저자는 올가 샤르마, 테레사 헌트 및 기술 지원에 대한 DZNE 현미경과 동물 시설의 직원을 감사드립니다.

동물을 포함한 모든 절차는 노르 트라 인베스트 팔렌 주 환경청 (LANUV)의 동물 보호위원회의 승인을 수행 하였다. 동물을 수용하고, 12 시간 광 / 암주기 및 음식과 물에 자유롭게 접근하는 표준 조건에 따라 든다고 하였다. 1. 동물 모델 <ol><li> Tg가 서로 엇 hemizygous (GFAP의 -luc +/-) hemizygous bigenic의 Tg (M83 +/- : GFAP의 -luc +/-) 생성 hemizygous의 Tg (M83 +/-) 마우스와 마우스 마우스 (15), (16). </li><li> 개똥 벌레 루시퍼 라제 (12), (17)을위한 트랜스 코딩 인간의 α- 시누 클레인의 표준 PCR과 존재를 실시간 PCR과 자손의 유전자형. </li><li> 여섯 - 투 - 8 주 시대에 동물을 접종한다. </li></ol> 2. 접종 제조 <ol><li> 미주리 준비로되어있는 한 20 mM 트리스 - 염산 (pH를 7.2) 150 mM의 NaCl을 함유하는 TBS 버퍼 (TBS) 완충액에 A53T 돌연변이와 nomeric 재조합 인간의 α- 시누 클레인은 앞서 설명한 14. </li><li> 800 rpm에서 5 일간 37 ℃에서 오비탈 진탕 단량체 재조합 인간의 α- 시누 클레인의 3 ㎍ / μL 교반하여 접종을 위해 섬유상의 α- 시누 클레인을 준비한다. </li><li> intraglossal 접종에 대한 복강 내 또는 2 ㎍ / μL 1 개 ㎍ / μL의 최종 농도에 도달하기 위해 인산 완충 용액 (PBS)에 브릴 어셈블리 희석. </li><li> 얼음 (각 펄스와 50 %로 설정된 진폭의 1 초 휴지 기간의 0.5 40 펄스)을 1 분 동안 초음파 처리와로드 알파 시누 클레인 브릴을 조각. 교차 접종에 의한 오염을 방지합니다. 다른 접종을 준비하는 깨끗한 초음파 프로브를 사용합니다. </li></ol> 3. Intraglossal 주사 <ol><li> intrape과 동물을 마취100 ㎎ / ㎏의 케타민과 10 밀리그램 / kg의 자일 라진 ritoneal 주입. 동물의 발가락을 꼬집어하고 적절한 마취을 보장하기 위해 자사의 사지를 철회하지 않는 것을 확인합니다. 마취 동안 건조를 방지하기 위해 동물의 눈에 수의사 연고를 사용합니다. </li><li> 신중 연구자 향 머리와 등쪽 누운 자세에서 가열 플레이트 상에 접착 테이프 narcotized 동물을 고정한다. </li><li> 초음파의 α- 시누 클레인 브릴 또는 PBS 5 μL와 27 G 일회용 피하 주사기를 채우기. </li><li> 동물의 입이 열려 개최 톱니 모양의 팁을 하나의 뭉툭한 코 엄지 손가락 집게를 사용하여 집게의 작은 제 2 쌍은주의 깊게 주입을위한 혀의 바닥면에 액세스 할 수 있도록하기 위해 혀를 당겨. </li><li> 설하 신경 부근의 혀의 오른쪽 또는 왼쪽 하단 측에 주사기 바늘을 주입한다. 천천히 5 초 후 접종을 주입. 천천히 접종 있도록 5 초 후 바늘을 철회조직을 침투하고 바늘을 철회하면서 손실되지 않습니다. </li><li> 그 고정물에서 동물을 해제하고 완전한 복구 될 때까지 지속적인 모니터링에서 가열 플레이트에 둡니다. </li></ol> 4. 복강 내 주사 <ol><li> 복강 내 주사를 위해, 2 L / min의 유량으로 2 %로 설정 증발기를 사용하여 이소 플루 란 / 산소 흡입 마취 챔버 내에 곧 동물을 마취시키다. 동물의 발가락을 꼬집어하고 적절한 마취을 보장하기 위해 자사의 사지를 철회하지 않는 것을 확인합니다. </li><li> 초음파의 α- 시누 클레인 브릴 또는 PBS 50 μL와 27 G 일회용 피하 주사기를 채우기. </li><li> 직접 마우스의 복강에 주사하고, 조사원으로부터 떨어져 대면하는 헤드와 등쪽 위치 동물 잡고 하향으로 대략 45 °로 복부 벽 뒤에있는 소장 또는 맹장 관통 피한다. 그들이 회복 될 때까지 동물을 모니터링마취에서. </li></ol> 5. 