Part of this work was supported by a Grant-in-Aid for Scientific Research (C) from the Japanese Society for the Promotion of Science (25350520, 22500401, 15K06105) and the US-ARMY ITC-PAC Research and Development Project (FA5209-15-P-0175).

동물 관리, 준비, 실험 프로토콜은 도쿄 농공 대학의 동물 연구위원회에 의해 승인되었다. 이 방법의 경우, 래트는 음식과 물을 사용할 수의와, 제어 된 환경 (24 ° C, 12 시간 광 / 암주기)에 수용된다. 광파 멀티 스펙트럼 확산 반사율 이미징 시스템의 1. 건설 <ol><li> 산의 중심 파장 협 아홉 광학 간섭 필터 500, 520, 540, 560, 570, 580, 600, 730, 및 전동 필터 휠의 필터 구멍 760 내지. </li><li> 광대역 백색 광원, 협 대역 간섭 필터들의 상기 세트, 광 가이드, 포집 렌즈, 비디오 줌 렌즈, 및 모노크롬 CCD 카메라와 전동 필터 휠을 사용하여 멀티 스펙트럼 영상 획득 시스템을 구축. 도 2에 도시 된 광학 구성 요소의 배치는 차에 대해 참조 할 수있다건설 절차입니다. 참고 : 조명 각도는 시료 표면에 대해 약 45 °입니다. </li><li> 할로겐 램프 광원을 켜서 간섭 필터, 광 가이드 및 수집 렌즈를 통해 시료 표면을 비 춥니 다. </li><li> CCD 카메라의 운영 소프트웨어를 엽니 다. </li></ol> 2. 동물 준비 참고 :이 프로토콜에서 쥐는 미래의 실험에 사용되지 않았으며 그것은 multispectral 이미지의 측정 직후 희생되었다. <ol><li> 유도 챔버의 입구 포트를 마취 기계의 출구 포트에 튜브로 연결하십시오. 유도 챔버의 출구 포트를 두 번째 튜브가있는 마취 기계의 흡입 포트에 연결하십시오. </li><li> 쥐를 유도 챔버에 넣고 5.0 % 이소 플루 란으로 마취를 유도하십시오. 쥐가 발가락 핀치에 반응하지 않도록 깊이 마취를 유지하십시오. 엘마취 기계의 회전식 손잡이를 사용하여 2.0 % isoflurane까지 세척하십시오. </li><li> stereotaxic 프레임에 쥐 머리를 수정. stereotaxic 프레임에 마 취용 마우스 피스를 연결합니다. </li><li> 마우스 피스의 입구 포트를 마취 기계의 출구 포트에 튜브로 연결하십시오. 마우스 피스의 출구 포트를 마취 기계의 흡입구에 튜브로 연결하십시오. </li><li> 피부 표면이 나타날 때까지 헤어 클리퍼를 사용하여 절개 부위를 넘어 머리 부위를 면도하십시오. </li><li> 외과 용 메스 ( 그림 1 (a) )를 사용하여 머리의 중간 선을 따라 약 20mm 길이로 세로 절개를하고 피하 결합 조직 ( 그림 1 (b) )을 노출 시키십시오. </li><li> 날카로운 curette 또는 협공을 사용하여 피하 결합 조직을 제거하고 두개골 뼈 ( 그림 1 (c) )를 노출하기 위해 머리의 양쪽으로 당겨. </li><li> 두개골 sutur 안에 두개골 뼈에 타원 도랑을 파다.es (coronal suture, sagittal suture, lambdoid 봉합사)를 사용 하였다 ( 그림 1 (d) ). </li><li> 고속 드릴을 사용하여 도랑 속의 두개골 뼈를 천천히 그리고 균일하게 굴착하십시오. </li><li> 뇌 혈관이 나타난 후 뼈의 두께와 강도를 추정하기 위해 협공의 끝으로 얇아진 두개골의 표면을 가볍게 누릅니다. 만약 얇아진 두개골 부위가 쉽게 눌려지면, 고속 드릴로 두개골 뼈의 축소를 종료하십시오. </li><li> 족집게 또는 작은 외과 용 가위를 사용하여 얇은 두개골 조각의 타원형 경계선을 자릅니다. </li><li> 협지를 사용하여 뇌 표면에서 얇은 두개골을 천천히 부드럽게 제거하십시오. </li><li> 생리 식염수로 두개골 창을 부드럽게 목욕하고 약 0.1mm 두께의 투명 유리판으로 덮습니다. </li></ol> 3. 