We would like to thank Dr. Lei Chen (Icahn School of Medicine at Mount Sinai, NY) who first established the use of this model in our laboratory, and to Dr. Gretchen Wolff (German Cancer Research Center, Heidelberg, Germany) for disseminating the technique in our laboratory. Supported in part by HL126559, DA039576, MH098891, MH63022, MH072567, DA027569, and NSC 2015/17/B/NZ7/02985.

다음 프로토콜에 관련된 절차는 마이애미 기관 동물 관리 및 사용위원회의 대학 (IACUC)에 의해 승인되었습니다. 또한, 모든 절차는 실험 동물 관리 국제 평가 및 인증 협회 (AAALAC)의 승인을 시설에서 진행되고있다. 외과 마우스 1. 준비 <ol><li> 실험실 마취 시스템을 이용하여, 산소와 혼합하여 이소 플루 란 마취 마우스. 2 L에 %와 산소 흐름 4-5 사이에 설정에서 이소 플루 란을 사용하여 / 상업 시스템에 분 (재료 표 참조). 실체 현미경 하에서 수술면에 동물을 전송하고, 노즈콘을 사용하여 마취를 유지 (- 2 L / 분으로 2.5 1.5 산소 유량 설정 이소 플루 란을 사용한다). <ol><li> 허걱없이 2 호흡 / 초 - 마우스 호흡 속도가 1 주위에 있는지 확인하십시오. 또한, 동물의 수염 자극 반응 페달 (R)가 발생하지 않도록eflex (발가락 핀치). , 수술 동안 적어도 매 5 분 호흡 속도와 노력을 모니터링합니다. 설치류 마취 모니터링을 위해 특정 기관 동물 관리 및 사용위원회 및 수의학 지침을 따르십시오. </li></ol></li><li> 건조 함을 예방하기 위해 멸균 면봉을 사용하여 각각의 눈에 대한 안과 용 윤활제 방울을 적용한다. 항 염증 및 통증을 완화하는 진통제의 투여는 권장합니다. </li><li> 동물의 뒷면에 누워, 코 콘을 사용하여 마취를 유지한다. </li><li> 지역을 에탄올 70 %와 클로르헥시딘로 3 회 닦아 동물의 목 영역을 청소합니다. (아래에 설명)을 면도칼을 사용하여 동물의 수술 영역을 면도. </li></ol> 경동맥 2. 해부 (CCA) <ol><li> 실체 현미경 하에서 전체 미세 절차를 수행합니다. 수술 가위를 사용하고 집게은 가슴 위에서 t 이하로, 목에 얕은 중간 선 절개를 수행그는 (약 3 ~ 4 cm)를 턱. </li><li> 집게를 조심스럽게 분리 지방 및 결합 조직을 사용하여 기관을 노출시킨다. </li><li> 더 영역을 노출, 목을 연장 마우스의 목에의 뒷면에 베개 (라운드 개체, 직경 약 0.5 cm)를 놓습니다. </li><li> 조직 권취 또는 후크를 사용하여 조직을 분리한다. </li><li> 기관의 동물의 왼쪽에서 조심스럽게 왼쪽 CCA를 노출 할 수있는 결합 조직을 분리 tweeze. </li><li> 사용 포셉 신중하게 CCA의 분기점을 노출하는 모든 결합 조직, 모두 외부 및 내부 경동맥의 시작을 제거합니다. </li></ol> 약물 주입을위한 CCA 3. 준비 <ol><li> 집게를 사용하여, 외부 경동맥에서 나일론 봉합사 (약 1cm 각)의 두 개의 세그먼트 (ECA)를 삽입합니다. </li><li> ECA의 가능한 가장 높은 지점에서 영구 매듭을 놓습니다. </li><li> ECA의 가능한 가장 낮은 지점, 바로 CCA의 분기점 위의 장소에서이동식 매듭. 이 매듭은 상부 매듭에 비해 느슨합니다. </li><li> 가장 낮은 지점에서, 용기 클립을 사용하여 CCA를 닫습니다. </li><li> 용기 클립을 사용하여 내 경동맥 (ICA)를 닫는다. </li><li> 사용하여 미세 절제 스프링 가위는 두 매듭 사이 ECA (약 2mm)에 작은 상처를 수행합니다. </li></ol> ICA를 통해 4. 약물 주입 <ol><li> 주입 시스템을 조립합니다. 모세관 튜브 6 인치 (이상적인 크기 : 2.5 mm X 1.2 mm)를 부착 물질의 250 μl를 포함하는 투베르쿨린 주사기는 (마약, 병원균, 다른 사람의 사이에서 세포 외 소포를) 주입 및 수행 절개로 부드럽게 모세관 팁을 삽입한다 단계 3.6 인치 </li><li> 가 분기와 CCA를 폐쇄 클립 사이의 중간 점에 도달 할 때까지 모세관을 아래로 슬라이드를 계속합니다. </li><li> 낮은 ECA 매듭을 묶어. 매듭은 모세관 유동을 허용 할 정도로 느슨하고 leakin을 방지하기에 충분히 단단한 지 확인지. </li><li> ICA에에서 클립을 제거합니다. </li><li> 조심스럽게 주사기 피스톤 허용 물질 주입 초당 약 10 μL에 압력을 적용합니다. </li></ol> 5. 사후 주입 절차 <ol><li> 장소는 ICA에 다시 클립. </li><li> 조심스럽게 모세관 튜브를 제거합니다. </li><li> 완전히 낮은 ECA 매듭을 묶어. </li><li> ICA에와 CCA에서 클립을 제거합니다. </li></ol> 6. 절개 결산 및 수술 후 케어 <ol><li> 트랙터 또는 조직 후크와 베개를 제거합니다. </li><li> 멸균 생리 식염수를 사용하여 청소 봉합 영역입니다. </li><li> 나일론 봉합사 / 바늘과 집게를 사용하여 절개를 닫습니다. </li><li> 항 염증 및 진통이 수술 후 불편 함을 완화하기 위해 관리 할 수 ​​있습니다. </li><li> 적어도 1 시간 모니터 복구를 위해 가열 패드에 배치 새장에 동물을 배치합니다. </li></ol>