생물 발광 영상 <ol><li> 이미지의 Tg (M83 +/- : GFAP의 -luc +/-) 마우스 생물 발광 이미징 시스템을 사용하는 모든 2-4주. </li><li> 곧 묘화 2 L / min의 유량으로 2 %로 기화기로 이소 플루 란 / 산소 챔버에서 쥐를 마취 전에. 동물의 발가락을 꼬집어하고 적절한 마취을 보장하기 위해 자사의 사지를 철회하지 않는 것을 확인합니다. </li><li> 면도와 제모 크림 쥐의 머리를 털을 뽑다. 불특정 생물 발광을 차단하는 비 자극 마커와 블랙의 귀를 색상. </li><li> 30 ㎎ / ㎖의 스톡 용액에 D-PBS 칼륨 염 루시페린, 루시 페라 제의 기판을 희석. -20 ° C에서 1 mL를 씩이 보관하십시오. 노광에서 용해 D-루시페린 보호. </li><li> 주입하기 전에 동물의 무게를. 150 mg / kg (체중)의 주입을위한 D-루시페린의 정확한 양을 계산한다. 복강 주사 D-luciferin 및 마취 챔버에 동물을 반환한다. </li><li> 이미징 소프트웨어를 시작합니다. 이미징 시스템이 도달하면 작동 온도 제어판에서 &#39;초기화&#39;버튼을 클릭하여 시스템을 초기화합니다. </li><li> 버튼 &#39;발광&#39;과 &#39;사진&#39;을 선택합니다. 60 초, &#39;보통&#39;의 F / 정지 &#39;1&#39;로 비닝 (binning)과 &#39;끄기&#39;로 EM 이득에 노출 시간을 설정합니다. </li><li> 여기 필터 &#39;열기&#39;을 &#39;차단&#39;하고, 발광 필터로 설정되어 있는지 확인하고, 1.50 cm 피사체 높이를 설정. </li><li> 텐 분 D-루시페린과 주사 후, 촬상 챔버 내의 가열 플레이트 상에 동물을 배치하고 그 총구 올바르게 마취 출구에 배치되도록. 흡입 챔버로부터 이소 플루 란 / 산소 유량을 닫고 0.25 L / min의 유량으로 2 %의 증착과 촬상 챔버에 대한 흐름을 연다. 이미징 챔버 홍보의 문을 닫습니다operly. </li><li> 60 초 걸리는 생물 발광을 측정하기 위해 &#39;획득&#39;버튼을 클릭합니다. 이소 플루 란의 흐름을 중지하고 완전히 자신의 새장에 다시 반환 후 회복 될 때까지 동물을 모니터링 할 수 있습니다. </li><li> 생물 발광을 정량화하기 위해 이미징 소프트웨어를 사용합니다. &#39;도구 팔레트&#39;내에서의 투자 수익 (ROI) 도구 &#39;를 선택합니다. 의 투자 수익 (ROI) 도구 &#39;에서&#39;유형 &#39;에서&#39;측정 투자 수익 (ROI) &#39;를 선택합니다. </li><li> 의 투자 수익 (ROI) 도구 &#39;내에서 &quot;원&quot;도구를 클릭하고 풀다운 메뉴에서 그릴 로아의 수를 선택 - 이상적으로&#39;3 &#39;세 마우스에 대해. </li><li> 이미지에서 각 ROI와 아래는 &#39;속성&#39;1.25 cm에 폭과 높이를 조정 마우스 오른쪽 단추로 클릭합니다. 정량화 뇌 영역에 걸쳐 각각의 투자 수익 (ROI)을 배치합니다. </li><li> 화상의 상부 좌측에, &quot;빛 (광자) &#39;단위로 패널을 설정. </li><li> &#39;도구 팔레트&#39;내 선택 &#39;이미지 보정&#39;최소를 조정하고0.20e6에서 1.00e6에 인스턴스의 &#39;색 눈금&#39;의 최대. </li><li> &#39;파일&#39;에서 &#39;저장&#39;을 사용하여 데이터를 저장합니다. </li></ol> 6. 생화학 분석 <ol><li> 설립 프로토콜 (18), (19)에 따라 뇌와 척수를 분리합니다. </li><li> 조직 균질 기에서 6,000 rpm으로 30 초 두 사이클로 (PBS에 1 배 희석), 프로테아제 및 포스파타제 억제제의 존재하에 별도로 PBS에서 전체 뇌와 척수 전체 샘플 균질화. 뇌와 척수 시료를 20 % (중량 / 부피)의 최종 농도로 균질화되었는지 확인. </li><li> 10 s의 일정한 펄스와 50 %로 설정된 진폭 얼음들을 유지하면서 두 샘플을 초음파 처리. </li><li> PBS 중 1.5 M NaCl로 1 : 1 그들 희석 PBS 750 mM의 NaCl을 10 %의 균질을 조정한다. </li><li> 세포 파편을 분리하기 위해 5 분으로 1,000 XG에서 균질 원심 분리4 ° C. 