이 연구는 충남 국립 대학, 연구원의 동양 의학 (KIOM)와 한국 건강 기술 연구의 보조금에 의해 지원 되었다 &#38; D 프로젝트 통해는 한국 보건 산업 개발 연구소 (진흥원), 보건 복지부에 의해 자금 &#38; 복지, 한국 (번호 부여: HI15C0007).

모든 실험 통증 연구에 대 한 국제 협회의 윤리 지침에 따라 실시 했다와 충남대 (에서 기관 동물 관리 및 사용 위원회에 의해 승인 되었다 대전, 한국).
 1. 좌 골 신경에 CCI 유도 <ol><li>집 남성 ICR 마우스 제어 실내 온도 (20-25 ° C에서 유지 하는)와 습도 (40-60%), 12 h 명암 주기에서 20-25 g 무게 무료 음식에 접근 하 고 물입니다. 수술 하기 전에 적어도 일주일의 동물 관리 실에서 쥐에 대 한 새 환경 순응 기간 허용.
	<ol><li>주택, 동안 모양 및 마우스의 동작을 관찰 하 고 비정상적인 운동 활동을 보여주는 마우스를 사용 하지 마십시오. 
		참고: CCI 수술 전에 1 일 폰 Frey 필 라 멘 트를 적용 하 여 뒷 발을의 기계적 감도 측정 하 고 정상적인 기준선 값 자동된 걸음 걸이 분석을 수행. 마우스 제어 및 실험적인 그룹에 무작위로 할당 다음,.</li>
	</ol></li> <li>수술 당일, 2,2,2-tribromoethanol (250 mg/kg, 복 (IP))를 주입 하 여 마우스 anesthetize. 
	참고: 외과 용 가운, 장갑, 마스크 등 개인 보호 장비를 착용 하십시오.
	<li><ol>2,2,2 tribromoethanol의 2.5 g의 무게와 5 mL의 최종 볼륨을 2-메 틸-2-buthanol를 추가 합니다. 멀리 빛 솔루션 유지 (예를 들어, 어두운 컨테이너를 사용 하 여 또는 호 일에 포장).</li>
		<li>40 ° C에가 열 하 고 10-30 분에 대 한 교 반에 의해 완전히 분해를 허용 시 약</li>
		<li>증류수 200 mL의 최종 볼륨을 추가 하 고 잘 혼합 때까지 저 어.</li>
		<li>4 ° C에서 aliquots를 저장 하 고 어둠 속에서 계속. 2 주 후, 마 취 새로운 aliquots로 대체 되어야 합니다.</li>
		<li>주사 1 g 몸 무게 당 20 µ L의 볼륨. 예를 들어 마우스의 몸 무게는 25 g, 500 µ L의 솔루션을 제공 합니다. 이 마 취의 일반적인 기간은 1 h. 
		참고:이 솔루션 빛에 노출 되 면 유독한 될 것입니다 하거나 열 빛과 열에 노출 하지 않도록 조심 해야 합니다. IP 주입 경로 사용 하지 않는 것이 좋습니다.</li>
	</ol></li> <li>마우스 깊은 마 취에 빠지고 때 (즉, 발생 하기 쉬운 위치)를 등 쪽으로 운영 테이블에 그것을 배치 하 고 70% 알코올 면봉으로 오른쪽의 외부 중반 허벅지 영역 소독. 
	참고: 꼬 집 거 나 뒷 발 또는 꼬리에 자극 압력에 응답의 부족을 확인 하 여 깊은 마 취 확인.</li>
	<li>마우스 오른쪽 좌 골 신경에 CCI 유도.
	<ol><li>메스 칼 날 길이가 1.0-1.5 cm의 중반 허벅지 주변 피부에 절 개를 만들기.</li>
		<li>노골적 노출 좌 골 신경에 마이크로-모기 hemostat와 허벅지 근육을 해 부.</li>
		<li>3 번 4-0 크롬 다 봉합 사를 사용 하 여 1.0-1.5 m m 간격으로 노출 된 좌 골 신경 선. 
		참고: 가벼운 떨고 동측 뒷 발에 나타날 때까지 좌 골 신경에서 매 여 느슨한 ligations를 확인 합니다. 쥐의 가짜 그룹 받은 신경 결 찰 하지 않고 제외 하 고 비슷한 조건에서 같은 외과 여.</li>
	</ol></li> <li>3-4 5-0 실크를 사용 하 여 간단한 중단 봉합 수술 열기를 닫고 povidone 요오드 수술의 바깥 영역의 살 균 소독.</li>
	<li>수술 후,가 열 패드에서 깨끗 한 장에 마우스 장소. 동물 마 취에서 회복은 때 케이지 그들의 가정 그들이 돌아갑니다. 
