이 작품은 (HPL에) NIH 보조금 R01 GM087609, (MTL에) (HPL에) 안토니 슈, NIH 보조금 U01의 HL099776의 명예에 잉그리드 라이 빌 슈에서 선물, Hagey 연구소에 대한로부터 연구비 일부 지원 소아 재생 의학 및 (MTL과 HPL에) 오크 재단. GGW 의학의 스탠포드 대학, 스탠포드 의료 과학자 훈련 프로그램에 의해 지원되며, NIGMS 훈련 보조금 GM07365했다. MSH는 CIRM 임상 연구원 교육 그랜트 TG2-01159에 의해 지원되었다. WXH가 노프 심장 혈관 재단 기금에 의해 지원되었다.

참고 :이 문서에 설명 된 모든 절차는 실험 동물 관리에 스탠포드 행정 패널 (APLAC)에 의해 설립 지침에 따라 수행된다. 1. 시간 제한 임신 - 과배란 기술 (그림 1) 참고 : 정확하게 E16.5 및 E18.5에서 태아 수술을 위해 마우스 배아의 임신 기간 타이밍은 매우 중요합니다. 과배란을 유도하기 위해 임신 한 암컷 혈청 (PMS) 및 인간 융모 성 성선 자극 호르몬 (HCG) 주사를 사용하여 마우스 임신 타이밍에 대한 우리의 프로토콜이 절 우리의 세부 사항. <ol><li> 1:00 하루 1 3:00 PM 사이에 100 μl의 PBS의 볼륨 PMS의 3.0-5.0 국제 단위 (IU)와 여성 마우스 (케이지 당 &lt;5) 복강 내 (IP)를 주입한다. </li><li> 12:00 3 일 (사십 오 PMS 주입 후 시간 47) 2:00시 사이에, 100 μl의 PBS의 볼륨 HCG의 3.0-5.0 IU와 여성 쥐 IP를 주입. 참고 : HCG 주입 유도한다<html> 배란 약 12​​ 시간 후 분사. </li><li> 즉시 HCG 주사 다음 8-16 주령의 수컷과 암컷을 짝짓기. 참고 : 우리는 일반적으로 개별 남성의 케이지에 두 여성을 배치합니다. </li><li> 태아 연령 E0.5으로 - (10:00 AM 6:00) 기록 4 일의 아침에 남성에서 분리 된 여성. 참고 : 질 플러그가이 시간에 확인할 수 있습니다; 그러나, 질 플러그의 관찰은 임신을 보장하지 않으며 어떠한 플러그가 관찰되지 않을 때 여성은 임신이 될 수 있습니다. 임신 4 일의 아침에 질 플러그 검사, 임신 나이 E16.5 및 E18.5에서 육안 검사 및 / 또는 촉진에 의해 일반적으로 관찰 할 수 있음을 감안할 때 꼭 필요한 것은. 경험상, 상기 균주에 따라 슈퍼 배란 여성의 약 30 % ~ 50 %가 여기에 설명 된 기술을 사용하여 임신. </li></ol>PG &quot;/&gt; 그림 과배란 기법에 대한 도식. 마우스에서 과배란 초과 임신에 대한 회로도를 보여주는 절차. <a href="https://www.jove.com/files/ftp_upload/52297/52297fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 2. 쥐과 태아 수술 E16.5 및 E18.5 배아에 (등쪽 부상) (그림 2) <ol><li> 절차 전에, 70 % 이소 프로필 알코올 수술실 및 장비의 모든 표면을 청소합니다. 또한,이를 오토 클레이브에 의해 절차에서 사용될 모든 수술 용품 및기구 소독. 일부 기관들은 뜨거운 비드 살균 이후의 사용을 허용 할 수 있습니다. 작업 당, 거즈 및 수술 도구를 포함 무균 팩을 사용합니다. </li><li> 분당 1 L에 유지 보수 마취 다음에 분당 2 L에서 2.5 % 이소 플루 란 / 산소 혼합물에서 임신 한 어머니의 마취 (태아 연령 E16.5 또는 E18.5)를 유도한다. </li><li> 사기하려면회사 적절한 마취, 마우스의 깊은 페달 반사가 억제되어 있는지 확인하고 발생하기 쉬운 위치에 마우스를 놓습니다. </li><li> Puralube이 절차를 수행하는 동안 눈에 자극이나 건조 함을 방지하기 위해 같은 수의사 안과 연고를 적용합니다. </li><li> 더 이상 30 초 (그림 2A)을 위해 제모 크림의 빛 응용 프로그램을 투여하여 복부를 준비합니다. </li><li> 포비돈 - 요오드 알코올 (그림 2B 및 2C) 무균 수술을 위해 복부를 준비합니다. </li><li> 미세 수술 가위 (그림 2D 및 2E)를 사용하여 현미경으로 중간 선 개복술을 수행합니다. </li><li> 조심스럽게 수술 (그림 2 층)으로 선택 자궁과 태아를 노출합니다. </li><li> 뭉툭한 팁 바늘을 사용하여 따뜻한 (38 ° C) 인산염 완충 식염수 (PBS)와 수술 필드를 관개 참고 : 하나는 조심스럽게 큰 구멍 바늘의 끝 부분을 구부려서 할 수있다. (그림 2G 및 2H). </li><li> 허용하는 방식으로 태아의 위치를배부에 대한 전체 액세스 권한을에요. </li><li> 의도 지느러미 상처 (그림 2I)의 사이트를 덮는 자궁을 통해 7-0 나일론 봉합사를 사용하여 지갑 문자열 스티치를 전달합니다. 