다음 단계의 상층 액을 유지하고 알약을 폐기합니다. </li><li> 제조업체의 지시에 따라 bicinchoninic 산 (BCA) 분석을 사용하여 균질의 단백질 농도를 측정한다. 얼음 위에서 15 분 동안 10 % (중량 / 부피)의 최종 농도에서 N-laurylsarcosyl 척수 균질의 뇌 균질 물에서 총 단백질 1.0 mg을 0.8 mg의 인큐베이션. </li><li> 증류수 10 % (중량 / 부피)의 3 mL의 슈 크로즈 쿠션 상에 균질 오버레이, 4 ° C에서 1 시간 동안 465,000 XG 그들을 초 원심 분리기. </li><li> 4 % 도데 실 황산나트륨 (SDS), 2 % β 메르 캅토 에탄올, 192 mM의 글리신, 25 개 mM 트리스, 5 % (중량 / 부피) 수 크로스를 함유하는 완충액에 재현 탁 펠릿. </li><li> 5 분 동안 100 ℃에서 샘플을 비등하고 morpholineethanesulfonic 술폰산 (MES) 버퍼 시스템에 대한 전기 SDS 폴리 아크릴 아미드 겔 상에 로딩들을. </li><li> 주사 전자 현미경 (SEM)으로 분리 된 폴리 비닐 리덴 디 플루오로 단백질 (PVDF) 멤브레인 전송시스템을 모래 바닥 idry. </li><li> 가교 35 RPM으로 로터에 실온에서 30 분 동안 PBS에서 (부피 / 부피) 포르말린 용액 멤브레인 단백질 0.4 %의 막을 인큐베이션. </li><li> 0.05 % (부피 / 부피의)을 포함하는 TBS 버퍼 트윈 20, 실온에서 1 시간 동안 5 % (중량 / 부피) 우유에서 막을 차단. </li><li> 4 ℃에서 밤새 블로킹 완충액에 일차 항체로 블롯을 인큐베이션. </li><li> TBS 버퍼 막 세 번 세척하고, 실온에서 1 시간 동안 TBS에서 페 록시 또는 형광 접합 된 이차 항체로 부화. </li><li> 확립 된 프로토콜 20에 따라 화학 발광 또는 형광으로 결합 이차 항체를 시각화. </li></ol> 7. 면역 형광 분석 <ol><li> 휴지 프로세싱 스테이션에서 해부 뇌 및 척수 탈수 및 파라핀 스테이션을 사용하여 파라핀에 그들을 삽입. </li><li> 파라핀 포매 TIS 컷8 개 μm의 두께 코로나 섹션 마이크로톰으로 고소와 유리 슬라이드에 섹션을 탑재합니다. </li><li> 10 분에서 5 개의 분리 크 실롤 조에이를 배양하여 조직 절편을 Deparaffinize 및 등급 에탄올 욕 (100 %, 90 %, 70 %, 50 %)의 시리즈를 통해 최종적으로 H 2 O로 재수 </li><li> 실온에서 5 분간 0.01 M 시트 레이트 완충액 (pH 6.0)의 섹션을 인큐베이션 추가적으로 마이크로파 오븐에서 10 분 동안 삶아. </li><li> 섹션을 실온까지 냉각하고, 내인성 퍼 옥시 다제를 억제하는 30 분 동안 3 % 과산화수소로 부화하자. </li><li> 20 % (부피 / 권) 정상 염소 혈청, 1 % (부피 / 부피) 소 혈청 알부민 (BSA), 0.5 % 트리톤 X-100을 실온에서 1 시간 동안 PBS에 함께 배양하여 조직 절편을 차단. </li><li> 1 % (부피 / 부피) 정상 염소 혈청, 1 % (부피 / 부피) BSA, 0.25 % 트리톤 X-PBS에서 밤새 100에서 4 ℃로 희석 차 항체와 함께 인큐베이션 섹션. </li> &lt;리&gt; 0.25 % (부피 / 부피의) 트리톤 X-100의 PBS 일단 PBS로 두 번 씻어 섹션. </li><li> 해당 형광 접합 된 이차 항체를 실온에서 1 시간 동안 1 % 희석 핵 염색 DAPI (용적 / 권) 정상 염소 혈청, 1 % (부피 / 부피) BSA 및 PBS와 섹션 얼룩. </li><li> 0.25 % (부피 / 부피의) 트리톤 X-100의 PBS 일단 PBS로 두 번 세척 한 후, 내장 미디어 슬라이드를 커버 슬립 및 공 초점 레이저 주사 현미경에 의한 염색을 시각화. </li></ol>