	참고:이 연구에서 항생제 안 사용 되었다. 일단 긍정적인 제어로 매일 50 mg/kg의 복용량에 CCI + GBP 받은 그룹 IP gabapentin (GBP).</li>
</ol> 2. 기계적인 용어인의 측정 (테스트 폰 Frey) 참고: 동측 뒷 발의 발바닥 표면에 폰 Frey 필 라 멘 트의 1 g를 사용 하 여 기계적 자극에 철수 응답의 주파수를 평가.
<ol><li>각 시험 당일 행동 테스트 룸에 마우스를가지고 하 고는 테스트 하기 전에 적어도 30 분 동안 그들의 자신의 가정 장에 마우스 적응 
	참고: 외과 용 가운, 장갑, 마스크 등 개인 보호 장비를 착용.</li>
	<li>금속 메쉬 바닥에 투명 한 아크릴 상자에 마우스를 놓고 마우스 30 분에 대 한 적응</li>
	<li>부드럽게 1 g 폰 Frey 필 라 멘 트의 뒷 발의 발바닥 표면에 적용 하는 필 라 멘 트 굴절 될 때까지.</li>
	<li>적용 필 라 멘 트 자극 동측 뒷 다리를 10 번 이상 10 s 간격으로 발 하 고 결과 기록. 
	참고:이 연구에서 10 타석에서 발 철수 응답 수 발 철수 주파수 (PWF, %)의 비율로 표시 됩니다. 폰 Frey 테스트 CCI 수술 후 매 2 일 수행한.</li>
</ol> 3. 걸음 걸이 분석 자동화 참고: 동물 걷고는 및 매개 변수를 자동으로 다양 한 걸음 걸이 분석 하는 동안 각 발 인쇄를 시각화 하는 걸음 걸이 분석 시스템, 등으로 발 인쇄, 강도, 보 폭 길이, 발 자세 위상, 스텝 시퀀스, 스윙, 스윙 속도, 등이 연구에서 우리는 발 인쇄 영역 및 백분율 데이터 변환 후 하나의 자세 변경 contralateral 사이 왼쪽 동측 오른쪽 뒷 발 대 보였다. 따라서, 50%의 결과 나타냅니다 발 인쇄의 크기와 바닥, 왼쪽과 오른쪽 사이의 접촉 발 지역 기간 동일한. 또한 0%에 접근 하는 더 낮은 데이터 값 표시 크기와 접촉의 기간 contralateral 측면에 비해 동측 뒷 발에 감소 (참조 그림 2의 B 패널 및 그림 3).
<ol><li>새 환경 순응, 계속 10 분 걸음 걸이 분석 장치 내에서 마우스 CCI 수술 전 5 일 하루에 한 번. 
	참고: 새 환경 순응을 포함 한 걸음 걸이 분석 테스트는 어둠 속에서 수행 되어야 합니다.</li>
	<li>걸음 걸이 분석을 위한 검사 실에 쥐 하 고는 테스트 하기 전에 적어도 30 분 동안 그들의 가정 장에 적응
	<ol><li>프로그램 메뉴의 설정 탭에서 설정는 &#34; 산책로 길이 &#34; 30 cm, 그리고 설정 하는 &#34; 기간을 실행 하는 최대 &#34; 5 s와 &#34; 변화를 실행 하는 최대 &#34; 50%.</li>
		<li>선택에서 등록 된 카메라는 &#39; 설치 &#39;의 탭은 &#39; 프로그램 &#39; 메뉴.</li>
		<li>선택 &#34; 수집 열 &#34;에서 &#39; 취득 &#39;의 탭에서 &#39; 프로그램 &#39; 메뉴.</li>
		<li>상태 메시지 다음 클릭은 &#39; 배경 스냅 &#39; (즉, 빈) 빈 보도의 배경 그림을 버튼.</li>
	</ol></li> <li>클릭는 &#39; 수집을 시작 &#39; 버튼 및 산책로;에 마우스를 놓고 녹음 마우스의 움직임에 따라 자동으로 시작 됩니다. 
	참고: 때 마우스는 명확 하 게 산책로에서 산책, 프로그램 자동으로 분류로이 이동 &#34; 준수 실행 &#34; 녹색 아이콘. 소프트웨어를 발 인쇄를 검색 하지 못했습니다, 경우는 실험 빨간색 아이콘을 볼 것 이다 하 고 녹음을 반복 해야 합니다. 적어도 5 개의 성공적인 준수 실행 분석을 위해 필요 하다.</li>
	<li>선택 테스트 후 &#39; 실행 분류 &#39;의 취득 탭에서는 &#39; 프로그램 &#39; 메뉴.</li>
	<li>선택 분석 및 클릭에 대 한 실행은 &#39; 자동 분류 &#39; 단추. 
	참고: 분류, 후 모든 통계 매개 변수는 자동으로 저장 하 고는 실험 클릭 하 여 결과 찾을 수 있습니다 &#34; 보기 실행 통계 &#34; 분석 메뉴에서.</li>
</ol>

