The authors would like to thank Autumn Peterson for assistance with mouse photography, Daniel Thoresen for assistance with mice, and Drs. Nicole Ward and Abdelmadjid Belkadi for assistance with surgical denervation. These studies were supported by funding from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (grants R00AR059796 and R01AR065409); the University of Michigan Department of Dermatology; the Biological Sciences Scholars Program; the Center for Organogenesis; the University of Michigan Comprehensive Cancer Center; and the John S. and Suzanne C. Munn Cancer Fund. S.C.P. was supported by funding from the National Institute of General Medical Sciences (grant T32 GM007315). This work was also supported by the NIH Intramural Research Program, Center for Cancer Research, National Cancer Institute.

이 프로토콜에 설명 된 모든 절차는 실험 동물 의학에 대한 미시간 단위의 대학 설립 규정에 따라 수행되었다. 1. 마우스의 유전자 재조합을 유도 참고 : Gli1 TM3 (CRE / ERT2) ALJ / J 마우스 변형 (Gli1-CreERT2) (13)는 상피를 TD하기 위해 타목시펜에 의한 유전자 재조합의 대상으로 할 수 있습니다. Gli1를 생성하는 B6.129S4-GT는 (ROSA) 26Sor tm1Sor / J 기자 마우스 (LacZ를) (22)이 변형 크로스, LacZ를 동물 전체 마운트 아래 염색에 의해 TD 세포를 시각화 할 수 있습니다. <ol><li> 옥수수 유 12.5 ㎎ / ㎖의 농도로 타목시펜 용액을 준비한다. <ol><li> 1.5 ML 튜브에서 결정 타목시펜의 20 ㎎, 옥수수 기름을 1 ml의까지 추가 할 수 있습니다. 타목시펜이 완전히 (2-4 시간)에 용해 될 때까지 단단히 같이 와류 믹서에 튜브 및 RT에서 가장 높은 설정에서 지속적으로 소용돌이를 테이프타목시펜 미립자의 부재에 대한 해부 현미경 튜브를 조사하여 확인 하였다. </li><li> 15 ㎖의 튜브에 용액을 옮기고 추가의 옥수수 오일 용액 12.5 밀리그램 / 최종 농도로 희석 타목시펜. 추가로 30 초 동안 점성 용액을 볼 텍싱하여 혼합한다. 어둠 속에서 4 ° C에서 최대 1 주일이 솔루션을 저장합니다. </li></ol></li><li> Gli1로 복강 타목시펜 용액을 주입, LacZ를 쥐, 마우스 체중 20g 당 200 ㎕의 부피로, 20g 마우스 체중 당 2.5 밀리그램의 유효 타목시펜 복용량. </li></ol> 2. 수확 피부 생검 참고 : 타목시펜 유도 후 주에 실험에 따라 수확 피부 생검 몇 일. 모든 수술를 들어, 멸균 장갑을 사용하여 수술 용 마스크 또는 머리 그물을 착용하고, 절차를 수행하는 동안 무균 수술 드레이프와 동물을 다루는 포함 설치류 수술을위한 표준 프로토콜을 따르십시오. <ol><li> 물에 90 ㎎ / ㎖ 케타민 6.5 ㎎ / ㎖ 자일 라진을 혼합하여 10 배 스톡 마취 솔루션을 준비합니다. 그냥 사용하기 전에 멸균 PBS로이 원액 1:10 희석하고, 최대 8 개월 동안 어둠 속에서 실온에서 보관하십시오. <ol><li> 또는, 완전 동물을 마취 산소와 4 %의 가스 농도를 시작으로 이소 플루 란 흡입에 의해 마우스를 마취하고 후속 절차 기간 동안 1 ~ 2 %이 저하. </li></ol></li><li> 20g의 마우스 체중 당 200 μL의 용량으로 복강 내 마취 용액을 주입한다. 동물이 발가락 핀치 분석에 의한 진정 작용의 적절한면에 도달 한 것을 확인하고, 그 마음을 확인하고 호흡 속도는 (각각 약 600 비트와 분당 160 호흡) 정상입니다. </li><li> , 지느러미 다시 피부에 생검의 사이트에서 머리를 제거하지 닉에주의하면서 또는 기본 피부를 손상 전기 클리퍼를 사용합니다. </li><li> 면도를 닦아 수술 부위를 준비Betadine과 알코올 잎사귀를 사용하여 전방 대 후방 방향으로 D 영역. 모든 머리를 잘라낸 사이트에서 제거해야합니다. </li><li> , 검은 색 마커를 사용하여 조직 검사 사이트 개요 무균 수술 영역에서 온난화 패드에 동물을 배치하고, 무균 수술 드레이프와 커버 (데모 목적을 위해, 무균 드레이프는 가시성을 높이기 위해 생략). 