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저자가 공개하는 게 없다.

Tg는 (M83 +/- : GFAP의 -luc +/-)의 복막 내로의 α- 시누 클레인 브릴의 말초 주입 마우스 synucleinopathies의 중요한 특성 요점을 되풀이하는 신경 염증을 동반 신경 질환을 유발하는 용이 한 방법을 나타낸다. 마찬가지로, 혀 주입 트랜스 제닉 마우스에서의 α- 시누 클레인의 prionoids 의해 neuroinvasion 대한 다른 경로를 나타내고 있지만, 덜 효율적이다. 우리는 주사 후 420일에서 실험을...

알파 - 시누 클레인은 특히 자기 씨앗 용량에, 프리온 단백질의 것과 유사 세포 사이의 연결 경로를 따라 미스 폴딩을 전파 특성을 가지고 성장 증거가있다. 알파 - 시누 클레인의이 속성은 &#39;프리온 같은&#39;또는 &#39;prionoid&#39;라고, 새로 건강한 뉴런 1, 2를 이식하는 병에 걸린 뉴런에서 잘못 접힌 알파 - 시누 클레인의 전달률을 제안 이식 실험에서 관찰에 의해 지원됩니다 3, 4. 또한 뇌 또는 주변부, 예 뒷다리 근육이나 장벽의 말단 부분의 α- 시누 클레인 병리의 확산의 결과로 잘못 폴딩의 α- 시누 클레인의 직접 분사 CNS의 5, 6, 7, &lt;SUP 클래스 = &quot;외부 참조&quot;&gt; 8, 9, 10. 우리는 더 상세히 주변 경로를 통해 알파 시누 클레인의 prionoids의 송신을 분석 미스 폴딩의 α- 시누 클레인은 단일 intraglossal 또는 복강 내 주사 한 후 이전 프리온 위해 아니라 잘못 폴딩 알파 위해 도시되었던 기능을 CNS를 neuroinvade 수 있는지 질문을 해결 시누 클레인. 혀에 프리온 주입 후 CNS의 neuroinvasion는 뇌간 (11)에있는 설하 신경의 핵 리드 혀 설하 신경을 따라 전달을 통해 달성된다. 하여 성상 세포의 활성화를 모니터 프리온 프로모터, 인간의 α- 시누 클레인의 A53T 변이체를 과발현하고 반딧불이 GFAP 프로모터의 제어 하에서 루시페라아제 마우스 : 마우스 모델로 우리의 Tg (GFAP의 -luc +/- M83를 +/-) 선택한 생물 발광, 이전의 뇌에서와 같이쥐 12 프리온은 감염. 우리의 손에 bigenic의 Tg (M83 +/- : GFAP의 -luc +/-) 다른 사람 (13)에 의해 도시 된 바와 같이 마우스는 시대의 23개월까지 질병을 개발하지 않았다. 뇌에서 병리학, Tg 및 척수와 intraglossal 또는 복강 내 경로 유도 된 신경 질환을 통해 인간의 α- 시누 클레인 브릴의 단일 주입 : 가설을지지 생쥐 (+/- +/- M83 GFAP의 -luc) 알파 - 시누 클레인의 prionoids 프리온 (14)와 중요한 특성을 공유합니다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>anti-actin antibody</td>
			<td>Merck Millipore</td>
			<td>MAB1501</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-alpha-synuclein, phospho S129 antibody [pSyn#64]</td>
			<td>Wako</td>
			<td>015-25191</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-alpha-synuclein, phospho S129 antibody [EP1536Y]</td>
			<td>Abcam</td>
			<td>ab51253</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-GFAP antibody</td>
			<td>Dako</td>
			<td>Z0334 01</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-IBA-1 antibody</td>
			<td>Wako</td>
			<td>019-19741</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Sequestosome-1 (p62) antibody</td>
			<td>Proteintech</td>
			<td>18420-1-AP</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-ubiquitin antibody [Ubi-1]</td>
			<td>Merck Millipore</td>
			<td>MAB1510</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Invitrogen</td>
			<td>14190169</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine&#160;</td>
			<td>Ratiopharm</td>
			<td></td>
			<td>100 mg/kg</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Ratiopharm</td>
			<td></td>
			<td>10 mg/kg</td>
		</tr>
		<tr>
			<td>27-gauge syringe</td>
			<td>VWR</td>
			<td>613-4900</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane&#160;</td>
			<td>Piramal Healthcare</td>
			<td>PZN&#160; 4831850</td>
			<td></td>
		</tr>
		<tr>
			<td>Depilatory cream</td>
			<td>Veet</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Secureline lab marker&#160;</td>
			<td>Neolab</td>
			<td>25040</td>
			<td></td>
		</tr>
		<tr>
			<td>D-luciferin potassium salt</td>
			<td>Acris</td>
			<td>LK10000</td>
			<td>30 mg/mL stock solution</td>
		</tr>
		<tr>
			<td>Thermomixer</td>
			<td>Eppendorf</td>
			<td>5776671</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonopuls Mini20 sonicator</td>
			<td>Bandelin</td>
			<td>3648</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Lumina II imaging system</td>
			<td>PerkinElmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Living Image 3.0 Software</td>
			<td>PerkinElmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tg(M83+/-) mice or B6;C3-Tg(Prnp-SNCA*A53T)83Vle/J mice</td>