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저자는 공개 없다.

현재 폰 Frey 필 라 멘 트를 사용 하 여 기계적 용어인의 측정은 가장 널리 방법 고통 동물 모델에서 보여 주기 위해 사용 촉각 과민 증. Neuropathic 고통에 대 한 동물 모델 개발을 계속, 감각 기능에 대 한 평가의 방법론이 되었습니다 또한 향상 된8,,910,15. 그 보고서에서 그것은 이러한 수정 이전 방법에 ?...

외상, 대사 장애, 염증, 감염, 허 혈에 의해 유도 된 신경의 병 적인 변화 또는 자가 면역 질환은 때때로 neuropathic 고통, 병 변 또는 질병의 직접적인 결과로 발생 하는 통증으로 정의 되는 결과 somatosensory 시스템1에 영향을 미치는. Neuropathic 고통을 일반적으로 견딜 수 이며 불행 하 게도, 기존의 진통제 일반적으로 생성 하지 않습니다 충분 한 고통 릴리프2. Neuropathic 고통의 주요 기능은 자연과 자극 갖는 (즉, 용어인 및 통) 통증을 포함합니다. 용어인 가벼운 터치 등 따뜻한 자극 일반적으로 비-고통 스러운 자극에 나타나는 nociceptive 응답 이다. 통 유해 기계적 또는 열 자극3에 향상 된 고통 응답을 나타냅니다. 비록이 두 증상 모두 비판적으로 환자의 삶의 질 저하, 기계적 용어인 부드러운 촉각 자극에 의해 불러 일으켰다는 가장 악화 증상 부드러운 접촉은 일상 생활에서 피하기 어렵다 때문입니다.
기본 메커니즘 및 neuropathic 고통의 처리를 위한 새로운 진통제를 조사, 고통 응답의 정확한 측정은 필수적입니다. 수많은 neuropathic 고통을 동물 모델 개발 nociceptive 답변은 뒷 다리에 때문에 그것의 높은 접근성4,5,,67지역 발. 따라서, 대부분 통증 응답 평가 폰 Frey 필 라 멘 트 같은 특별 한 악기를 사용 하 여 기계적 자극을 적용 하 여 뒷 발 발바닥 또는 등 쪽 표면에 수행 되었습니다. 가장 자주 사용 되는 방법 중 하나는 최대 이며 딕슨에 의해 설명 하는 방법을 아래로8 그리고 나중 수정 버전9,10. 그러나, 매우 숙련 된, 경험 있는 경험 폰 Frey 테스트를 수행 하는 데 필요한 되며 결과 주관적 일 수 있습니다.
자동된 보 행 분석 시스템은 자유롭게 이동 하는 설치류에서 걷기의 다양 한 매개 변수를 측정 하 여 신경 및 신경 근육 학 장애를 조사할 수 있습니다. 다양 한 신경 손상 동물 모델에서에서 nociception 정도와 여러 치료의 antinociceptive 효과 평가 될 수는 고통 자극11,12,13, 를 추가 하지 않고 14.이 분석 시스템 같은 정적 및 동적 걸음 걸이 매개 변수를 검색할 수 있습니다: 인쇄 영역 (바닥 연락처 전체 발 인쇄의 영역)를 발, 발 강도 (접촉 발 영역의 평균 강도), 길이 (보 폭 같은 발의 연속 게재 위치 사이의 거리), 자세 단계 (단일 뒷 발에 대 한 지상 접촉의 기간), 단계 시퀀스 (순서는 4 발 바닥에 배치 됩니다), 스윙 (스윙 단계 기간), 그리고 스윙 속도 (계산 기간에서 보 폭 길이 스윙과 초당 픽셀으로 표시). 이 종이 사용 분석 시스템 및 하 고 만성 수축 부상 (CCI) neuropathic 마우스를 사용 하 여 폰 Frey 테스트와 함께 데이터의 간단한 비교를 제공 합니다.