위치 지갑 왼쪽 또는 척수의 오른쪽에 배부의 영역을 통해 문자열, 큰 혈관이없는 자궁 벽의 영역에서. </li><li> 지갑 문자열 (그림 2J)의 중심에 자궁 벽과 양막을 통해 3mm의 절개를합니다. </li><li> 따뜻한 (38 ° C) PBS와 절개 사이트를 관개. </li><li> 미세 수술 가위를 사용하여 태아의 등쪽에서, 하나의 전층 상처 절제 길이 약 1mm 잘라. </li><li> 부드럽게 얼룩 절개 사이트는면 팁 어플리케이터를 건조. </li><li> 상처의 위치 (그림 2K)를 표시하기 위해 상처 부위에 3 μL 볼륨 인도 잉크 피하 주입한다. </li><li> 잉크가 상처 사이트 내에서 유지 된 보장하기 위해 따뜻한 (38 ° C) PBS로 세척하십시오. </li><li> 수술 보조 난 되세요nject 지갑 문자열로 양막에 무딘 팁 10 G 주사기를 통해 따뜻한 (38 ° C) PBS (그림 2L)를 폐쇄한다. 지갑 문자열 폐쇄 완료 (그림 2M)에 가까워지면서 주사기를 집어 넣으십시오. </li><li> 조심스럽게 복강 (그림 2N)로 자궁을 반환합니다. </li><li> 에버트 피부와 복막. </li><li> 따뜻한 (38 ° C) PBS로 복강 수술 보조 관개했다. </li><li> 피부와 복막을 스테이플 링으로 빠르게 닫기 복부 (그림 2O)을 마감했다. 표준 클로저는 두 층에서 수행된다; 복막과 제 2 층에서의 하나의 층, 피하 조직 및 피부 복근. 즉시 희생과 태아의 수확을 위해, 우리의 시위는 한 층에 폐쇄를 보여줍니다. </li><li> 30 분 동안이나 동물이 흉골 드러 누움을 유지하기에 충분한 의식을 회복 할 때까지 37 ° C로 설정 따뜻한 인큐베이터에서 관찰에서 동물을 놓습니다. </li><li> 마지막 ret하지 마십시오완전히 절차에서 회복 될 때까지 다른 동물의 회사에 동물을 항아리. </li><li> 마취에서 그 이후 48 시간 동안 각성시, 부 프레 노르 핀의 피하 주사 (0.05 ㎎ / ㎏) 통증 평가에 따라 필요에 따라 진통제마다 12 시간을 관리 할 수​​ 있습니다. 필요에 따라 추가 수술 후 통증 완화를 위해 피하 주사를 통해 카프로 펜 (5 ㎎ / ㎏)을 관리합니다. </li><li> 케이지에 동물을 반환하고 물과식이로 제공합니다. </li><li> 통증의 양상 면밀히 모니터링합니다. </li><li> 48 시간의 수술 후는, 이소 플루 란과 수확 부상 태아의 과다 복용으로 임신 한 어머니를 희생. 이를 위해, 5 % 이상으로 이소 플루 란 농도를 조정하고 호흡의 중단 후 1 분 동안 노출을 유지한다. 자궁 경부 전위와 안락사를 확인합니다. 연령대 제어를위한 unwounded 배아를 수확. 늦은 배아는 같은 IACUC의 권고와 일치 안락사의 별도의 방법이 있어야 드인두세, 자궁 전위, 또는 화학 물질 주입. </li></ol><img alt="그림 2" src="/files/ftp_upload/52297/52297fig2highres.jpg" /> 그림 2. 회로도 쥐과 태아 외과. E16.5 및 E18.5 마우스 배아에 상처를 입는 등의 일반 단계를 반복합니다. (A) 마우스 복부의 탈모. (B 및 C) 마우스 복부의 준비. (D) 현미경 수술에 사용 자궁을 통해 지갑 문자열 봉합의 과정. (E) 중간 선 개복술. (F) 자궁의 노출. 뭉툭한 팁 바늘 (G) 작성. 따뜻한 식염수와 자궁의 (H) 관개. (I) 작성. (J) 절개 벽과 1mm 전체 두께 절제 상처 발생. (K) 인도 잉크의 피하 주사. (L과 M) 지갑 문자열 봉합의 폐쇄. (N과 O)복부의 폐쇄. <a href="https://www.jove.com/files/ftp_upload/52297/52297fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