참고 : 모낭의 길이 방향의 부분을 얻기 위해 생검의 긴 가장자리에, 전방 - 후방 방향으로 parasagital를 (모낭의 방향에 평행)을 실행해야합니다 (가장자리가 ~ 1cm가, 조직학에 대한 구분한다) 지느러미 중간 선 (그림 1A). </li><li> 기본 근육의 근막을 손상시키지 않고 표시된 지역에 따라 전체 두께 절제술을 만들기 위해 멸균 # 11 메스를 사용합니다. 참고 : 적출 피부 조직은 표피, 진피, 피하 지방과 panniculus의 카르노 서스를 포함한다. 출혈은 일반적으로 최소이다. </li><li> t을 평평그는 마른 종이 타월에 피부 샘플을 적출, 진피, 아래로 옆 초과 종이 타월을 멀리 트림 및 기타 샘플을 수집해야 할 경우 최대 1 시간 동안 차가운 PBS에서 샘플을 저장합니다. 준비가되면 조직 검사를위한 샘플, 또는 전체 마운트 β - 갈 락토시다 제 (LacZ를) 염색을위한 4 단계를 처리하는 단계 3.1 또는 3.2로 진행합니다. </li><li> 봉합사는 약 3mm 이격 간단한 인터럽트 패턴에 6-0 나일론 봉합사를 사용하는 생검 부위를 닫는다. </li><li> 전체 복구 후까지 다른 동물과 같은 케이지에 수술을받은 쥐를 반환하지 않습니다. </li><li> 수술 영역은 일반적으로 일주일 내, 치유 될 때까지 그들은 매일 또한 의식을 되 찾을 때까지 즉시 수술 후 동물을 모니터링합니다. 마우스는 통증이나 고통의 흔적을 나타내는 경우 지정 기관 동물 관리 및 사용 지침에 따라 진통제를 사용합니다. 수술 후 7 ~ 10 일이 봉합사를 제거합니다. 주 : 필요한 경우, (50 ㎎ / ㎖ 스톡 오디오 솔루션 카프로 펜 희석하여 용액을 제조 진통이온) 1 : 멸균 100. 20g 체중 당 200 μL (5 ㎎ / ㎏의 마우스 체중)의 투여 량으로, 목 목덜미 근처 견갑골 사이의 용액을 피하 주사. </li></ol> 조직학 3. 프로세스 샘플 해결하려면 및 적출 조직을 처리, 응용 프로그램에 따라 아래의 방법 중 하나를 사용합니다. <ol><li> 파라핀 조직 학적 샘플을 생성하려면 최대 2 주 동안 70 % 에탄올에서 RT에서 PBS O / N에서 3.7 % 포르말린에 피부와 저장소를 수정합니다. 파라핀에 삽입하기 전에 종이 타월을 제거합니다. </li><li> 냉동 조직 학적 샘플을 생성하기 위해, PBS 차가운 4 % 파라 포름 알데히드에서 조직 잠수함 천천히 1 시간 동안 흔들어. 솔루션을 제거하고 PBS 3 변화, 약 5 분 각각에 샘플을 씻는다. 다음으로, ( &quot;크로스 침몰&quot;) 조직을 cryoprotect하는 PBS 30 % 수크로오스에서 샘플 잠수함. </li><li> 4 ° C에서 부드럽게 흔들어 O / N로 품어. 다음 날, 종이 견인을 제거EL은 피부의 진피 측으로부터 과량의 지방 조직을 잘라 내고. 직접 10월에 조직을 포함하고 -80 ° C에서 냉동 블록을 저장합니다. 참고 : 이전에 5,19을 설명한대로 절편 한 후, 파라핀 또는 냉동 샘플 중 하나는, TDs를, 메르켈 세포 각각 케라틴 17, 각질 8 신경 미세 섬유,에 대한 항체를 사용하여 신경을 식별하기 위해 면역 조직 화학 염색으로 염색 할 수 있습니다. </li></ol> 전체 마운트 LacZ를 염색 4. 시각화 샘플 <ol><li> X-여자의 염색 액을 준비합니다. <ol><li> 멸균 수 250 mL에 0.94 g의 인산 나트륨 일 염기, 2.6 g 인산 나트륨을 결합한다. 7.3의 pH를 조정합니다. 여기에, 1 M 염화 마그네슘 0.5 ㎖, 페로 시안화 칼륨 0.528 g의 칼륨 페리시 아나이드의 0.412 g을 추가한다. 옥틸 - 폴리에틸렌 글리콜 250 μL 및 데 옥시 콜레이트 125 mg을 추가합니다. 염기 용액을 어둠 속에서 최대 6 개월 동안 4 ℃에서 저장 될 수있다. </li><li> 50 배 재고 X-여자의 soluti 준비50 ㎎ / ㎖ 솔루션을 생성하기 위해 X-여자의 재고 병에 디메틸 포름 아미드를 추가하여합니다. 어둠 속에서 -20 ° C에서이 솔루션을 저장합니다. </li><li> 염색 솔루션을 생성하기 위해 X-여자 기반 솔루션에 재고의 X-여자 솔루션 1:50 희석 사용하기 직전에. 작은 생검 (&lt;1cm 2), 샘플 당 염색 액의 나누어지는 1-2 ml의하십시오. </li></ol></li><li> 부드럽게 진탕 얼음 30 분 동안 차가운 PBS로 2 % 파라 포름 알데히드 / 0.2 % 글루 타르 알데히드를 함유하는 용액의 단계 2.7에서 수집 된 피부 샘플을 고정한다. 작은 생검 (&lt;1cm 2)의 경우, 샘플 당 정착액 솔루션의 1-2 ml를 사용합니다. 참고 : 또는 만 2-4% 파라 포름 알데히드에 샘플을 수정, 또는 0.5 % 글루 타르 알데히드에서만. 