<table>
	<tbody>
		<tr>
			<td>0.9% Saline</td>
			<td>JW Pharmaceutical</td>
			<td>N/A</td>
			<td>Vehicle for drugs</td>
		</tr>
		<tr>
			<td>1ml syringe</td>
			<td>BD Plastipak</td>
			<td>300013</td>
			<td>Injecting device</td>
		</tr>
		<tr>
			<td>2, 2, 2-tribromoethanol (97% purity)</td>
			<td>Sigma</td>
			<td>T48402</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>2-methyl-2-buthanol (99% purity)</td>
			<td>Sigma</td>
			<td>152463</td>
			<td>Solvent for 2, 2, 2-tribromoethanol</td>
		</tr>
		<tr>
			<td>Catwalk Automated gait analysis system</td>
			<td>Noldus</td>
			<td>N/A</td>
			<td>Automatic analysis software of aniaml gait</td>
		</tr>
		<tr>
			<td>Chromic catgut (4-0 thickness)</td>
			<td>AILEE</td>
			<td>C442</td>
			<td>Ligature to make chronic constriction injury on the sciatic nerve</td>
		</tr>
		<tr>
			<td>Gabapentin</td>
			<td>Sigma</td>
			<td>Y0001280</td>
			<td>Analgeisc, Used as a positive control drug in this study</td>
		</tr>
		<tr>
			<td>Graefe Forceps</td>
			<td>F.S.T</td>
			<td>11051-10</td>
			<td>Surgical instrument</td>
		</tr>
		<tr>
			<td>Heating Pad</td>
			<td>DAESHIN ELECTRONICS</td>
			<td>M-303AT</td>
			<td>Regulation of body temperature</td>
		</tr>
		<tr>
			<td>ICR Mouse</td>
			<td>Samtaco</td>
			<td>N/A</td>
			<td>Experimental animal</td>
		</tr>
		<tr>
			<td>Mersilk (3-0 thickness)</td>
			<td>ETHICON</td>
			<td>W598H</td>
			<td>Suture material for surgical closure of skin</td>
		</tr>
		<tr>
			<td>Micro-Mosquito</td>
			<td>F.S.T</td>
			<td>13010-12</td>
			<td>Surgical instrument</td>
		</tr>
		<tr>
			<td>Micro-scissors</td>
			<td>F.S.T</td>
			<td>14090-09</td>
			<td>Surgical instrument</td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>F.S.T</td>
			<td>12002-12</td>
			<td>Surgical instrument</td>
		</tr>
		<tr>
			<td>Povidone Iodine</td>
			<td>Firson</td>
			<td>N/A</td>
			<td>Disinfectant to prevent infection after surgery</td>
		</tr>
		<tr>
			<td>Scalpel blade</td>
			<td>F.S.T</td>
			<td>10010-00 (#10)</td>
			<td>Surgical instrument to make an incision</td>
		</tr>
		<tr>
			<td>Scalpel handle</td>
			<td>F.S.T</td>
			<td>10003-12 (#3)</td>
			<td>Surgical instrument to make an incision</td>
		</tr>
		<tr>
			<td>Von-Frey filaments</td>
			<td>North Coast</td>
			<td>NC12775-99</td>
			<td>Measurement device to test sensory function for mechanical stimulation</td>
		</tr>
	</tbody>
</table>