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The authors have nothing to disclose.

여기에 제시된 수술 프로토콜은 먼저 우리의 실험실 (10)에 의해 2006 년에 출판 된 태아 쥐의 흉터 치유 절제 모델에 대해 설명합니다. 절제 상처 (11)의 다른 모델의 확립 외에, 뮤린 태아 흉터, 창상 치유의 모델 (12, 13)뿐만 아니라 존재한다. 원숭이, 양, 토끼, 쥐, 쥐 태아 흉터 상처 치유의 조사는 14 ~ 17보고되었다. 그러나 마우스는 그들의 비교적 낮은 당 일일 케이...

태아 피부 상처 임신 1 늦게까지 신속하고 scarlessly 치유. 태아 흉터 상처 수리는 정상 조직의 구조와 기능의 재생이 특징입니다. 흉터 표현형에 흉터에서의 전환은 인간 및 마우스 2,3에 배아 일 18 (E18)의 주위에 임신의 세 번째 임신에서 발생합니다. 성인에 비해, 상처 복구 태아 급속한 상피화, 결합 조직의 증착, 및 섬유 아세포 이동을 특징으로한다. 많은 연구 초기 태아의 개발 기간 동안 흉터 상처 치유의 현상에 대한 가능한 설명을 제안했다. 염증은 성인 상처 수리의 기본 구성 요소입니다; 그러나, 태아의 상처는 급성 염증 (4)의 부족을 특징으로한다. 이 단계 동안 여부 태아 면역계의 작용 미성숙 결과는 불분명하다. 최근 연구 제안이 풍부, 매트의 차이urity 및 E18 태아의 피부 대 E15에서 비만 세포의 기능 이상 마우스 3 흉터 표현형으로의 전환에 대한 책임이 있습니다. 다른 연구는 속성과 태아와 성인 상처 대 식세포의 풍요 로움의 차이가 태아의 상처 수리 (5) 동안 정상 세포 외 기질 (ECM)의 개혁에 대한 책임이 있음을 가정하는. 태아 및 성인 개발시 환경 요인의 차이도 상처 복구에 영향을 미칠 수 있습니다. Longaker와 동료들은 태아의 상처 액이 6 유체 상처 성인 없음에 비해 히알루 론산 자극 활동의 높은 수준을 보유하고 있음을 보여 주었다. 따라서, 태아 상처 환경에서 히알루 론산, 세포 운동성 및 확산에 도움이 미세 환경을 촉진하는 글리코 사 미노 글리 칸, 높은 수준의 초기 태아의 개발 과정에서 볼 수있는 흉터의 표현형에 대한 책임을 할 수있다. 태아는 사실에 근거 포인트의 다른 라인L 상처 환경은 상대적으로 저산소증과 성장 인자 7 풍부한 무균 양수에 잠겨있다. 그러나, 확실한 대답은 섬유 성 수리에 흉터 재생으로의 전환을 트리거 배아 중 중요한 사건이나 요인에 제공 없습니다. 태아의 흉터 치유 메커니즘 책임을 이해하는 것은 정확하고 재현성 모델을 필요로한다. 여기 세부 E16.5 (흉터) 및 E18.5 (흉터) 마우스 배아의 배부 태아 흉터 상처 치유의 재현 모델. 또한,이 모델의 경미한 변화는 이러한 태아 피부 및 상처 8,9의 유전자 발현 분석과 같은 추가의 연구들을 수행하는데 이용 될 수있다. 정확하게 시간이 초과 임신이 태아의 흉터 상처 치유 모델의 성공적인 반복 설에 중요한 점을 감안, 우리 과배란 초과 임신에 대해서도 상세하게 우리의 프로토콜입니다.