최적의 정착 조건은 조직 LacZ를 발현 및 응용 정도에 의존한다. </li><li> 정착액 솔루션을 제거하고 실온에서 진탕에 PBS의 3 변경, 5 분마다로 샘플을 씻어. </li><li> 샘플 아래에 종이 타월을 제거하고 exces를 절단무딘 집게로 지방을 파지 및 해부 가위 트리밍에 의해 피부의 진피 측면에서 지방 조직을이야. </li><li> X-여자의 염색 액의 샘플 잠수함, 37 ° CO / N에 품어. LacZ를 발현은 해부 현미경 (그림 1B)에서 파란색 얼룩으로 표시됩니다. 주 : 신호 강도가 약한 경우에는 염색 액 다음날 교체 및 O / N 항온 처리를 반복한다. 백그라운드 염색이 너무 강한 경우에는, 염색의 시간을 줄이거 나, C ° 37 RT에서 샘플을 부화 대신. </li><li> 염색 액을 제거하고 PBS 부드럽게 RT 진탕, 약 5 분 동안 3 % DMSO를 포함하는 3 변경에 샘플을 씻는다. </li><li> 5 분 각 70 % 에탄올 2-3 변화 워시 샘플. 70 % 에탄올에 보관 샘플. </li></ol> 5. 수술 탈 신경 <ol><li> 단계 2.2에서와 같이 동물을 마취하고 전체 등쪽 피부를 면도. </li><li> 수술 영역 (을)를 준비Betadine 및 알코올 잎사귀 및 무균 수술 드레이프와 동물 다루 사용하여 다시 피부 F (데모 목적을 멸균 드레이프는 가시성을 높이기 위해 생략). 무균 수술 영역에서 작동하는 동안 가열 패드를 사용하여 동물을 따뜻하게 유지. </li><li> 꼬리 위의 약 0.5 cm에 목 기지에서 지느러미 중간 선을 따라 멸균 # 11 메스를 사용하여 절개를합니다. </li><li> 무딘 집게를 사용하여 부드럽게 단지 뒷다리 위에서 목 근처 견갑골 지방 패드의 하부 조직을 시각화 떨어진 측면에서 좌측 피부를 반영한​​다. 참고 : 지느러미 피부 신경 (그림 2) 날카로운 굴곡을 피부 아래에 느슨한 결합 조직에 들어가기 전에 트렁크 벽의 반투명 밴드를 통해 꼬리 쪽 여행으로 흰색 가닥 나타납니다. </li><li> 해부 현미경 하에서 광 초미립자 집게를 사용하여 애니 왼쪽에서 독점적 신경 제거어디에서 따 버릴에 의해 해부학 사이트 T3-12에 위치한 리터 피부에 입국 사이트에 트렁크 벽 세그먼트 벤드 (그림 2). <ol><li> 동양 집게 수직 및 스트레칭과 주변 조직 (- E 그림 2C)에서 분리 신경을 일으키는 원인이 자신의 굴곡 사이트 아래 약 0.5 cm를 잡고 위쪽으로 잡아 당겨 신경을 제거합니다. 인접한 혈관을 파열 않도록주의하십시오. </li><li> 피부에 트렁크 벽으로부터 연장 모든 신경이 제거 될 때까지 계속합니다. 트렁크 벽의 밀도 밴드 내에서 신경을 방해하지 마십시오. 주기적 멸균 0.9 % 식염수 용액의 방울을 적용하여 절차 내내 습윤 티슈 유지. </li><li> 또한, 집게와 트렁크 벽 근처에 자신의 근위 끝을 잡고 미세 가위로 자르는 것은에 의해 신경을 제거합니다. 그 후, 피부에 가까운 말단부를 절단 (도 2F - H). 마지막으로, INT를 제거ervening 신경 세그먼트 (그림 2I). </li></ol></li><li> 단계 5.4에 노출 된 피부 플랩에서 모든 신경을 제거합니다. 이 섬유는 등쪽 피부 신경의 말단 가지를 포함하고, 피부 플랩의 피부 측 (도 2J - K)에 결합 조직 내에서 산발적으로 위치한 흰색 분기 가닥으로 나타납니다. <ol><li> 이 좋은 지점을 제거하려면 미세 집게이 약은 피부 표면에 평행 신경을 파악하고 혈관을 방해하고 피부를 천공 방지하기 위해 위쪽으로 뽑아 놓습니다. 보이는 모든 신경이 제거 될 때까지 계속합니다. </li></ol></li><li> 무딘 절개를 사용 등쪽 정중선 절개 오른쪽의 피부를 반영하지만, 신경 제거되지 않는다. 이 반대측 가짜로 작동하는 제어 될 것입니다. </li><li> 간단한 중단 패턴에서 지느러미 중간 선을 따라 봉합은 절개를 닫습니다. 복구 및 사후 운영자 동안 동물을 모니터링atively로 이전 (단계 2.8-10) 보여 주었다. 수술 후 7 ~ 10 일이 봉합사를 제거합니다. </li><li> 기능적으로 수술 후 몇 주 안정 탈 신경을 위로 평가하기 위해, 전기 클리퍼를 사용하는 등의 피부에서 머리를 제거합니다. </li><li> 부드럽게 피하 주사 바늘을 사용하여 denervated 피부 영역을 찌를하고, 동물이 일반적으로 몸서리 또는 머리를 돌려, 응답 여부를 확인합니다. 피부 영역이 안정적 denervated 된 경우, 동물은 거의 또는 전혀 반응을 나타낼 것이다. </li><li> 검정색 마커를 사용하여, 반응의 영역뿐만 아니라, 반대측 모의 측 유사한 크기 및 위치의 영역을 설명합니다. </li><li> 단계 분석을위한 2.1-2.9에서 이러한 사이트에서 조직 검사를 수집합니다. 참고 : 또는 denervated 및 가짜로 작동하는 지역을 포함한 전체 지느러미 다시 피부를, 4 단계에 설명 된대로 유사한 전체 마운트 염색에 대한 한 장으로 제거 할 수 있습니다. </li></ol>

