에 반복 - 4 헤파린 (20 U / ㎖, 0.1 ㎖ / 일) 동안 주입 일주일에 5 P​​E-50 카테터 외부 경정맥을 시행 하였다 주 (4)과 헤파린 주사 후 5. 5 분, [14 C] -IAP가 주입, 희생 마취?...

Watch this Scientific Journal Video about Autoradiographic Measurements of [14C]-Iodoantipyrine in Rat Brain Following Central Post-Stroke Pain at JoVE.com

Autoradiographic Measurements of [14C]-Iodoantipyrine in Rat Brain Following Central Post-Stroke Pain

We present a protocol to measure [14C]-iodoantipyrine (IAP) uptake and assess the activation of neural substrates that are involved in central post-stroke pain (CPSP) in a rodent model.

의 방사 법적 측정 [<sup&gt; (14)</sup&gt; 중앙 후 스트로크 통증에 따라 쥐의 뇌에서 C] -Iodoantipyrine

Approximately 8% of stroke patients present symptoms of central post-stroke pain (CPSP). CPSP is associated with allodynia and hypersensitivity to ...

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Medicine

Autoradiographic Measurements of [14C]-Iodoantipyrine in Rat Brain Following Central Post-Stroke Pain

8.7K 조회수.  Fo Guang University. We present a protocol to measure [14C]-iodoantipyrine (IAP) uptake and assess the activation of neural substrates that are involved in central post-stroke pain (CPSP) in a rodent model.

비디오: Autoradiographic Measurements of [14C]-Iodoantipyrine in Rat Brain Following Central Post-Stroke Pain

2-Vessel Occlusion/Hypotension: A Rat Model of Global Brain Ischemia

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ischemia produces damage to specific brain regions shown to be highly sensitive to ischemia 1. Hippocampal neurons have higher sensitivity to ischemic insults compared to other cell populations, and specifically, the CA1 region of the hippocampus is particularly vulnerable to ischemia/reperfusion 2.
The design of therapeutic interventions, or study of mechanisms involved in cerebral damage, requires a model that produces damage similar to the clinical condition and in a reproducible manner. Bilateral carotid vessel occlusion with hypotension (2VOH) is a model that produces reversible forebrain ischemia, emulating the cerebral events that can occur during cardiac arrest and resuscitation. We describe a model modified from Smith et al. (1984) 2, as first presented in its current form in Sanderson, et al. (2008) 3, which produces reproducible injury to selectively vulnerable brain regions 3-6. The reliability of this model is dictated by precise control of systemic blood pressure during applied hypotension, the duration of ischemia, close temperature control, a specific anesthesia regimen, and diligent post-operative care. An 8-minute ischemic insult produces cell death of CA1 hippocampal neurons that progresses over the course of 6 to 24 hr of reperfusion, while less vulnerable brain regions are spared. This progressive cell death is easily quantified after 7-14 days of reperfusion, as a near complete loss of CA1 neurons is evident at this time.
In addition to this brain injury model, we present a method for CA1 damage quantification using a simple, yet thorough, methodology. Importantly, quantification can be accomplished using a simple camera-mounted microscope, and a free ImageJ (NIH) software plugin, obviating the need for cost-prohibitive stereology software programs and a motorized microscopic stage for damage assessment.

Bilateral carotid occlusion coupled with systemic hypotension produces global brain ischemia in the rat, resulting in damage to the hippocampus with reproducible severity. Animal subjects are impaired with predictable patterns of brain damage, they recover expediently, and mortality rates are comparatively low.

2 혈관 폐색 / 저혈압 : 세계적인 두뇌 국소 빈혈의 쥐 모델

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ...

연구

의학

A Middle Cerebral Artery Occlusion Technique for Inducing Post-stroke Depression in Rats

Post-stroke depression (PSD) is the most recurrent of all psychiatric complications resulting from an ischemic stroke. A greater majority (about 60%) of all ischemic stroke patients suffer from PSD, a disorder considered to be an ischemic stroke-related precursor for increased death and degradation in health. The pathophysiology of PSD is still obscure. To study the mechanism of development and occurrence of PSD further, and to find out a therapy, we attempted to develop a new protocol that requires occluding the middle cerebral artery (MCA) via the internal carotid artery (ICA) in rats. This protocol describes a model of PSD induced in rats through the middle cerebral artery occlusion (MCAO). Also used in the experiment are the Porsolt forced swim test and the sucrose preference test to confirm and evaluate the depressive mood of the rats under investigation. Rather than inserting the catheter through the external carotid artery (ECA), as stipulated for the original procedure, this MCAO technique has the monofilament passing directly through the ICA. This MCAO technique was developed a few years ago and leads to a reduction in mortality and variability. It is generally accepted that the criteria used are preferred in the selection of biological models. The data obtained with this protocol show that this model of MCAO could be a way of inducing PSD in rats and could potentially lead to the understanding of the pathophysiology and the future development of new drugs and other neuroprotective agents.