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			<td height="20" style="height:20px;">7-O MONOSOF Suture</td>
			<td>eSuture</td>
			<td>SN-1647G</td>
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			<td height="20" style="height:20px;">Surgical Forceps</td>
			<td>Kent Scientific</td>
			<td>INS650916</td>
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			<td height="20" style="height:20px;">Micro-scissors</td>
			<td>Kent Scientific</td>
			<td>INS600127</td>
			<td></td>
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			<td height="20" style="height:20px;">Autoclip 9mm</td>
			<td>Texas Scientific Instruments</td>
			<td>205060</td>
			<td></td>
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			<td height="20" style="height:20px;">Insulin Syringe</td>
			<td>Thermo Fisher Scientific</td>
			<td>22-272-382</td>
			<td></td>
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			<td height="20" style="height:20px;">Black Pigment</td>
			<td>AIMS</td>
			<td>242</td>
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			<td height="20" style="height:20px;">BD Safety-Lok 3ml Syringe</td>
			<td>BD Biosciences</td>
			<td>309596</td>
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			<td height="20" style="height:20px;">Phosphate Buffered Saline</td>
			<td>Life Technologies</td>
			<td>10010-049</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">OPMI-MD Surgical Microscope</td>
			<td>Carl Zeiss Surgical Inc</td>
			<td></td>
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			<td height="20" style="height:20px;">Pregnant Mares Serum (PMS)</td>
			<td>Millipore</td>
			<td>367222</td>
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			<td height="20" style="height:20px;">Human Chorionic Gonadotropin (HCG)</td>
			<td>Sigma-Aldrich</td>
			<td>CG10</td>
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			<td height="20" style="height:20px;">Povidone Iodine Prep Solution</td>
			<td>Dynarex</td>
			<td>1415</td>
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			<td height="20" style="height:20px;">Nair (depilatory cream)</td>
			<td>Church and Dwight Co.</td>
			<td>22600267058</td>
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a mouse fetal skin model of scarless wound repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

조직 학적 분석을 위해, E16.5 및 E18.5 마우스 배아의 등 피부에 피부의 상처는 48 시간의 PFA 4 %에 고정 후 상처, 및 파라핀을 수확해야합니다. 형광 형질 전환 모델에서, 빛 간섭 단층 냉동 보존이 적합 할 수 있습니다. 세포 및 결합 조직 구조를 시각화하는데 이용 될 수있는 몇 가지 얼룩이있다. 헤 마톡 실린 및 에오신 파란색 핵 및 산성 구조 (즉., 세포질과 세포 외 콜라겐) 레드, 핑크, 오렌?...

Watch this Scientific Journal Video about A Mouse Fetal Skin Model of Scarless Wound Repair at JoVE.com

A Mouse Fetal Skin Model of Scarless Wound Repair

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

흉터 상처 수리의 마우스 태아의 피부 모델

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

a-mouse-fetal-skin-model-of-scarless-wound-repair

Research

JoVE Journal

Medicine

12.2K 조회수.  Stanford University School of Medicine. During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

비디오: A Mouse Fetal Skin Model of Scarless Wound Repair

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.1 Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.
Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5&deg; (animals rotated at 22.5&deg;), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.
The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

무한 호라이즌 임팩터을 사용하여 일방적 경부 척수의 부상의 Contusive 모델

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of ...

연구

의학

A Simplified Technique for Producing an Ischemic Wound Model

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work well in non-ischemic wounds may not work in human diabetic wounds because vasculopathy is one major factor that hinders healing of these wounds. We published an article in 2007 describing a rabbit ear ischemic wound model created by a minimally invasive surgical technique. Since then, we have further simplified the procedure for easier operation. On one ear, three small skin incisions were made on the vascular pedicles, 1-2 cm from the ear base. The central artery was ligated and cut along with the nerve. The whole cranial bundle was cut and ligated, leaving only the caudal branch intact. A circumferential subcutaneous tunnel was made through the incisions, to cut subcutaneous tissues, muscles, nerves, and small vessels. The other ear was used as a non-ischemic control. Four wounds were made on the ventral side of each ear. This technique produces 4 ischemic wounds and 4 non-ischemic wounds in one animal for paired comparisons. After surgery, the ischemic ear was cool and cyanotic, and showed reduced movement and a lack of pulse in the ear artery. Skin temperature of the ischemic ear was 1-10 &deg;C lower than that on the normal ear and this difference was maintained for more than one month. Ear tissue high-energy phosphate contents were lower in the ischemic ear than the control ear. Wound healing times were longer in the ischemic ear than in the non-ischemic ear when the same treatment was used. The technique has now been used on more than 80 rabbits in which 23 were diabetic (diabetes time ranging from 2 weeks to 2 years). No single rabbit has developed any surgical complications such as bleeding, infection, or rupture in the skin incisions. The model has many advantages, such as little skin disruption, longer ischemic time, and higher success rate, when compared to many other models. It can be safely used in animals with reduced resistance, and can also be modified to meet different testing requirements.