automated gait analysis in mice with chronic constriction injury

폰 Frey 테스트 neuropathic 고통 동물의 감각 기능 검사에 널리 이용 되는 고전적인 방법 이다. 그러나, 그것은 주관적인 데이터와 같은 몇 가지 단점이 고 숙련 된, 경험 있는 실험의 요구 사항이 있다. 날짜 하려면, 다양 한 수정 폰 Frey 방법 개선 하지만 여전히 몇 가지 한계가 있다. 최근 보고서는 걸음 걸이 분석 neuropathic 동물에서 더 정확 하 고 객관적인 데이터를 생성 하는 건의 했다. 이 프로토콜을 쥐에 neuropathic 고통의 결정 자동화 된 걸음 걸이 분석을 수행 하는 방법을 보여 줍니다. 새 환경 순응의 몇 일 후 쥐 발자국을 밝히는 유리 바닥에 자유롭게 걸을 허용 되었다. 발자국과 걸음 걸이의 정량화 발 인쇄, 그네 시간, 발, 등 의 각도의 영역 등 다양 한 걷는 매개 변수 자동 분석 비디오 클립을 통해 수행 된 다음,
이 연구의 주요 목적은 자동화 된 걸음 걸이 분석의 방법론을 설명 하 고 간단히 폰 Frey 필 라 멘 트를 사용 하 여 고전적인 감각 테스트에서 데이터와 비교 하는 것입니다.

우리 폰 Frey 테스트를 수행 하 고 CCI 수술 후 10 일 이내 CCI 쥐에서 걸음 걸이 분석을 자동화. 통계 분석에 대 한 양방향 분산 분석 (ANOVA)의 반복된 측정 전반적인 효과 결정 하 고 Dunnett의 게시물-특별 분석 실험 그룹 중 p-값을 결정 하기 위해 수행 합니다.
그림 1 에 표시 된 결과 고전 폰 Frey 테스트 ?...

Watch this Scientific Journal Video about Automated Gait Analysis in Mice with Chronic Constriction Injury at JoVE.com

Automated Gait Analysis in Mice with Chronic Constriction Injury

Neuropathic 동물 모델에서 고통 응답의 정확한 평가 통증 질환의 병 태 생리학을 조사 하 고 새로운 진통제 개발에 중요 한. 우리는 뒷 다리 발 자동된 보 행 분석 시스템에 의해 설치류의 감각 기능을 결정 하는 과민 하 고 객관적인 방법을 제시.

만성 수축 부상으로 쥐에 있는 자동화 된 걸음 걸이 분석

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some ...

automated-gait-analysis-in-mice-with-chronic-constriction-injury

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

10.0K 조회수.  Chungnam National University. Neuropathic 동물 모델에서 고통 응답의 정확한 평가 통증 질환의 병 태 생리학을 조사 하 고 새로운 진통제 개발에 중요 한. 우리는 뒷 다리 발 자동된 보 행 분석 시스템에 의해 설치류의 감각 기능을 결정 하는 과민 하 고 객관적인 방법을 제시.

비디오: Automated Gait Analysis in Mice with Chronic Constriction Injury

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3. Specifically, it affects the ability of neurons to receive inputs from other cells2 and contributes to the propagation of action potentials4,5. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network6,7. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease8, schizophrenia9,10, and mental retardation11. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.
Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations12.
The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.

We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed.

여러 스케일에서 디지털의 연결 형태론의 자동 숄 분석

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

연구

신경과학

Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences. For instance, addiction to nicotine via tobacco smoking is a leading cause of premature death worldwide (World Health Organization) and is likely caused by an alteration of ion channel distribution in the brain1. Chronic nicotine exposure in both rodents and humans results in increased numbers of nicotinic acetylcholine receptors (nAChRs) in brain tissue1-3. Similarly, alterations in the glutamatergic GluN1 or GluA1 channels have been implicated in triggering sensitization to other addictive drugs such as cocaine, amphetamines and opiates4-6.