Here we present a protocol to induce post-stroke depression in rats by occluding the middle cerebral artery via the internal carotid artery. We use the Porsolt forced swim test and the sucrose preference test to confirm and evaluate induced depressive moods.

쥐의 사후 뇌졸중 우울증을 유도 하기 위한 중간 대뇌 동맥 폐색 기술

Post-stroke depression (PSD) is the most recurrent of all psychiatric complications resulting from an ischemic stroke. A greater majority (about 60%) of ...

행동분석학

A Dual Tracer PET-MRI Protocol for the Quantitative Measure of Regional Brain Energy Substrates Uptake in the Rat

We present a method for comparing the uptake of the brain&#39;s two key energy substrates: glucose and ketones (acetoacetate [AcAc] in this case) in the rat. The developed method is a small-animal positron emission tomography (PET) protocol, in which&#160;11C-AcAc and 18F-fluorodeoxyglucose (18F-FDG) are injected sequentially in each animal. This dual tracer PET acquisition is possible because of the short half-life of 11C (20.4 min). The rats also undergo a magnetic resonance imaging (MRI) acquisition seven days before the PET protocol. Prior to image analysis, PET and MRI images are coregistered to allow the measurement of regional cerebral uptake (cortex, hippocampus, striatum, and cerebellum). A quantitative measure of 11C-AcAc and 18F-FDG brain uptake (cerebral metabolic rate; &#956;mol/100 g/min) is determined by kinetic modeling using the image-derived input function (IDIF) method. Our new dual tracer PET protocol is robust and flexible; the two tracers used can be replaced by different radiotracers to evaluate other processes in the brain. Moreover, our protocol is applicable to the study of brain fuel supply in multiple conditions such as normal aging and neurodegenerative pathologies such as Alzheimer&#39;s and Parkinson&#39;s diseases.

Small-animal positron emission tomography enables the assessment of the brain&#39;s two main energy substrates:&#160;glucose and ketones. In the present method, 11C-acetoacetate and 18F-fluorodeoxyglucose are injected sequentially in each animal, and their uptake is measured quantitatively in specific brain regions determined from the magnetic resonance images.

We present a method for comparing the uptake of the brain&#39;s two key energy substrates: glucose and ketones (acetoacetate [AcAc] in this case) in the ...

신경과학

Measurement of Glutamate Uptake using Radiolabeled L-[3H]-Glutamate in Acute Transverse Slices Obtained from Rodent Resected Hippocampus

Glutamate removal from the extracellular space by high-affinity Na+-dependent transporters is essential to ensure that the brain&#39;s intrinsic connectivity mechanisms work properly and homeostasis is maintained. The hippocampus is a unique brain structure that manages higher cognitive functions, and is the subject of several studies regarding neurologic diseases. The investigation of physiological and pathological mechanisms in rodent models can benefit from acute hippocampal slice (AHS) preparations. AHS has the advantage of providing reliable information on cell function since the cytoarchitecture and synaptic circuits are preserved. Although AHS preparations are commonly used in neurochemistry laboratories, it is possible to find some methodological differences in the literature. Considering that distinctive slice preparation protocols might change the hippocampal regions analyzed, this current protocol proposes a standard technique for obtaining transverse AHS from resected hippocampus. This simple-to-perform protocol may be used in mice and rats&#39; experimental models and allow several ex vivo approaches investigating neurochemical dynamics (in dorsal, intermediate and ventral hippocampus) in different backgrounds (e.g., transgenic manipulations) or after in vivo manipulations (e.g., pharmacological treatments or suitable rodent models to study clinical disorders). After dissecting the hippocampus from the rodent brain, transverse slices along the septo-temporal axis (300 &#181;m thick) were obtained. These AHS contain distinct parts of the hippocampus and were subjected to an individual neurochemical investigation (as an example: neurotransmitter transporters using their respective substrates). As the hippocampus presents a high density of excitatory synapses, and glutamate is the most important neurotransmitter in the brain, the glutamatergic system is an interesting target for in vivo observed phenomena. Thus, the current protocol provides detailed steps to explore glutamate uptake in ex vivo AHS using L-[3H]-Glutamate. Using this protocol to investigate hippocampal function may help to better understand the influence of glutamate metabolism on mechanisms of neuroprotection or neurotoxicity.