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The authors have nothing to disclose.

신경은 감각에서뿐만 아니라 포유류의 장기 개발, 유지 보수 및 재생 13,24-27에서뿐만 아니라 중요한 기능을 제공합니다. 신경 최근 다양한 피부 질환에 연루되어있는 바와 같이, 여기에 설명 된 기술은 동물 질병 모델의 다양한 신경 분포에 대한 요구 사항을 연구하는데 사용될 수있다. 사실, 일방적 탈 신경 기술은 동일한 마우스에서 손상 또는 파괴 신경 중 하나와 피부의 직접적인 비교...

Over the past few years, there has been a widening appreciation for the influence of nerves on diseases not typically regarded as classical neuropathies1-4. In the skin, recent experimental evidence has suggested that sensory nerves can modulate diverse pathologies ranging from psoriasis to cancer5-9. This has been demonstrated using techniques such as surgical denervation and pharmacological inhibition of neural function in rodents. In the case of psoriasis, these studies have provided a mechanistic framework for understanding why human psoriatic plaques regress following loss of neural function7,10-12.
Cutaneous nerves can also affect gene expression13,14 and are critical for mechanosensing in normal skin15. In particular, touch dome (TD) epithelia are comprised of a patch of columnar epidermal cells in juxtaposition with neuroendocrine Merkel cells innervated by slowly adapting type 1 (SA1) nerve fibers16-18. TDs mediate light touch sensation and have been shown to display Hedgehog pathway activity5,19. TD maintenance depends on innervation20,21, as nerves secrete Hedgehog ligands to sustain normal TDs and their associated Merkel cells19. In addition, innervation promotes Hedgehog-dependent tumor formation from TD epithelia5. Together, these studies reinforce the notion that intricate molecular interactions occurring between nerves and the surrounding cells in their niche are crucial for normal TD physiology as well as disease.
To interrogate the nature of these interactions, we describe here a series of in vivo techniques for manipulating gene expression in the TD, as well as for harvesting skin biopsies for TD visualization after lineage tracing. Finally, we describe procedures for performing unilateral surgical denervation, wherein nerves are removed from one side of the mouse dorsal skin, while leaving the contralateral side intact as a sham internal control. Several weeks after surgery, denervated and sham control skin are compared to assess changes that occur when nerves are ablated. Although these techniques are described in the context of normal TDs, the denervation procedure has been used to examine the requirement for nerves in mouse models of psoriasis6, wound healing13 and tumorigenesis5. Finally, since the skin is amenable to repeated biopsies, this provides an opportunity to monitor the long-term fates of labeled cells or to assess disease progression over multiple time points.