			<td>The Jackson Laboratory</td>
			<td>004479</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard pattern forceps</td>
			<td>Fine Science Tools</td>
			<td>11000-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Narrow pattern forceps</td>
			<td>Fine Science Tools</td>
			<td>11002-12</td>
			<td></td>
		</tr>
		<tr>
			<td>N-laurylasarcosyl</td>
			<td>Sigma</td>
			<td>L5125-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Optima Max-XP ultracentrifuge&#160;</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>TLA-110 rotor&#160;</td>
		</tr>
		<tr>
			<td>Thickwall polycarbonate tubes</td>
			<td>Beckman Coulter</td>
			<td>362305</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAG Novex 4-12% Bis-Tris Midi Protein Gels</td>
			<td>Thermo Fisher Scientific</td>
			<td>WG1401BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP conjugated antibody</td>
			<td>Cayman</td>
			<td>Cay10004301-1</td>
			<td></td>
		</tr>
		<tr>
			<td>IR Dye 680 conjugated antibody&#160;</td>
			<td>LI-COR Biosciences</td>
			<td>926-68070</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperSignal West Dura Extended Duration Substrate</td>
			<td>Thermo Fisher Scientific</td>
			<td>34075</td>
			<td></td>
		</tr>
		<tr>
			<td>Stella 3200 imaging system</td>
			<td>Raytest</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Odyssey infrared imaging system&#160;</td>
			<td>LI-COR Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20&#160;</td>
			<td>MP Biomedicals</td>
			<td>TWEEN201</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>SA/T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Immobilon-FL PVDF membrane</td>
			<td>Merck Millipore</td>
			<td>IPFL00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylol</td>
			<td>Sigma</td>
			<td>Roth</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide</td>
			<td>Sigma</td>
			<td>SA/00216763/000500</td>
			<td>working solution 3%</td>
		</tr>
		<tr>
			<td>Bovine serum albumine (BSA)&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>A3294-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>PCN5000</td>
			<td></td>
		</tr>
		<tr>
			<td>4,6-diamidino-2-phenylindole (DAPI)</td>
			<td>Thermo Fisher Scientific</td>
			<td>D1306</td>
			<td>working dilution 1:50,000</td>
		</tr>
		<tr>
			<td>Fluoromount media&#160;</td>
			<td>Omnilab</td>
			<td>SA/F4680/000025</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM700 confocal laser scanning microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HALT protease and phosphatase inhibitors</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>10516495</td>
			<td></td>
		</tr>
		<tr>
			<td>Precellys 24-Dual homogenizer&#160;</td>
			<td>Peqlab</td>
			<td>91-PCS24D</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 conjugated antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>A31619</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 conjugated antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11005</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce&#160;BCA&#160;Protein&#160;Assay&#160;Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>10741395</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome RM2255</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LSM700 confocal laser scanning microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

bioluminescence imaging of neuroinflammation in transgenic mice after peripheral inoculation of alpha-synuclein fibrils