영감을받은 산소의 분율 조절 참고 : 호흡기 상태기 영감 산소의 분율 (FIO 2)를 조절하여 변경할 수있다. <ol><li> 튜브를 사용하여, 다른 Y 형 튜브 커넥터 (커넥터 (2))의 제 포트에 Y 형 튜브 커넥터 (커넥터 (1))의 첫 번째 포트를 연결한다. </li><li> 튜브 커넥터 (1)의 두 번째 포트에 마우스 피스의 입구를 연결한다. </li><li> 튜브를 사용하여, 산소 농도를 모니터 장치에 튜브 커넥터 (1)의 제 3 포트를 연결한다. </li><li> 튜브를 통해 마취 기계의 출구 포트 튜브 커넥터 (2)의 두 번째 포트를 연결한다. </li><li> 튜브를 사용하는 가스 혼합물을 장치의 출구 포트 튜브 커넥터 (2)의 제 3 포트를 연결한다. </li><li> 5 %의 튜브를 사용하여 CO2 가스 실린더 - 고압 95 % O 2 가스 혼합 장치의 하나 개의 입구 포트를 연결한다. </li><li> 튜브를 사용하여 5 % CO 2 가스 실린더 - 고압 95 % N 2 가스 혼합물을 장치의 다른 입구 포트를 연결한다. </li><li> 오 가스 유량을 변경가스 혼합물을 장치에 로터리 노브를 사용하여 F의 O 2 N 2. </li><li> 확인 및 산소 농도를 모니터 장치를 사용하여 2 FIO을 조절한다. </li></ol> 멀티 스펙트럼 확산 반사율 이미지 4. 취득 <ol><li> 참조 이미지의 취득 참고 : 같은 광원, 광학 섬유 등이 실험에 사용 된 광학 부품, 및 감지기는 자신의 스펙트럼 특성을 가지고있다. 따라서, 이들 구성 요소를 통과하는 빛의 강도가 참조 화상으로서 기록한다. 참조 화상은 광원으로부터의 광으로 조명하는 표준 화이트 확산기로 찍은 이미지이다. <ol><li> 수평으로 무대에 표준 흰색 확산을 넣습니다. </li><li> 배럴의 줌 링을 돌려 흰색 확산 판의 표면에 카메라 렌즈를 초점을 맞 춥니 다. </li><li> 로부터 적절한 값을 선택하여 카메라의 적분 시간을 조정드롭 다운 통합 시간의리스트를 카메라의 운영 소프트웨어에서 광의 최대량이 최대 카운트의 약 75 % 인 신호를 생성하도록. 화소 값의 히스토그램을 보면서 신호 강도 레벨이 최대 카운트의 약 75 %가 될 때까지 적분 시간을 조정한다. </li><li> 파일에 이미지를 저장하려면 파일 메뉴에서 &quot;저장&quot;명령을 선택합니다. </li><li> 필터 휠을 돌려 필터 위치를 변경합니다. </li><li> 상술 한 방법에 따라, 다른 파장의 이미지를 저장한다. 파일 이름은 시료와 사용 파장 (예를 들어, W500, W520, W540 ... W760)를 식별해야합니다. </li></ol></li><li> 샘플 이미지의 취득 참고 : 구 파장에 노출 된 쥐의 뇌의 확산 반사 된 빛의 세기의 이미지 캡처와 같은 인수 조건을 사용하여 개인 컴퓨터의 하드 드라이브에 저장됩니다. <ol><li> 조심스럽게 연구를 배치카메라가 쥐의 뇌의 표면에 초점을 맞출 수 있도록 무대에서 천천히 무대 레벨을 조절합니다. </li><li> 파일에 이미지를 저장하려면 파일 메뉴에서 &quot;저장&quot;명령을 선택합니다. </li><li> 필터 휠을 돌려 필터 위치를 변경합니다. </li><li> 상술 한 방법에 따라, 다른 파장의 이미지를 저장한다. 파일 이름은 샘플과 파장 (예를 들어, R500, R520, R540 ... R760)를 식별해야합니다. </li></ol></li><li> 어두운 이미지의 취득 주 : CCD 카메라는 전기 신호에 따라 광 세기를 생성 할 수있다. 그러나, 광이 검출기에 입력하지 않더라도 때문에 전기 회로와 검출기의 잡음에 약간의 출력이있다; 이 어두운 전류 잡음이라고합니다. 정확하게 광의 분광 강도를 측정하기 위해, 암전류 성분이 어두운 화상으로서 기록하고 측정 된 신호로부터 감산한다. 