<table><tbody><tr><td>Alcohol prep pads</td><td>PDI</td><td>B339</td><td></td></tr><tr><td>AnaSed (Xylazine)</td><td>Lloyd</td><td>NADA 139-236</td><td></td></tr><tr><td>Antibody, anti-Keratin 8</td><td>Developmental Studies Hybridoma Bank</td><td>TROMA-I</td><td>rat antibody, use at 1:500 concentration</td></tr><tr><td>Antibody, anti-Keratin 17</td><td>Cell Signaling</td><td>#4543</td><td>rabbit antibody, use at 1:1,000 concentration</td></tr><tr><td>Antibody, anti-Neurofilament</td><td>Cell Signaling</td><td>C28E10</td><td>rabbit antibody, use at 1:500 concentration</td></tr><tr><td>Betadine prep pads</td><td>Medline</td><td>MDS093917</td><td></td></tr><tr><td>Carprofen (Rimadyl)</td><td>Zoetis</td><td></td><td></td></tr><tr><td>Cordless rechargable clipper</td><td>Wahl</td><td>trimmer model 8900</td><td></td></tr><tr><td>Corn Oil</td><td>Sigma-Aldrich</td><td>C8267</td><td></td></tr><tr><td>Cryostat</td><td>Leica</td><td>CM1860</td><td></td></tr><tr><td>DAPI</td><td>ThermoFisher Scientific</td><td>D1306</td><td>use at 1:1,000 concentration</td></tr><tr><td>Deoxycholate</td><td>Sigma-Aldrich</td><td>D6750</td><td></td></tr><tr><td>Depilatory Cream</td><td>Nair</td><td>N/A</td><td></td></tr><tr><td>Dimethylforamide</td><td>Sigma-Aldrich</td><td>319937</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D8418</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Sigma-Aldrich</td><td>G5882</td><td></td></tr><tr><td>ImmEdge Pen</td><td>Vector Laboratories</td><td>H-4000</td><td></td></tr><tr><td>Ketamine HCl</td><td>Hospira</td><td>NDC 0409-2051-05</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma</td><td>M8266</td><td></td></tr><tr><td>Micro cover glass</td><td>VWR</td><td>48404-454</td><td></td></tr><tr><td>Micro Slides</td><td>VWR</td><td>48311-703</td><td></td></tr><tr><td>10% Neutral Buffered Formalin</td><td>VWR</td><td>BDH0502-4LP</td><td></td></tr><tr><td>6-0 nylon sutures</td><td>DemeTECH</td><td>NL166012F4P</td><td></td></tr><tr><td>Octylphenyl-polyethylene glycol</td><td>Sigma-Aldrich</td><td>I8896</td><td></td></tr><tr><td>O.C.T. Compound</td><td>Sakura Tissue-Tek</td><td>4583</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>Pottasium ferrocyanide</td><td>Sigma-Aldrich</td><td>P9387</td><td></td></tr><tr><td>Pottasium ferricyanide</td><td>Sigma-Aldrich</td><td>702587</td><td></td></tr><tr><td>Sodium phosphate monobasic</td><td>Sigma-Aldrich</td><td>P9791</td><td></td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma-Aldrich</td><td>S5136</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>84097</td><td></td></tr><tr><td>Tamoxifen</td><td>Sigma-Aldrich</td><td>T5648-1G</td><td></td></tr><tr><td>Ultra fine forceps</td><td>Dumont</td><td>0103-5-PO</td><td></td></tr><tr><td>Vectashield</td><td>Vector Laboratories</td><td>H1000</td><td></td></tr><tr><td>X-gal</td><td>Roche</td><td>10 651 745 001</td><td>Disolve in dimethylforamide to create 50x stock prior to use</td></tr></tbody></table>

cutaneous surgical denervation:  a method for testing the requirement for nerves in mouse models of skin disease

Cutaneous somatosensory nerves function to detect diverse stimuli that act upon the skin. In addition to their established sensory roles, recent studies have suggested that nerves may also modulate skin disorders including atopic dermatitis, psoriasis and cancer. Here, we describe protocols for testing the requirement for nerves in maintaining a cutaneous mechanosensory organ, the touch dome (TD). Specifically, we discuss methods for genetically labeling, harvesting and visualizing TDs by whole-mount staining, and for performing unilateral surgical denervation on mouse dorsal back skin. Together, these approaches can be used to directly compare TD morphology and gene expression in denervated as well as sham-operated skin from the same animal. These methods can also be readily adapted to examine the requirement for nerves in mouse models of skin pathology. Finally, the ability to repeatedly sample the skin provides an opportunity to monitor disease progression at different stages and times after initiation.

생성 타목시펜 - 유도 Gli1 CreERT2 - 발현 생쥐와 LacZ를 리포터 대립함으로써, TD 상피를 시각화하고 시간이 지남에 따라 이들 세포의 운명을 추적 할 수있다. 전체 탈 신경 절차는 통상적으로, 마우스 당 1 시간 내에 완료 될 수 있고, 동물에게 최소의 고통을 야기한다. 5,19 - 우리의 이전 연구는 신경이 정상 T...

Watch this Scientific Journal Video about Cutaneous Surgical Denervation:  A Method for Testing the Requirement for Nerves in Mouse Models of Skin Disease at JoVE.com

Cutaneous Surgical Denervation:  A Method for Testing the Requirement for Nerves in Mouse Models of Skin Disease

This article includes detailed protocols for genetic labeling of mouse skin, surgical denervation, skin biopsy and visualizing labeled epithelia by whole-mount &#946;-galactosidase staining. These methods can be used to test the requirement for nerves in mouse models of normal and pathological skin.

탈 신경 외과 피부 : 피부 질환의 마우스 모델에서 신경에 대한 요구를 테스트하기위한 방법

Cutaneous somatosensory nerves function to detect diverse stimuli that act upon the skin. In addition to their established sensory roles, recent studies ...

cutaneous-surgical-denervation-method-for-testing-requirement-for

Research

JoVE Journal

Medicine

9.7K 조회수.  University of Michigan. This article includes detailed protocols for genetic labeling of mouse skin, surgical denervation, skin biopsy and visualizing labeled epithelia by whole-mount β-galactosidase staining. These methods can be used to test the requirement for nerves in mouse models of normal and pathological skin.