잘못 접힌 알파 - 시누 클레인의 프리온 같은 동작을 연구하기 위해 마우스 모델은 중추 신경계 (CNS)에서 신경 병리학을 일으킬 알파 - 시누 클레인의 prionoids의 빠르고 간단하게 전송을 허용하는 것이 필요하다. 마우스, 프리온 단백질 프로모터로부터 A53T 돌연변이와 인간의 α- 시누 클레인 과발현과 반딧불에서 루시퍼 : 여기에서는 bigenic의 Tg (GFAP의 -luc +/- M83 +/-)으로의 α- 시누 클레인 브릴의 intraglossal 또는 복강 내 주사를 설명 글 리아 섬유 성 산성 단백질 프로모터 (GFAP), 신경 병리학 적 질환을 유발하기에 충분하다. 동물 그들이 도달 할 때까지 자연 질환 무료 남아 8 개월 이형의 Tg (GFAP의 -luc +/- M83 +/-)의 나이에 시작 심한 신경 학적 증상이 동형 접합의 Tg (M83 + / +) 마우스와 비교 22개월의 나이. 흥미롭게 intraperitone 통해 알파 시누 클레인 브릴 주입229 ± 십칠일의 중간 배양 시간과 마우스 : 알 경로가 다섯의 Tg (GFAP의 -luc +/- M83 +/-) 네 마비와 신경 학적 질병을 유발. 병에 걸린 동물은 뇌와 척수에서 인산화 된 알파 - 시누 클레인의 심각한 예금을 보였다. 알파 시누 클레인의 축적은 sarkosyl 불용성이었고 유비퀴틴과 P62와 colocalized 및 성상 신경교 증 및 microgliosis 결과 염증 반응을 수반 하였다. 놀랍게도, 혀에 알파 - 시누 클레인 원 섬유의 접종 285 일 후에 알파 - 시누 클레인 병리를 보여주는 다섯 주입 동물의 한으로 질병을 일으키는 덜 효과적이었다. 우리의 연구 결과는 (+/- M83을 : GFAP의 -luc를 +/-) 복강 내 경로를 통해 intraglossal 노선과 더욱 그렇다 통해 접종의 Tg에 synucleinopathies의 관련 특징과 신경 학적 질병을 유도하기 적합하다는 것을 보여 쥐를. 이 유도 프리온 같은 기전 연구를위한 새로운 모델을 제공합니다보다 상세히의 α- 시누 클레인의 prionoids d 개의.

마우스 (표 1 및도 1)은 혀 또는 bigenic의 Tg의 CNS (GFAP의 -luc +/- +/- M83)의 복막 유도 병리학 통해 알파 시누 클레인의 prionoids 주변의 주입. 알파 - 시누 클레인 섬유소와 하나의 복강 내 주사 한 후, 다섯 마우스의 네 229 ± 십칠일의 중간 배양 시간과 신경 학적 질환을 개발했다. 놀랍게도, 단지 1 ~ 5의 마우스 285 일 후 알파 - 시누...

Watch this Scientific Journal Video about Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils at JoVE.com

Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils

(+/- M83 : GFAP의 -luc +/-)의 Tg는 복막 내로 또는 혀의 α- 시누 클레인 브릴의 말초 주입 가족적 A53T 돌연변이와 반딧불이 루시퍼 라제와 인간의 α- 시누 클레인 표현 마우스, 신경 염증을 포함한 신경 병리학을 유도 할 그들의 중추 신경계한다.

알파 - 시누 클레인 브릴의 주변 접종 후 형질 전환 마우스의 신경 염증의 생물 발광 영상

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein ...

bioluminescence-imaging-neuroinflammation-transgenic-mice-after

Research

JoVE Journal

Neuroscience

8.5K 조회수.  German Center for Neurodegenerative Diseases (DZNE).  (+/- M83 : GFAP의 -luc +/-)의 Tg는 복막 내로 또는 혀의 α- 시누 클레인 브릴의 말초 주입 가족적 A53T 돌연변이와 반딧불이 루시퍼 라제와 인간의 α- 시누 클레인 표현 마우스, 신경 염증을 포함한 신경 병리학을 유도 할 그들의 중추 신경계한다. 