We have developed a minimally invasive technique to create a rabbit ischemic ear wound model by dividing the central artery and nerve and the cranial neurovascular bundle. A subcutaneous tunnel then cuts all subcutaneous tissues. This procedure causes minimal skin disruption and can be safely used in diabetic animals.

허혈성 상처 모델 생산을위한 단순화된 테크닉

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work ...

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

태아 알코올 노출의 Zebrafish 모델에서 Teratogenic 변경 사항 평가

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

태아 마우스 모델에서 Bioactive 에이전트의 Intratracheal 관리를위한 소설 외과 접근

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

상처 치유의 생쥐 모델

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ultrasound and fetal echocardiography. Increasingly, these imaging modalities are being employed clinically to examine cardiac function and assess wellbeing in utero, thereby guiding timing of birth decisions. Here, we used a rabbit model of IUGR that allows analysis of cardiac function in a clinically relevant way. Using isoflurane induced anesthesia, IUGR is surgically created at gestational age day 25 by performing a laparotomy, exposing the bicornuate uterus and then ligating 40-50% of uteroplacental vessels supplying each gestational sac in a single uterine horn. The other horn in the rabbit bicornuate uterus serves as internal control fetuses. Then, after recovery at gestational age day 30 (full term), the same rabbit undergoes examination of fetal cardiac function. Anesthesia is induced with ketamine and xylazine intramuscularly, then maintained by a continuous intravenous infusion of ketamine and xylazine to minimize iatrogenic effects on fetal cardiac function. A repeat laparotomy is performed to expose each gestational sac and a microultrasound examination (VisualSonics VEVO 2100) of fetal cardiac function is performed. Placental insufficiency is evident by a raised pulsatility index or an absent or reversed end diastolic flow of the umbilical artery Doppler waveform. The ductus venosus and middle cerebral artery Doppler is then examined. Fetal echocardiography is performed by recording B mode, M mode and flow velocity waveforms in lateral and apical views. Offline calculations determine standard M-mode cardiac variables, tricuspid and mitral annular plane systolic excursion, speckle tracking and strain analysis, modified myocardial performance index and vascular flow velocity waveforms of interest. This small animal model of IUGR therefore affords examination of in utero cardiac function that is consistent with current clinical practice and is therefore useful in a translational research setting.

We describe examination of fetal cardiac function with contemporary functional fetal echocardiography and fetoplacental Doppler ultrasound using the VisualSonics VEVO 2100 microultrasound in a surgically induced model of intrauterine fetal growth restriction in a rabbit.

자궁 내 성장 제한의 토끼 모델에서 태아 심 초음파 및 펄스 파 도플러 초음파

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ...

A New Murine Model of Endovascular Aortic Aneurysm Repair

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of Abdominal aortic aneurysm (AAA) pathophysiology. There is no abdominal aortic aneurysm endograft exclusion model cheap and reproducible, which would allow deep investigations of AAA before and after treatment. We hereby describe how to induce, and then to exclude with a covered coronary stentgraft an abdominal aortic aneurysm in a rat. The well known elastase induced AAA model was first reported in 19901 in a rat, then described in mice2. Elastin degradation leads to dilation of the aorta with inflammatory infiltration of the abdominal wall and intra luminal thrombus, matching with human AAA. Endovascular exclusion with small covered stentgraft is then performed, excluding any interactions between circulating blood and the aneurysm thrombus. Appropriate exclusion and stentgraft patency is confirmed before euthanasia by an angiography thought the left carotid artery. Partial control of elastase diffusion makes aneurysm shape different for each animal. It is difficult to create an aneurysm, which will allow an appropriate length of aorta below the aneurysm for an easy stentgraft introduction, and with adequate proximal and distal neck to prevent endoleaks. Lots of failure can result to stentgraft introduction which sometimes lead to aorta tear with pain and troubles to stitch it, and endothelial damage with post op aorta thrombosis. Giving aspirin to rats before stentgraft implantation decreases failure rate without major hemorrhage. Clamping time activates neutrophils, endothelium and platelets, and may interfere with biological analysis.