비디오: Cutaneous Surgical Denervation:  A Method for Testing the Requirement for Nerves in Mouse Models of Skin Disease

The Spared Nerve Injury (SNI) Model of Induced Mechanical Allodynia in Mice

Peripheral neuropathic pain is a severe chronic pain condition which may result from trauma to sensory nerves in the peripheral nervous system. The spared nerve injury (SNI) model induces symptoms of neuropathic pain such as mechanical allodynia i.e. pain due to tactile stimuli that do not normally provoke a painful response [1]. 
The SNI mouse model involves ligation of two of the three branches of the sciatic nerve (the tibial nerve and the common peroneal nerve), while the sural nerve is left intact [2]. The lesion results in marked hypersensitivity in the lateral area of the paw, which is innervated by the spared sural nerve. The non-operated side of the mouse can be used as a control. The advantages of the SNI model are the robustness of the response and that it doesn&#x2019;t require expert microsurgical skills.
The threshold for mechanical pain response is determined by testing with von Frey filaments of increasing bending force, which are repetitively pressed against the lateral area of the paw [3], [4]. A positive pain reaction is defined as sudden paw withdrawal, flinching and/or paw licking induced by the filament. A positive response in three out of five repetitive stimuli is defined as the pain threshold. 
As demonstrated in the video protocol, C57BL/6 mice experience profound allodynia as early as the day following surgery and maintain this for several weeks.

The Spared Nerve Injury SNI Model of Induced Mechanical Allodynia in Mice

The Spared Nerve Injury animal model is described here as a mouse model of peripheral neuropathic pain following partial denervation of the sciatic nerve by lesioning the tibial and common peroneal nerve branches, leaving the remaining sural nerve intact. Behavioral modification resulting from mechanical allodynia is quantified by von Frey filaments.

마우스에서 유도된 기계 Allodynia의 겪었던 신경 상해 (스니) 모델

Peripheral neuropathic pain is a severe chronic pain condition which may result from trauma to sensory nerves in the peripheral nervous system. The spared ...

연구

의학

Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. Although originally developed and used extensively in the rat due to its larger size, the tibial nerve in mice is big enough that it can be easily manipulated with either crush or transection, leaving the peroneal and sural nerve branches of the sciatic nerve intact and thereby preserving their target muscles. Thus, this model offers the advantages of inducing less morbidity and impediment of ambulation than the sciatic nerve transection model and also allows investigators to study the physiologic, cellular and molecular biologic mechanisms regulating the process of muscle atrophy in genetically engineered mice. The tibial nerve supplies the gastrocnemius, soleus and plantaris muscles, so its transection permits the study of denervated skeletal muscle composed of fast twitch type II fibers and/or slow twitch type I fibers. Here we demonstrate the tibial nerve transection model in the C57Black6 mouse. We assess the atrophy of the gastrocnemius muscle, as a representative muscle, at 1, 2, and 4 weeks post-denervation by measuring muscle weights and fiber type specific cross-sectional area on paraffin-embedded histologic sections immunostained for fast twitch myosin.

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of skeletal muscle atrophy. The model surgical protocol is described and demonstrated in C57Black6 mice. &#160;

경골 신경 절개 - 마우스의 탈 신경에 의한 골격근의 위축에 대한 표준 모델

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. ...

Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and either cut or crush it to induce peripheral nerve injury. Advantages of this surgery are its simplicity, high reproducibility, and the lack of effect on vital functions or mobility from the subsequent facial paralysis, thus resulting in a relatively mild surgical outcome compared to other nerve injury models. A major advantage of using a cranial nerve injury model is that the motoneurons reside in a relatively homogenous population in the facial motor nucleus in the pons, simplifying the study of the motoneuron cell bodies. Because of the symmetrical nature of facial nerve innervation and the lack of crosstalk between the facial motor nuclei, the operation can be performed unilaterally with the unaxotomized side serving as a paired internal control. A variety of analyses can be performed postoperatively to assess the physiologic response, details of which are beyond the scope of this article. For example, recovery of muscle function can serve as a behavioral marker for reinnervation, or the motoneurons can be quantified to measure cell survival. Additionally, the motoneurons can be accurately captured using laser microdissection for molecular analysis. Because the facial nerve axotomy is minimally invasive and well tolerated, it can be utilized on a wide variety of genetically modified mice. Also, this surgery model can be used to analyze the effectiveness of peripheral nerve injury treatments. Facial nerve injury provides a means for investigating not only motoneurons, but also the responses of the central and peripheral glial microenvironment, immune system, and target musculature. The facial nerve injury model is a widely accepted peripheral nerve injury model that serves as a powerful tool for studying nerve injury and regeneration.

We present a surgical protocol detailing how to perform a cut or crush axotomy on the facial nerve in the mouse. The facial nerve axotomy can be employed to study the physiological response to nerve injury and test therapeutic techniques.

마우스의 얼굴 신경 Axotomy : 부상으로 운동 신경 응답을 연구하는 모델

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and ...