비디오: Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or thinned-skull 3 preparations. While the open-skull technique is very versatile, it is not optimal for studying microglia because it is invasive and can cause microglial activation. Even though the thinned-skull approach is minimally invasive, the repeated re-thinning of skull required for chronic imaging increases the risks of tissue injury and microglial activation and allows for a limited number of imaging sessions. Here we present a chronic thin-skull window method for monitoring murine microglia in vivo over an extended period of time using two-photon microscopy. We demonstrate how to prepare a stable, accessible, thinned-skull cortical window (TSCW) with an apposed glass coverslip that remains translucent over the course of three weeks of intermittent observation. This TSCW preparation is far more immunologically inert with respect to microglial activation than open craniotomy or repeated skull thinning and allows an arbitrary number of imaging sessions during a time period of weeks. We prepare TSCW in CX3CR1 GFP/+ mice 4 to visualize microglia with enhanced green fluorescent protein to &le;150 &mu;m beneath the pial surface. We also show that this preparation can be used in conjunction with stereotactic brain injections of the HIV-1 neurotoxic protein Tat, adjacent to the TSCW, which is capable of inducing durable microgliosis. Therefore, this method is extremely useful for examining changes in microglial morphology and motility over time in the living brain in models of HIV Associated Neurocognitive Disorder (HAND) and other neurodegenerative diseases with a neuroinflammatory component.

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat.

만성 두 광자를위한 박막 해골 창 기법 생체내에서</emNeuroinflammation의 모델 Murine Microglia의> 이미징

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

연구

신경과학

Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, the olfactory bulb (OB), the dorsal motor nucleus of the vagus (DMV)3, the intermediolateral nucleus of the spinal cord 4 and the substantia nigra, providing the basis for the neuropathological staging of the disease4,5. The ENS and the OB are the most exposed nervous structures and the first ones to be affected. Interestingly, PD has been related to pesticide exposure6-8. Here we show in detail two methods used in our previous study 9. In order to analyze the effects of rotenone acting locally on the ENS, we administered rotenone using a gavage to one-year old C57/BL6 mice. Rotenone is a widely used pesticide that strongly inhibits mitochondrial Complex I 10. It is highly lipophylic and poorly absorbed in the gastrointestinal tract 11. Our results showed that the administration of 5 mg/kg of rotenone did not inhibit mitochondrial Complex I activity in the muscle or the brain. Thus, suggesting that using our administration method rotenone did not cross the hepatoportal system and was acting solely on the ENS. Here we show a method to administer pesticides using a gavage and the image analysis protocol used to analyze the effects of the pesticide in alpha-synuclein accumulation in the ENS. The first part shows a method that allows intragastric administration of pesticides (rotenone) at a desired precise concentration. The second method shows a semi-automatic image analysis protocol to analyze alpha-synuclein accumulation in the ENS using an image analysis software.

Parkinson's disease has been related to the exposure to pesticides. Here we show a method to deliver pesticides using a gastric tube at the desired concentration and a method to analyze their effect in alpha-synuclein accumulation in the enteric nervous system.

장용 신경계의 알파 synuclein의 흠도의 Gavage 및 이미지 분석을 사용하여 Rotenone의 구강 관리

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, ...

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

Rhizotomy 후 도설 루트 Axons의 라이브 영상

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. 
 For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. 
 Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. 
 Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at &ge;6 months after birth.
 We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

몸에 붙이기 형질 전환 쥐의 발생에 의해<em&gt; 자궁 내</em&gt; 일렉트로 및<em&gt; 생체 내</em&gt; 생물 발광 상영

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

말초 신경 손상 후 기능 회복을 평가하기 위해 후각 Ensheathing 세포의 이식

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

하는 세포 칼슘의 형광 및 생물 발광 영상<sup&gt; 2 +</sup&gt; 노인 해마 신경 세포에서

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, oligomers and fibrils. During disease, &#945;-syn undergoes conformational changes to form oligomers and high molecular weight aggregates that tend to make the protein more insoluble. Abnormally aggregated &#945;-syn is a neuropathological feature of Parkinson&#39;s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Biochemical characterization and analysis of insoluble &#945;-syn using buffers with increasing detergent strength and high-speed ultracentrifugation provides a powerful tool to determine the development of &#945;-syn pathology associated with disease progression. This protocol describes the isolation of increasingly insoluble/aggregated &#945;-syn from post-mortem human brain tissue. This methodology can be adapted with modifications to studies of normal and abnormal &#945;-syn biology in transgenic animal models harbouring different &#945;-syn mutations as well as in other neurodegenerative diseases that feature aberrant fibrillar deposits of proteins related to their respective pathologies.