Measurement of Glutamate Uptake using Radiolabeled L-[3H]-Glutamate in Acute Transverse Slices Obtained from Rodent Resected Hippocampus

This article describes a reliable and simple way to obtain ex vivo acute hippocampal transverse slices from mice and rats using a tissue chopper. Slices obtained from resected hippocampi can be submitted for functional glutamate uptake analysis to investigate glutamatergic system homeostasis.

Glutamate removal from the extracellular space by high-affinity Na+-dependent transporters is essential to ensure that the brain&#39;s intrinsic ...

생화학

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to return to normal in a large majority of patients2, which is mainly due to current lack of clinical treatment for acute stroke. This necessitates understanding the physiological effects of cerebral ischemia on brain tissue over time and is a major area of active research. Towards this end, experimental progress has been made using rats as a preclinical model for stroke, particularly, using non-invasive methods such as 18F-fluorodeoxyglucose (FDG) coupled with Positron Emission Tomography (PET) imaging3,10,17. Here we present a strategy for inducing cerebral ischemia in rats by middle cerebral artery occlusion (MCAO) that mimics focal cerebral ischemia in humans, and imaging its effects over 24 hr using FDG-PET coupled with X-ray computed tomography (CT) with an Albira PET-CT instrument. A VOI template atlas was subsequently fused to the cerebral rat data to enable a unbiased analysis of the brain and its sub-regions4. In addition, a method for 3D visualization of the FDG-PET-CT time course is presented. In summary, we present a detailed protocol for initiating, quantifying, and visualizing an induced ischemic stroke event in a living Sprague-Dawley rat in three dimensions using FDG-PET.

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Brain damage resulting from cerebral ischemia may be non-invasively imaged and studied in rats using pre-clinical positron emission tomography coupled with the injectable radioactive probe, 18F-fluorodeoxyglucose. Further, the use of modern software tools that include volume of interest (VOI) brain templates dramatically increase the quantitative information gleaned from these studies.

과 양전자 방출 단층 촬영을 이용하여 비 침습적 영상 및 뇌허혈의 분석 생활 쥐<sup&gt; (18)</sup&gt; F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to ...

Intracranial Pharmacotherapy and Pain Assays in Rodents

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the development of effective therapeutics. Intracranial pharmacology presents an important tool for understanding the molecular and cellular mechanisms of pain in the brain, as well as for novel treatments. Here we present a protocol that integrates intracranial pharmacology with pain behavior testing. Specifically, we show how to infuse analgesic drugs into a select brain region, which may be responsible for pain modulation. Furthermore, to determine the effect of the candidate drug in the central nerve system, pain assays are performed after intracranial treatment. Our results demonstrate that intracranial administration of analgesic drugs in a targeted region can provide relief of pain in rodents. Thus, our protocol successfully demonstrates that intracranial pharmacology, combined with pain behavior testing, can be a powerful tool for the study of pain mechanisms in the brain.

Here we present a protocol to perform intracranial pharmacological experiments followed by pain behavior assays in rodents. This protocol allows researchers to deliver molecular and cellular targets in the brain, for pharmacologic agents in the treatment of pain.

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the ...

Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it is thought that dysregulation of protein synthesis in the nervous system may be a core phenotype in some developmental disorders. Accurate measurement of rates of cerebral protein synthesis in animal models is important for understanding these disorders. The method that we have developed was designed to be applied to the study of awake, behaving animals. It is a quantitative autoradiographic method, so it can yield rates in all regions of the brain simultaneously. The method is based on the use of a tracer amino acid, L-[1-14C]-leucine, and a kinetic model of the behavior of L-leucine in the brain. We chose L-[1-14C]-leucine as the tracer because it does not lead to extraneous labeled metabolic products. It is either incorporated into protein or rapidly metabolized to yield 14CO2 which is diluted in a large pool of unlabeled CO2 in the brain. The method and the model also allow for the contribution of unlabeled leucine derived from tissue proteolysis to the tissue precursor pool for protein synthesis. The method has the spatial resolution to determine protein synthesis rates in cell and neuropil layers, as well as hypothalamic and cranial nerve nuclei. To obtain reliable and reproducible quantitative data, it is important to adhere to procedural details. Here we present the detailed procedures of the quantitative autoradiographic L-[1-14C]-leucine method for the determination of regional rates of protein synthesis in vivo.