어두운 화상은 화상 포획이다N 개의 광로로 막았다. <ol><li> 할로겐 램프 광원을 끕니다. </li><li> 차폐 판을 이용하여 CCD 카메라 시스템의 광 경로를 차단. </li><li> 파일에 이미지를 저장하려면 파일 메뉴에서 &quot;저장&quot;명령을 선택합니다. 파일 이름은 샘플 (예를 들어, 다크)를 식별해야합니다. </li></ol></li></ol> 5. 헤모글로빈 콘텐츠 및 빛 산란 매개 변수를 시각화 참고 : 다중 스펙트럼 확산 반사율 이미지의 집합은 개인용 컴퓨터의 하드 드라이브에 저장하고 오프라인으로 분석된다. 아홉 개 파장들 (500, 520, 540, 560, 570, 580, 600, 730, 및 760 ㎚)에서 멀티 스펙트럼 확산 반사율 영상의 몬테카를로 시뮬레이션 (19)를 이용한 다중 회귀 분석은 다음 두 차원 시각화 수행 산소화 헤모글로빈 농도, 탈산 소화 헤모글로빈 농도, 총 헤모글로빈 농도 지역 뇌 산소 포화도, 및 배설물의지도전원을 tering. 자세한 알고리즘은 문헌 17, 18 년에 출판되었다. <ol><li> 참조 화상과 각 파장에서의 샘플 화상 모두로부터 어두운 화상을 뺀다. </li><li> 각 파장 λ에서 기준 화상에 의한 샘플 화상을 정규화한다. 확산 반사율 화상 R로서 표준화 이미지를 처리한다. </li><li> 각 파장 λ에서의 확산 반사율 화상 R의 역수의 대수를 취함으로써 흡광도 (또는 광학 밀도) 영상 A를 계산한다 : <img alt="수학 식 1" src="/files/ftp_upload/55399/55399eq1.jpg" /> (1) </li><li> y 평면 뇌 표면 얻어진 구조 정보를 표시하고, Z는시킴으로써 행한다 스펙트럼 정보를 표시 -은 X가 파장의 순서에서 흡광도 이미지를 적층하여 입체 매트릭스를 생성한다. </li><li> 체육각각의 XY 좌표를 흡광도 스펙트럼 A (λ)에 대한 다중 회귀 분석을 rform. </li><li> 단계 5.5 독립 변수와 종속 변수 HBO (λ) ε 산소화 헤모글로빈의 몰 흡광 계수 스펙트럼 및 탈산 소화 헤모글로빈 ε의 HBR (λ)로 흡광도 스펙트럼 A (λ)를 사용하여 (HBO ε의 값을 문서 (λ) 및 ε HBR (λ)의) 표 1에 제공된다. </li><li> 세 회귀의 2 차원지도 (화상)을 확인하는 것은 HBO하는 HBR 및 0 계수. </li><li> 순서에 HBO하는 HBR, 및 0, 회귀 계수의 이미지를 적층하여 입체 매트릭스를 생성 어디 <em&gt; X - Y 평면 뇌 표면의 Z시킴으로써 행한다 대해 얻어진 구조 정보를 나타낸다는 회귀 계수를 나타낸다. </li><li> 산소화 헤모글로빈 농도 C HBO, 탈산 소화 헤모글로빈 농도 C의 HBR, 각각 XY에서 HBO하는 HBR 및 0 계수 그가 β 값 (다음의 실험식을 이용하여 좌표 회귀 세트로부터 산란 전원 (B)을 계산 HBO, I, β HBR, I 및 β 0, I는 (i = 0,1,2,3)는 표 2에 제공된다) : <img alt="수학 식 2" src="/files/ftp_upload/55399/55399eq2.jpg" /> (2) <img alt="식 (3)" src="/files/ftp_upload/55399/55399eq3.jpg" /> (삼) <img alt="수학 식 4" src="/files/ftp_upload/55399/55399eq4.jpg" /> (4) </li><li> 산소화 헤모글로빈 농도 C HBO, 탈산 소화 헤모글로빈 농도 C HBR 및 산란 전원 (B)의 2 차원지도 (화상)을 확인한다. </li><li> 각 X에서 C 및 C HBO HBr을 합산하여 총 헤모글로빈 농도 C의 HBT의 2 차원지도를 계산 - y 좌표. </li><li> Y 좌표 - 각각 X에서 총 헤모글로빈 농도 C의 HBT하여 산소화 헤모글로빈 농도 C HBO 나누어 지역 뇌 산소 포화도 RSO 2의 2 차원지도를 계산한다. </li></ol>