Here, we present a protocol for the isolation of increasingly insoluble/aggregated alpha-synuclein (&#945;-syn) from post-mortem human brain tissue. Through the utilization of buffers with increasing detergent strength and high-speed ultracentrifugation techniques, the variable properties of &#945;-syn aggregation in diseased and non-diseased tissue can be examined.

파킨슨 뇌에서, 수용성 알파 시누 클레인의 순차 추출

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, ...

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 ...

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the study of &#945;S aggregation in Parkinson&#39;s Disease (PD). Recently, the development of new animal models through the exogenous inoculation of brain extracts from diseased &#945;S transgenic mice or PD patients has given new hopes to the possibility of creating more adequate cell models of &#945;S aggregation. Unfortunately, when it comes to cells in cultures, administration of raw brain extracts has not proven as successful as in mice and the source of choice of exogenous aggregates is still in vitro preformed &#945;S fibrils.
We have developed a method to induce the formation of intracellular &#945;S inclusions in primary neurons through the exogenous administration of native microsomes-associated &#945;S aggregates, a highly toxic &#945;S species isolated from diseased areas of transgenic mice. This fraction of &#945;S aggregates that is associated with the microsomes vesicles, is efficiently internalized and induces the formation of intracellular inclusions positive for aggregated and phosphorylated &#945;S. Compared to in vitro-preformed fibrils which are made from recombinant &#945;S, our method is faster and guarantees that the pathogenic seeding is made with authentic &#945;S aggregates extracted from diseased animal models of PD, mimicking more closely the type of inclusions obtained in vivo. As a result, availability of tissues rich in &#945;S inclusions is mandatory.
We believe that this method will provide a versatile cell-based model to study the microscopic aspects of &#945;S aggregation and the related cellular pathophysiology in vivo and will be a starting point for the creation of more accurate and sophisticated cell paradigm of PD.

The goal of this protocol is to provide a cell-based system that replicates the formation of alpha-synuclein aggregates in vivo. Intracellular alpha-synuclein inclusions are seeded in primary neurons by the internalization and propagation of exogenous administered native microsomes-associated alpha-synuclein aggregates isolated from diseased alpha-synuclein transgenic mice.

소 대형의 강력한 셀 모델로 기본 신경 하 Microsomes 관련 알파 synuclein 집계의 외 인 관리

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the ...

Targeting Alpha Synuclein Aggregates in Cutaneous Peripheral Nerve Fibers by Free-floating Immunofluorescence Assay

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the diagnosis still relies only on clinical signs of motor involvement that appear later on in the disease course, when most of the dopaminergic neurons are already lost. Hence, there is a strong need for a biomarker that can identify patients at the beginning of disease or at the risk of developing it. Over the last few years, skin biopsy has proved to be an excellent research and diagnostic tool for peripheral nerve diseases such as small fiber neuropathy. Interestingly, a small fiber neuropathy and alpha synuclein (&#945;Syn) neural deposits have been shown by skin biopsy in PD patients. Indeed, skin biopsy has the great advantage of being an easily accessible, minimally invasive and painless procedure that allows the analysis of peripheral nervous tissue prone to the pathology. Moreover, the possibility of repeating the skin biopsy in the course of the follow-up of the same patient allows studying the longitudinal correlation with the disease progression. We set up a standardized reliable protocol to investigate the presence of &#945;Syn aggregates in skin nerve fibers of the PD patient. This protocol involves few short fixation steps, a cryotome sectioning and then a free-floating immunofluorescence double-staining with two specific antibodies: anti Protein Gene Product 9.5 (PGP9.5) to mark the cutaneous nerve fibers and anti 5G4 for detecting &#945;Syn aggregates. It is a versatile, sensitive and easy to perform protocol that can also be applied for targeting other proteins of interest in skin nerves. The ability to mark &#945;Syn aggregates is another step forward to the use of skin biopsy as a tool for establishing a pre-mortem histopathological diagnosis of PD.

Here, we present a protocol for a free-floating indirect immunofluorescence assay on skin biopsy sections that allows for the identification of disease specific conformation variants of alpha synuclein involved in Parkinson disease and multiple proteins of the peripheral nervous system.

자유 부동 면역 형광 분석에 의하여 절단 말초 신경 섬유에 있는 알파 Synuclein 응집체를 표적으로 하는

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the ...