Protein synthesis is&#160;a critical biological process for cells. In brain, it is required for adaptive changes. Measurement of rates of protein synthesis in the intact brain requires careful methodological considerations. Here we present the L-[1-14C]-leucine quantitative autoradiographic method for determination of regional rates of cerebral protein synthesis in vivo.

생체 내 뇌 단백질 합성의 지역 비율 측정을 위한 정량적 자가 방사선 측정 방법

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it ...

PET Imaging of Neuroinflammation Using [11C]DPA-713 in a Mouse Model of Ischemic Stroke

Neuroinflammation is central to the pathological cascade following ischemic stroke. Non-invasive molecular imaging methods have the potential to provide critical insights into the temporal dynamics and role of certain neuroimmune interactions in stroke. Specifically, Positron Emission Tomography (PET) imaging of translocator protein 18 kDa (TSPO), a marker of activated microglia and peripheral myeloid-lineage cells, provides a means to detect and track neuroinflammation in vivo. Here, we present a method to accurately quantify neuroinflammation using [11C]N,N-Diethyl-2-[2-(4-methoxyphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl]acetamide ([11C]DPA-713), a promising second generation TSPO-PET radiotracer, in distal middle cerebral artery occlusion (dMCAO) compared to sham-operated mice. MRI was performed 2 days post-dMCAO surgery to confirm stroke and define the infarct location and volume. PET/Computed Tomography (CT) imaging was carried out 6 days post-dMCAO to capture the peak increase in TSPO levels following stroke. Quantitation of PET images was conducted to assess the uptake of [11C]DPA-713 in the brain and spleen of dMCAO and sham mice to assess central and peripheral levels of inflammation. In vivo [11C]DPA-713 brain uptake was confirmed using ex vivo autoradiography.

PET Imaging of Neuroinflammation Using [11C]DPA-713 in a Mouse Model of Ischemic Stroke

Positron Emission Tomography (PET) imaging of translocator protein 18 kDa (TSPO) provides a non-invasive means to visualize the dynamic role of neuroinflammation in the development and progression of brain diseases. This protocol describes TSPO-PET and ex vivo autoradiography to detect neuroinflammation in a mouse model of ischemic stroke.

애완 동물 Neuroinflammation [11C] DPA-713을 사용 하 여 허 혈 성 뇌졸중의 마우스 모델에서의 영상

Neuroinflammation is central to the pathological cascade following ischemic stroke. Non-invasive molecular imaging methods have the potential to provide ...

Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The method is based on the specific binding of a radioligand to its biological target. Therefore, frozen tissue sections are incubated with radioligand solution, and the binding to the target is subsequently localized by the detection of radioactive decay, for example, by using photosensitive film or phosphor imaging plates. Resulting digital autoradiograms display remarkable spatial resolution, which enables quantification and localization of radioligand binding in distinct anatomical structures. Moreover, quantification allows for the pharmacological characterization of ligand affinity by means of dissociation constants (Kd), inhibition constants (Ki) as well as the density of binding sites (Bmax) in selected tissues. Thus, the method provides information about both target localization and ligand selectivity. Here, the technique is exemplified with autoradiographic characterization of the high-affinity &#947;-hydroxybutyric acid (GHB) binding sites in mammalian brain tissue, with special emphasis on methodological considerations regarding the binding assay parameters, the choice of the radioligand and the detection method.

The method of autoradiography is routinely used to study binding of radioligands to tissue sections for determination of qualitative or quantitative pharmacology.

시각화 및 약리 대상의 특성에 대 한 간단 하 고 강력한 방법으로 autoradiography

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The ...

Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke involves middle cerebral artery occlusion (MCAO). Infarct zone, brain edema and blood-brain barrier (BBB) breakdown are measured as parameters that reflect the extent of brain injury after MCAO. A significant limitation to this method is that these measurements are normally obtained in different rat brain samples, leading to ethical and financial burdens due to the large number of rats that need to be euthanized for an appropriate sample size. Here we present a method to accurately assess brain injury following MCAO by measuring infarct zone, brain edema and BBB permeability in the same set of rat brains. This novel technique provides a more efficient way to evaluate the pathophysiology of stroke.

This protocol describes a novel technique of measuring the three most important parameters of ischemic brain injury on the same set of rodent brain samples. Using only one brain sample is highly advantageous in terms of ethical and economic costs.

설치류 뇌 샘플의 단일 세트에서 뇌졸중 후 뇌 부종, 경색 영역 및 혈액 뇌 장벽 고장 측정

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke ...

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