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The authors have nothing to disclose.

이 프로토콜에서 가장 중요한 단계는 두개골 창을 만들 수있는 얇아 두개골 지역의 제거이다; 이 예기치 않은 출혈을 방지하기 위해주의 깊게 수행해야합니다. 이 단계는 높은 정밀도로 반사 이미지를 확산 다중 분광 높은 품질을 얻는 것이 중요하다. 실체 현미경의 사용은 수술 가능한 경우에 권장됩니다. 젤라틴 스폰지의 작은 조각 지혈하는 데 유용합니다. 이 문서에서 ?...

멀티 스펙트럼 확산 반사율 영상은 피질 조직에서 고유의 광 신호 (손실이 최소화)의 공간지도를 획득하기위한 가장 일반적인 방법이다. 형태 학적 변화에 의해 유발 된 광 산란 특성의 광 흡수 변화 의한 피질 혈류 역학에 대한 산란 성질, 감소 또는 미토콘드리아 내의 사이토 크롬의 산화에 따라 흡수 편차 및 변형 : 손실이 최소화 주로 세 현상에 기인하는 생체의 뇌에서 관찰 1. 근적외선 (NIR) 분광 범위 (VIS)에 보이는 빛을 효과적으로 흡수 및 생체 조직에 의해 산란된다. 생체의 뇌의 확산 반사율 스펙트럼은 흡수 및 산란 스펙트럼을 특징으로한다. 감소 된 스 캐터링 계수는 단조로운 산란 스펙트럼 전시에 VIS 투 NIR 파장 범위의 결과 뇌 조직들 &#39;μ긴 파장에서 더 작은 크기를 보내고. 감소 된 산란 스펙트럼 계수 μ는 S &#39;(λ)는 s의 μ로서 멱 함수 (2), (3)의 형태로 근사 할 수있는&#39;(λ)는 λ × -b =. 산란 파워는 조직 B (2, 3) 살아있는 생물에서 산란의 크기에 관련된다. 조직 및 대뇌 피질의 생체 조직의 생존을 감소 형태 학적 변화는 생물학적 산란 4, 5, 6, 7, 8, 9의 크기에 영향을 미칠 수있다. 멀티 스펙트럼 확산 반사율 촬상 용 광학 시스템을 용이 백열 리로 구성 될 수있다GHT 원본, 간단한 광학 구성 요소 및 단색 충전 결합 소자 (CCD). 따라서, 다양한 알고리즘과 멀티 스펙트럼 확산 반사율 촬상 광학 시스템은 피질 혈류 역학 및 / 또는 티슈 형태 10, 11, 12, 13, 14, 15, 16, 17, 18을 평가하기 위해 사용되었다. 본 문서에 기재된 방법은 혈류 역학과 종래의 멀티 스펙트럼 확산 반사율 이미징 시스템을 사용하여 생체 내에서 래트 뇌 조직의 산란 특성 모두를 시각화하는데 사용된다. 대안 기술을 통해이 방법의 장점은 대뇌 혈류 역학과 대뇌 피질의 조직 모두의 시공간적 변화를 평가 할 수있는 기능입니다형태뿐만 아니라 다양한 뇌 기능 장애의 동물 모델에의 적용. 따라서, 방법은 외상성 뇌 손상, 간질 발작, 뇌졸중, 허혈의 조사에 적합합니다.