Histological and biochemical modifications after aneurysm endograft exclusion are still unclear. We describe a new model of endograft implantation on a murine aneurysm. Through thrombus analysis with and without persistant circulating blood stream interface, and new endovascular biomaterials evaluation, this model will help to better understand endovascular aneurysm exclusion pathobiology.

혈관 동맥류 수리의 새로운 쥐과 모델

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of ...

Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are still occurring, particularly in reconstructive flap surgery. Multiple experimental flap models have been developed to analyze underlying causes and mechanisms and to investigate treatment strategies to prevent ischemic complications. The limiting factor of most models is the lacking possibility to directly and repetitively visualize microvascular architecture and hemodynamics. The goal of the protocol was to present a well-established mouse model affiliating these before mentioned lacking elements. Harder et al. have developed a model of a musculocutaneous flap with a random perfusion pattern that undergoes acute persistent ischemia and results in ~50% necrosis after 10 days if kept untreated. With the aid of intravital epi-fluorescence microscopy, this chamber model allows repetitive visualization of morphology and hemodynamics in different regions of interest over time. Associated processes such as apoptosis, inflammation, microvascular leakage and angiogenesis can be investigated and correlated to immunohistochemical and molecular protein assays. To date, the model has proven feasibility and reproducibility in several published experimental studies investigating the effect of pre-, peri- and postconditioning of ischemically challenged tissue.

The window of the murine dorsal skinfold chamber presented visualizes a zone of acute persistent ischemia of a musculocutaneous flap. Intravital epi-fluorescence microscopy permits for direct and repetitive assessment of the microvasculature and quantification of hemodynamics. Morphologic and hemodynamic results can further be correlated with histological and molecular analyses.

마우스의 등쪽 피하 지방 상공 회의소에서 허혈성 조직 손상 : 피부 플랩 모델은 급성 영구 허혈을 조사하기

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are ...

A Surgical Procedure for Resecting the Mouse Rib: A Model for Large-Scale Long Bone Repair

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation of the animal for surgery, opening the thoracic body wall, exposing the desired rib from the surrounding intercostal muscles, excising the desired section of rib without inducing a pneumothorax, and closing the incisions. Compared to the bones of the appendicular skeleton, the ribs are highly accessible. In addition, no internal or external fixator is necessary since the adjacent ribs provide a natural fixation. The surgery uses commercially available supplies, is straightforward to learn, and well-tolerated by the animal. The procedure can be carried out with or without removing the surrounding periosteum, and therefore the contribution of the periosteum to repair can be assessed. Results indicate that if the periosteum is retained, robust repair occurs in 1 - 2 months. We expect that use of this protocol will stimulate research into rib repair and that the findings will facilitate the development of new ways to stimulate bone repair in other locations around the body.

The overall goal of this procedure is to successfully resect a portion of bone from the rib of a mouse. The procedure was developed as a model to study large-scale long bone repair.

마우스 리브를 절제하는 수술 : 대규모 긴 뼈 수리에 대한 모델

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation ...

Demonstration of the Rat Ischemic Skin Wound Model

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and unrelieved pressure due to immobility. Animal models have been developed that attempt to mimic these conditions for the purpose of furthering our understanding of the complexity of chronic wounds. The model described herein is a rat ischemic skin flap model that permits a prolonged reduction of blood flow resulting in wounds that become ischemic and resemble a chronic wound phenotype (reduced vascularization, increased inflammation and delayed wound closure). It consists of a bipedicled dorsal flap with 2 ischemic wounds placed centrally and 2 non-ischemic wounds lateral to the flap as controls. A novel addition to this ischemic skin flap model is the placement of a silicone sheet beneath the flap that functions as a barrier and a splint to prevent revascularization and reduce contraction as the wounds heal. Despite the debate of using rats for wound healing studies due to their quite distinct anatomic and physiologic differences compared to humans (i.e., the presence of a panniculus carnosus muscle, short life-span, increased number of hair follicles, and their ability to heal infected wounds) the modifications employed in this model make it a valuable alternative to previously developed ischemic skin flap models.

The rat, due to its size, availability, and rather docile behavior, has been utilized as a research model for many years. The goal of this protocol is to utilize the rat as an ischemic skin wound healing model to provide valuable insight into the pathophysiology of chronic wounds.