Consequently, the ability to map and quantify distribution and expression patterns of specific ion channels is critically important to understanding the mechanisms of addiction. The study of brain region-specific effects of individual drugs was advanced by the advent of techniques such as radioactive ligands. However, the low spatial resolution of radioactive ligand binding prevents the ability to quantify ligand-gated ion channels in specific subtypes of neurons. 

Genetically encoded fluorescent reporters, such as green fluorescent protein (GFP) and its many color variants, have revolutionized the field of biology7.By genetically tagging a fluorescent reporter to an endogenous protein one can visualize proteins in vivo7-10. One advantage of fluorescently tagging proteins with a probe is the elimination of antibody use, which have issues of nonspecificity and accessibility to the target protein. We have used this strategy to fluorescently label nAChRs, which enabled the study of receptor assembly using F&ouml;rster Resonance Energy Transfer (FRET) in transfected cultured cells11.More recently, we have used the knock-in approach to engineer mice with yellow fluorescent protein tagged &alpha;4 nAChR subunits (&alpha;4YFP), enabling precise quantification of the receptor ex vivo at submicrometer resolution in CNS neurons via spectral confocal microscopy12. The targeted fluorescent knock-in mutation is incorporated in the endogenous locus and under control of its native promoter, producing normal levels of expression and regulation of the receptor when compared to untagged receptors in wildtype mice. This knock-in approach can be extended to fluorescently tag other ion channels and offers a powerful approach of visualizing and quantifying receptors in the CNS.

In this paper we describe a methodology to quantify changes in nAChR expression in specific CNS neurons after exposure to chronic nicotine. Our methods include mini-osmotic pump implantation, intracardiac perfusion fixation, imaging and analysis of fluorescently tagged nicotinic receptor subunits from &alpha;4YFP knock-in mice (Fig. 1). We have optimized the fixation technique to minimize autofluorescence from fixed brain tissue.We describe in detail our imaging methodology using a spectral confocal microscope in conjunction with a linear spectral unmixing algorithm to subtract autofluoresent signal in order to accurately obtain &alpha;4YFP fluorescence signal. Finally, we show results of chronic nicotine-induced upregulation of &alpha;4YFP receptors in the medial perforant path of the hippocampus.

We have developed a novel technique of quantifying nicotinic acetylcholine receptor changes within subcellular regions of specific subtypes of CNS neurons to better understand the mechanisms of nicotine addiction by using a combination of approaches including fluorescent protein tagging of the receptor using the knock-in approach and spectral confocal imaging.

만성 니코틴 관리와 마우스 노크 인의 Fluorescently 태그를 Nicotinic 수용체의 분광 공촛점 이미징

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences.  For ...

Sagittal Plane Kinematic Gait Analysis in C57BL/6 Mice Subjected to MOG35-55 Induced Experimental Autoimmune Encephalomyelitis

Kinematic gait analysis in the sagittal plane has frequently been used to characterize motor deficits in multiple sclerosis (MS). We describe the application of these techniques to identify gait deficits in a mouse model of MS, known as experimental autoimmune encephalomyelitis (EAE). Paralysis and motor deficits in mice subjected to EAE are typically assessed using a clinical scoring scale. However, this scale yields only ordinal data that provides little information about the precise nature of the motor deficits. EAE disease severity has also been assessed by rotarod performance, which provides a measure of general motor coordination. By contrast, kinematic gait analysis of the hind limb in the sagittal plane generates highly precise information about how movement is impaired. To perform this procedure, reflective markers are placed on a hind limb to detect joint movement while a mouse is walking on a treadmill. Motion analysis software is used to measure movement of the markers during walking. Kinematic gait parameters are then derived from the resultant data. We show how these gait parameters can be used to quantify impaired movements of the hip, knee, and ankle joints in EAE. These techniques may be used to better understand disease mechanisms and identify potential treatments for MS and other neurodegenerative disorders that impair mobility.