<table border="0" cellpadding="0" cellspacing="0" width="856">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="36">
			<td height="36" style="height:36px;width:289px;">150-W halogen-lamp light source</td>
			<td style="width:281px;">Hayashi Watch Works Co., Ltd, Tokyo, Japan</td>
			<td style="width:119px;">LA-150SAE</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;">Light guide</td>
			<td style="width:281px;">Hayashi Watch Works Co., Ltd, Tokyo, Japan</td>
			<td>LGC1-5L1000</td>
			<td></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;">Collecting lens</td>
			<td style="width:281px;">Hayashi Watch Works Co., Ltd, Tokyo, Japan</td>
			<td>SH-F16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Interference filters l@500nm</td>
			<td>Edmund Optics Japan Ltd, Tokyo, Japan</td>
			<td>#65088</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Interference filters l@520nm</td>
			<td>Edmund Optics Japan Ltd, Tokyo, Japan</td>
			<td>#65093</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Interference filters l@540nm</td>
			<td>Edmund Optics Japan Ltd, Tokyo, Japan</td>
			<td>#65096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Interference filters l@560nm</td>
			<td>Edmund Optics Japan Ltd, Tokyo, Japan</td>
			<td>#67766</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Interference filters l@570nm</td>
			<td>Edmund Optics Japan Ltd, Tokyo, Japan</td>
			<td>#67767</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Interference filters l@580nm</td>
			<td>Edmund Optics Japan Ltd, Tokyo, Japan</td>
			<td>#65646</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Interference filters l@600nm</td>
			<td>Edmund Optics Japan Ltd, Tokyo, Japan</td>
			<td>#65102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Interference filters l@730nm</td>
			<td>Edmund Optics Japan Ltd, Tokyo, Japan</td>
			<td>#65115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Interference filters l@760nm</td>
			<td>Edmund Optics Japan Ltd, Tokyo, Japan</td>
			<td>#67777</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Motorized filter wheel&#160;</td>
			<td>Andover Corporation, NH, USA</td>
			<td>FW-MOT-12.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">16-bit cooled CCD camera</td>
			<td>Bitran, Japan</td>
			<td>BS-40</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Video zoom lens</td>
			<td>Edmund Optics Japan Ltd, Tokyo, Japan</td>
			<td>VZMTM300i</td>
			<td></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:289px;">Spectralon white standard with 99% diffuse reflectance</td>
			<td>Labsphere Incorporated, North Sutton, NH, USA</td>
			<td>SRS-99-020</td>
			<td></td>
		</tr>
	</tbody>
</table>

simultaneous evaluation of cerebral hemodynamics and light scattering properties of the in vivo rat brain using multispectral diffuse reflectance imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system. This system is constructed from a broadband white light source, a motorized filter wheel with a set of narrowband interference filters, a light guide, a collecting lens, a video zoom lens, and a monochromatic charged-coupled device (CCD) camera. An ellipsoidal cranial window is made in the skull bone of a rat under isoflurane anesthesia to capture in vivo multispectral diffuse reflectance images of the cortical surface. Regulation of the fraction of inspired oxygen using a gas mixture device enables the induction of different respiratory states such as normoxia, hyperoxia, and anoxia. A Monte Carlo simulation-based multiple regression analysis for the measured multispectral diffuse reflectance images at nine wavelengths (500, 520, 540, 560, 570, 580, 600, 730, and 760 nm) is then performed to visualize the two-dimensional maps of hemodynamics and the light scattering properties of the in vivo rat brain.

생체 래트 뇌로부터 취득한 확산 반사율의 대표적인 분광 화상 500, 520, 540, 560, 570에서도 3 이미지 표시되었으며 580 명확 대뇌 피질에서 혈관의 밀도 네트워크 시각화 nm의. 혈관 (600), (730)에서 이미지에 주변 조직 관찰, 및 760 nm의 사이의 콘트라스트의 저하는 이상과 NIR 파장의 광에 의한 헤모글로빈의 낮은 흡수를 반영한다. <p class="jove_content" fo:keep-to...

Watch this Scientific Journal Video about Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging at J

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

뇌 혈류 역학 및 생체 내 쥐의 뇌 조직의 산란 특성의 동시 평가는 종래의 멀티 스펙트럼 확산 반사율 촬상 시스템을 이용하여 설명된다.

대뇌 혈류와의 광 산란 특성을 동시에 평가<em&gt; 생체</em&gt; 다중 스펙트럼 확산 반사 이미징을 이용한 쥐 뇌

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional ...

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Research

JoVE Journal

Neuroscience

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

7.6K 조회수.  Tokyo University of Agriculture & Technology.  뇌 혈류 역학 및 생체 내 쥐의 뇌 조직의 산란 특성의 동시 평가는 종래의 멀티 스펙트럼 확산 반사율 촬상 시스템을 이용하여 설명된다. 

비디오: Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

쥐 두뇌에 동시 fMRI와 전기 생리학

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

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Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

초음파 혈액 - 뇌 장벽 파괴 및 망간 향상된 MRI를 사용하여 기능성 Neuroimaging

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.

Functional studies of the auditory system in mammals have traditionally been conducted using spatially-focused techniques such as electrophysiological recordings. The following protocol describes a method of visualizing large-scale patterns of evoked hemodynamic activity in the cat auditory cortex using functional magnetic resonance imaging.