Establishing a Mouse Model of a Pure Small Fiber Neuropathy with the Ultrapotent Agonist of Transient Receptor Potential Vanilloid Type 1

Patients with diabetes mellitus (DM) or those experiencing the neurotoxic effects of chemotherapeutic agents may develop sensation disorders due to degeneration and injury of small-diameter sensory neurons, referred to as small fiber neuropathy. Present animal models of small fiber neuropathy affect both large- and small-diameter sensory fibers and thus create a neuropathology too complex to properly assess the effects of injured small-diameter sensory fibers. Therefore, it is necessary to develop an experimental model of pure small fiber neuropathy to adequately examine these issues. This protocol describes an experimental model of small fiber neuropathy specifically affecting small-diameter sensory nerves with resiniferatoxin (RTX), an ultrapotent agonist of transient receptor potential vanilloid type 1 (TRPV1), through a single dose of intraperitoneal injection, referred to as RTX neuropathy. This RTX neuropathy showed pathological manifestations and behavioral abnormalities that mimic the clinical characteristics of patients with small fiber neuropathy, including intraepidermal nerve fiber (IENF) degeneration, specifically injury in small-diameter neurons, and induction of thermal hypoalgesia and mechanical allodynia. This protocol tested three doses of RTX (200, 50, and 10 &#181;g/kg, respectively) and concluded that a critical dose of RTX (50 &#181;g/kg) is required for the development of typical small fiber neuropathy manifestations, and prepared a modified immunostaining procedure to investigate IENF degeneration and neuronal soma injury. The modified procedure is fast, systematic, and economic. Behavioral evaluation of neuropathic pain is critical to reveal the function of small-diameter sensory nerves. The evaluation of mechanical thresholds in experimental rodents is particularly challenging and this protocol describes a customized metal mesh that is suitable for this type of assessment in rodents. In summary, RTX neuropathy is a new and easily established experimental model to evaluate the molecular significance and intervention underlying neuropathic pain for the development of therapeutic agents.

This study establishes an experimental model of pure small fiber neuropathy with resiniferatoxin (RTX). A unique dose of RTX (50 &#181;g/kg) is optimal for developing a small fiber neuropathy model that mimics patient characteristics and could help investigate the nociceptive molecular significance underlying neuropathic pain.

일시적인 수용 체 잠재력 Vanilloid 타입-1의 Ultrapotent 주 작동 근의 순수한 작은 섬유 신경 병 마우스 모델 수립

Patients with diabetes mellitus (DM) or those experiencing the neurotoxic effects of chemotherapeutic agents may develop sensation disorders due to ...

신경과학

Protocol to Create Chronic Wounds in Diabetic Mice

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ~6.5M people and cost ~$40B/year in the US alone. Although a significant effort has been invested in understanding how chronic wounds develop in humans, fundamental questions remain unanswered. Recently, we developed a novel mouse model for diabetic chronic wounds that have many characteristics of human chronic wounds. Using db/db-/- mice, we can generate chronic wounds by inducing high levels of oxidative stress (OS) in the wound tissue immediately after wounding, using a one-time treatment with inhibitors specific to the antioxidant enzymes catalase and glutathione peroxidase. These wounds have high levels of OS, develop biofilm naturally, become fully chronic within 20 days after treatment and can remain open more for more than 60 days. This novel model has many features of diabetic chronic wounds in humans and therefore can contribute significantly to advancing fundamental understanding of how wounds become chronic. This is a major breakthrough because chronic wounds in humans cause significant pain and distress to patients and result in amputation if unresolved. Moreover, these wounds are very expensive and time-consuming to treat, and lead to significant loss of personal income to patients. Advancements in this field of study through the use of our chronic wound model can significantly improve health care for millions who suffer under this debilitating condition. In this protocol, we describe in great detail the procedure to cause acute wounds to become chronic, which has not been done before.

Chronic wounds are developed from acute wounds on a diabetic mouse model by inducing high levels of oxidative stress after a full-thickness cutaneous wound. The wound is treated with inhibitors for catalase and glutathione peroxidase, resulting in impaired healing and biofilm development by bacteria present in the skin microbiome.

당뇨병 마우스에서 만성 상처를 만드는 프로토콜

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ...

Modified Spared Nerve Injury Surgery Model of Neuropathic Pain in Mice

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular mechanism of neuropathic pain in mice and rats. Currently, there are two types of SNI model, one to cut and ligate the common peroneal and the tibial nerves with intact sural nerve, which is defined as SNIs in this study, and another to cut and ligate the common peroneal and the sural nerves with intact tibial nerve, which is defined as SNIt in this study. Because the sural nerve is purely sensory whereas the tibial nerve contains both motor and sensory fibers, the SNIt model has much less motor deficit than the SNIs model. In the traditional SNIt mouse model, the common peroneal and the sural nerves are cut and ligated separately. Here a modified SNIt surgery method is described to damage both common peroneal and sural nerves with only one ligation and one cut with a shorter procedure time, which is easier to perform and reduces the potential risk of stretching the sciatic or tibial nerves, and produces similar mechanical hypersensitivity as the traditional SNIt model.