Kinematic gait analysis in the sagittal plane yields highly precise information about how movement is executed. We describe the application of these techniques to identify gait deficits for mice subjected to autoimmune-mediated demyelination. These methods may also be used to characterize gait deficits for other mouse models featuring impaired locomotion.

MOG35-55를 받게 하는 C57BL/6 쥐에 화살 평면 운동학 걸음 걸이 분석 실험적인 자기 면역 뇌 유발

Kinematic gait analysis in the sagittal plane has frequently been used to characterize motor deficits in multiple sclerosis (MS). We describe the ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

Thy1를 사용 하 여 기본 Somatosensory 피 질에서 신경 활동을 이미징-GCaMP6s 유전자 변형 쥐

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

일반적인 마모 셋에 전체 피 질 Electrocorticographic 배열의 만성 이식

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

Investigating Alterations in Caecum Microbiota After Traumatic Brain Injury in Mice

Increasing evidence shows that the microbiota-gut-brain axis plays an important role in the pathogenesis of brain diseases. Several studies also demonstrate that traumatic brain injuries cause changes to the gut microbiota. However, mechanisms underlying the bidirectional regulation of the brain-gut axis remain unknown. Currently, few models exist for studying the changes in gut microbiota after traumatic brain injury. Therefore, the presented study combines protocols for inducing traumatic brain injury using a lateral fluid percussion device and analysis of caecum samples following injury for investigating alterations in the gut microbiome. Alterations of the gut microbiota composition after traumatic brain injury are determined using 16S-rDNA sequencing. This protocol provides an effective method for studying the relationships between enteric microorganisms and traumatic brain injury.

Presented here is a protocol to induce diffuse traumatic brain injury using a lateral fluid percussion device followed by the collection of the caecum content for gut microbiome analysis.

쥐에서 외상성 뇌 손상 후 Caecum Microbiota에서 변경 을 조사

Increasing evidence shows that the microbiota-gut-brain axis plays an important role in the pathogenesis of brain diseases. Several studies also ...

Automated Gait Analysis to Assess Functional Recovery in Rodents with Peripheral Nerve or Spinal Cord Contusion Injury

Peripheral and central nerve injuries are mostly studied in rodents, especially rats, given the fact that these animal models are both cost-effective and a lot of comparative data has been published in the literature. This includes a multitude of assessment methods to study functional recovery following nerve injury and repair. Besides evaluation of nerve regeneration by means of histology, electrophysiology, and other in vivo and in vitro assessment techniques, functional recovery is the most important criterion to determine the degree of neural regeneration. Automated gait analysis allows recording of a vast quantity of gait-related parameters such as Paw Print Area and Paw Swing Speed as well as measures of inter-limb coordination. Additionally, the method provides digital data of the rats&#8217; paws after neuronal damage and during nerve regeneration, adding to our understanding of how peripheral and central nervous injuries affect their locomotor behavior. Besides the predominantly used sciatic nerve injury model, other models of peripheral nerve injury such as the femoral nerve can be studied by means of this method. In addition to injuries of the peripheral nervous systems, lesions of the central nervous system, e.g., spinal cord contusion can be evaluated. Valid and reproducible data assessment is strongly dependent on meticulous adjustment of the hard- and software settings prior to data acquisition. Additionally, proper training of the experimental animals is of crucial importance. This work aims to illustrate the use of computerized automated gait analysis to assess functional recovery in different animal models of peripheral nerve injury as well as spinal cord contusion injury. It also emphasizes the method&#8217;s limitations, e.g., evaluation of nerve regeneration in rats with sciatic nerve neurotmesis due to limited functional recovery. Therefore, this protocol is thought to help researchers interested in peripheral and central nervous injuries to assess functional recovery in rodent models.

Automated gait analysis is a feasible tool to evaluate functional recovery in rodent models of peripheral nerve injury and spinal cord contusion injury. While it requires only one setup to assess locomotor function in various experimental models, meticulous hard- and soft-ware adjustment and training of the animals is highly important.

주변 신경 또는 척수 타박상 부상으로 설치류의 기능적 회복을 평가하기 위한 자동 걸음걸이 분석

Peripheral and central nerve injuries are mostly studied in rodents, especially rats, given the fact that these animal models are both cost-effective and ...