하이 필드의 fMRI를 사용하여 성인 고양이의 청각 피질의 기능적 이미징

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

설치류에 동시 fMRI를 깊은 뇌 자극

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

동시<em&gt; 생체</em두 망막의&gt; 기능 테스트로<em&gt; 생체</em&gt; 전위도 시스템

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

동시에 두 광자<em&gt; 생체</em&gt; 마우스 Retrosplenial 코어 텍스에서 시냅틱 입력의 이미징 및 시냅스 대상

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types that comprise the BBB; these cells have a very high-energy demand, which requires optimal mitochondrial function. In the case of disease or injury, the mitochondrial function in these cells can be altered, resulting in disease or&#160;the opening of the BBB. In this manuscript, we introduce a method to measure mitochondrial function in CVE cells by using whole, intact cells and a bioanalyzer. A mito-stress assay is used to challenge the cells that have been perturbed, either physically or chemically, and evaluate their bioenergetic function. Additionally, this method also provides a useful way to screen new therapeutics that have direct effects on mitochondrial function. We have optimized the cell density necessary to yield oxygen consumption rates that allow for the calculation of a variety of mitochondrial parameters, including ATP production, maximal respiration, and spare capacity. We also show the sensitivity of the assay by demonstrating that the introduction of the&#160;microRNA, miR-34a, leads to a pronounced and detectable decrease in mitochondrial activity. While the data shown in this paper is optimized for the bEnd.3 cell line, we have also optimized the protocol for primary CVE cells, further suggesting the utility in preclinical and clinical models.

Endothelial cell mitochondria are critical to maintain blood-brain-barrier integrity. We introduce a protocol to measure bioenergetic function in cerebral vascular endothelial cells.

대뇌 혈관 내피 세포의 생체 에너지 기능의 평가

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

2D 문화 시스템을 사용 하 여 기본 대뇌 피 질 세포의 영상 라이브

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

Diffuse Reflectance Spectroscopy: Getting the Capillary Refill Test Under One's Thumb

The capillary refill test was introduced in 1947 to help estimate circulatory status in critically ill patients. Guidelines commonly state that refill should occur within 2 s after releasing 5 s of firm pressure (e.g., by the physician&#39;s finger) in the normal healthy supine patient. A slower refill time indicates poor skin perfusion, which can be caused by conditions including sepsis, blood loss, hypoperfusion, and hypothermia. Since its introduction, the clinical usefulness of the test has been debated. Advocates point out its feasibility and simplicity and claim that it can indicate changes in vascular status earlier than changes in vital signs such as heart rate. Critics, on the other hand, stress that the lack of standardization in how the test is performed and the highly subjective nature of the naked eye assessment, as well as the test&#39;s susceptibility to ambient factors, markedly lowers the clinical value. The aim of the present work is to describe in detail the course of the refill event and to suggest potentially more objective and exact endpoint values for the capillary refill test using diffuse polarization spectroscopy.

This protocol describes how the use of diffuse polarization spectroscopy can improve the clinical usefulness of the capillary refill test. We suggest a more detailed analysis of the course of the capillary refill in healthy volunteers using diffuse reflectance spectroscopy videos and new informatic endpoints.

The capillary refill test was introduced in 1947 to help estimate circulatory status in critically ill patients. Guidelines commonly state that refill ...

Induction of Diffuse Axonal Brain Injury in Rats Based on Rotational Acceleration

Traumatic brain injury (TBI) is a major cause of death and disability. Diffuse axonal injury (DAI) is the predominant mechanism of injury in a large percentage of TBI patients requiring hospitalization. DAI involves widespread axonal damage from shaking, rotation or blast injury, leading to rapid axonal stretch injury and secondary axonal changes that are associated with a long-lasting impact on functional recovery. Historically, experimental models of DAI without focal injury have been difficult to design. Here we validate a simple, reproducible and reliable rodent model of DAI that causes widespread white matter damage without skull fractures or contusions.

This protocol validates a reliable, easy-to-perform and reproducible rodent model of brain diffuse axonal injury (DAI) that induces widespread white matter damage without skull fractures or contusions.

회전 가속도에 따라 쥐에 확산 축 차 뇌 손상의 유도

Traumatic brain injury (TBI) is a major cause of death and disability. Diffuse axonal injury (DAI) is the predominant mechanism of injury in a large ...

대뇌 혈류와의 광 산란 특성을 동시에 평가

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