<meta charset="utf8"></head><body>마우스의 신경병증성 통증에 대한 수정된 보존 신경 손상 수술 모델

The modified surgery is a simplified method for mouse or rat spared nerve injury model that requires only one ligation and one cut to injure both common peroneal and sural nerves.

마우스의 신경병증성 통증에 대한 수정된 보존 신경 손상 수술 모델

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular ...

Partial Sciatic Nerve Ligation: A Mouse Model of Chronic Neuropathic Pain to Study the Antinociceptive Effect of Novel Therapies

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. Chronic neuropathic pain results from lesions or diseases in the somatosensory system. To investigate potential therapies with reduced side effects, animal pain models are the gold standard in preclinical studies. Therefore, well-characterized and well-described models are crucial for the development and validation of innovative therapies. 
Partial ligation of the sciatic nerve (pSNL) is a procedure that induces chronic neuropathic pain in mice, characterized by mechanical and thermal hypersensitivity, ongoing pain, and changes in limb temperature, making this model a great fit to study neuropathic pain preclinically. pSNL is an advantageous model to study neuropathic pain as it reproduces many symptoms observed in humans with neuropathic pain. Furthermore, the surgical procedure is relatively fast and straightforward to perform. Unilateral pSNL of one limb allows for comparison between the ipsilateral and contralateral paws, as well as evaluation of central sensitization. 
To induce chronic neuropathic hypersensitivity, a 9-0 non-absorbable nylon thread is used to ligate the dorsal third of the sciatic nerve. This article describes the surgical procedure and characterizes the development of chronic neuropathic pain through multiple commonly used behavioral tests. As a plethora of innovative therapies are now being investigated to treat chronic pain, this article provides crucial concepts for standardization and an accurate description of surgeries required to induce neuropathic pain.

Partial sciatic nerve ligation induces long-lasting chronic neuropathic pain, characterized by exaggerated responses to thermal and mechanical stimuli. This mouse model of neuropathic pain is commonly used to study innovative therapies for pain management. This article describes in detail the surgical procedure to improve standardization and reproducibility.

부분 좌골 신경 결찰: 새로운 치료법의 항통각수용 효과를 연구하기 위한 만성 신경병성 통증의 마우스 모델

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. ...

마우스 교감신경 신경 세포 표적화: 요추 교감신경 절제술 접근법

Surgical Lumbar Sympathectomy in Mice

Peripheral nerve injuries are common, and full functional recovery after injury is achieved in only 10% of patients. The sympathetic nervous system plays many critical roles in maintaining bodily homeostasis, but it has rarely been studied in the context of peripheral nerve injury. The extent of postganglionic sympathetic neuronal functions in distal targets in the periphery is currently unclear. To better explore the role of sympathetic innervation of peripheral targets, a surgical &#34;knock-out&#34; model provides an alternative approach. Although this can be achieved chemically, chemical destruction of postganglionic sympathetic neurons can be nonspecific and dose-dependent. The use of a surgical lumbar sympathectomy in mice, once thought to be &#34;virtually not practicable&#34; in small animals, allows for specific targeting of postganglionic sympathetic neurons that innervate the hind limbs. This manuscript describes how to surgically remove the L2-L5 lumbar sympathetic ganglia from a mouse as a survival surgery, which reliably decreases the hind paw sweat response and the number of sympathetic axons in the sciatic nerve.

저자 스포트라이트: 교감신경 뉴런의 가소성 탐구

This manuscript presents a protocol for surgically removing the postganglionic lumbar sympathetic neurons from a mouse. This procedure will facilitate a multitude of studies aimed at investigating the role of sympathetic innervation in distal tissue targets.

생쥐의 외과적 요추 교감신경 절제술

Peripheral nerve injuries are common, and full functional recovery after injury is achieved in only 10% of patients. The sympathetic nervous system plays ...

탈 신경 외과 피부 : 피부 질환의 마우스 모델에서 신경에 대한 요구를 테스트하기위한 방법

요약

더 많은 비디오 탐색

이 비디오의 챕터

마우스에서 유도된 기계 Allodynia의 겪었던 신경 상해 (스니) 모델

경골 신경 절개 - 마우스의 탈 신경에 의한 골격근의 위축에 대한 표준 모델

마우스의 얼굴 신경 Axotomy : 부상으로 운동 신경 응답을 연구하는 모델

일시적인 수용 체 잠재력 Vanilloid 타입-1의 Ultrapotent 주 작동 근의 순수한 작은 섬유 신경 병 마우스 모델 수립

당뇨병 마우스에서 만성 상처를 만드는 프로토콜

마우스의 신경병증성 통증에 대한 수정된 보존 신경 손상 수술 모델

마우스의 신경병증성 통증에 대한 수정된 보존 신경 손상 수술 모델

부분 좌골 신경 결찰: 새로운 치료법의 항통각수용 효과를 연구하기 위한 만성 신경병성 통증의 마우스 모델

생쥐의 외과적 요추 교감신경 절제술

생쥐의 외과적 요추 교감신경 절제술