Motor Dual-Tasks for Gait Analysis and Evaluation in Post-Stroke Patients

Eighteen stroke patients were recruited for this study involving the evaluation of cognition and walking ability and multitask gait analysis. Multitask gait analysis consisted of a single walking task (Task 0), a simple motor dual-task (water-holding, Task 1), and a complex motor dual-task (crossing obstacles, Task 2). The task of crossing obstacles was considered to be equivalent to the combination of a simple walking task and a complex motor task as it involved more nervous system, skeletal movement, and cognitive resources. To eliminate heterogeneity in the results of the gait analysis of the stroke patients, the dual-task gait cost values were calculated for various kinematic parameters. The major differences were observed in the proximal joint angles, especially in the angles of the trunk, pelvis, and hip joints, which were significantly larger in the dual motor tasks than in the single walking task. This research protocol aims to provide a basis for the clinical diagnosis of gait function and an in-depth study of motor control in stroke patients with motor control deficits through the analyses of dual-motor walking tasks.

This paper presents a protocol specifically for dual motor task gait analysis in stroke patients with motor control deficits.

뇌졸중 후 환자의 보행 분석 및 평가를 위한 운동 이중 작업

Eighteen stroke patients were recruited for this study involving the evaluation of cognition and walking ability and multitask gait analysis. Multitask ...

Comprehensive Understanding of Inactivity-Induced Gait Alteration in Rodents

It is well known that disuse affects neural systems and that joint motions become altered; however, which outcomes properly exhibit these characteristics is still unclear. The present study describes a motion analysis approach that utilizes three-dimensional (3D) reconstruction from video captures. Using this technology, disuse-evoked alterations of walking performances were observed in rodents exposed to a simulated microgravity environment by unloading their hindlimb by their tail. After 2 weeks of unloading, the rats walked on a treadmill, and their gait motions were captured with four charge-coupled device (CCD) cameras. 3D motion profiles were reconstructed and compared to those of control subjects using the image processing software. The reconstructed outcome measures successfully portrayed distinct aspects of distorted gait motion: hyperextension of the knee and ankle joints and higher position of the hip joints during the stance phase. Motion analysis is useful for several reasons. First, it enables quantitative behavioral evaluations instead of subjective observations (e.g., pass/fail in certain tasks). Second, multiple parameters can be extracted to fit specific needs once the fundamental datasets are obtained. Despite hurdles for broader application, the disadvantages of this method, including labor intensity and cost, may be alleviated by determining comprehensive measurements and experimental procedures.

The present protocol describes three-dimensional motion tracking/evaluation to depict gait motion alteration of rats after exposure to a simulated disuse environment.

설치류의 비활동으로 인한 보행 변화에 대한 포괄적인 이해

It is well known that disuse affects neural systems and that joint motions become altered; however, which outcomes properly exhibit these characteristics ...

Electromagnetic Controlled Closed-Head Model of Mild Traumatic Brain Injury in Mice

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and understanding the mechanisms of how a TBI alters brain function. The availability of multiple animal models of TBI is necessary to model the different aspects and severities of TBI seen in people. This manuscript describes the use of a midline closed head injury (CHI) to develop a mouse model of mild TBI. The model is considered mild because it does not produce structural brain lesions based on neuroimaging or gross neuronal loss. However, a single impact creates enough pathology that cognitive impairment is measurable at least 1 month after injury. A step-by-step protocol to induce a CHI in mice using a stereotaxically guided electromagnetic impactor is defined in the paper. The benefits of the mild midline CHI model include the reproducibility of the injury-induced changes with low mortality. The model has been temporally characterized up to 1 year after the injury for neuroimaging, neurochemical, neuropathological, and behavioral changes. The model is complementary to open skull models of controlled cortical impact using the same impactor device. Thus, labs can model both mild diffuse TBI and focal moderate-to-severe TBI with the same impactor.

The protocol describes mild traumatic brain injury in a mouse model. In particular, a step-by-step protocol to induce a mild midline closed head injury and the characterization of the animal model is fully explained.

생쥐의 경미한 외상성 뇌 손상에 대한 전자기 제어 폐쇄 머리 모델

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and ...