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The authors have nothing to disclose.

여기에 설명 된 주입 미세는 본체 (1), (12)을 통해 원치 않는 확산을 방지 CNS에 다양한 생물학적 기능 외인성 물질을 전달하는 매우 성공적인 것으로 판명되었습니다. 혈액 - 뇌 장벽을 중단 여러 CNS 관련 질환의 병리학 적 특징이다; 따라서 혈뇌 장벽 외인성 물질의 관계를 평가하는 것은 매우 중요하고 흥미 롭다. 선물이 수술 모델 동물에 제한 외상의 원인과 매우 낮은 사망률 (1), (12)와 연결되어 있습니다. 또한, 상기 절차는 CNS 기능 또는 뇌 혈류 1 (12)과 간섭하지 않는다. 이 방법의 가장 중요한 측면만을 허용해야하는 상기 ECA 올바른 절단을 수행 할 수있다모세관 튜브의 삽입. 더 넓은 컷 반대 사이트의 다른 ECA 수술을 필요로하는, 물질의 누출이 주입되는 원인이됩니다. 이러한 상황을 방지하기 위해, 모세관 튜브는 상처보다 더 큰 직경을 가져야하며 또한 혈관을 통해 입구를 용이하게하기 위하여 날카로운 에지를 가져야한다. 기술적 특징에 대해 완전히 숙련 된 기술자는 20 분 내에이 수술을 수행 할 수있다. 이 기술의 주요 제한은 동일한 ICA 통해 한 번만 주입의 가능성이다. CNS에 사용되어야하며 전술 한 여기에 설명 된 하나 국지적 기술이 BBB 내의 새로운 양 및 음의 상호 작용에 대한 연구를 지원하기 위해,뿐만 아니라 병원균을 제공하는 잠재력을 가지고있다 microport 용기 내로 반복 물질 배달 약물 및 뇌에 생리 에이전트.

중추 신경계의 생체 내 모델 (CNS) 질환은 뇌에 이러한 약물, 병원균 또는 엑소 좀과 같은 외래 물질의 효과적인 전달을 필요로한다. 따라서, 이상적인 전달 방법은 동물에게 최소의 외상을 유발하는 신경 네트워크의 무결성을 유지하고, 뇌 한 높은 약물 농도를 달성한다. 지방 물질 전달의 여러 가지 수술 방법은 인트라 - 시스, 뇌내, 뇌 실내 주사 나 주입, 2, 3, 4, 5를 포함하여 기술되었다. 이러한 방법은 그러나, CNS 외상으로 간주하고, 관심 물질의 낮은 양의 관리를 허용한다. 또한, 외인성 물질이 빠르게 뇌척수액 (6)에 의해 제거 할 수 있다고 제안되어왔다 </s업&gt; 및 상술 한 기술이 사용될 때 뇌 실질 저 투과 범위는 7 관찰되었다. 그들은 CNS에 물질 전달이 낮은 효과를 발휘하지만 경구, 폐, 피하, 정맥 내 경로 등의 전신 전달 방법은 흔히 다른 기관 (8), (9)의 흡수로 인해, 동물 모델에서 이용된다. 따라서, 전달 노선들은 부작용 및 독성이 10, 11의 위험을 증가 투여 물질의 높은 투여 량을 필요로한다. 여기서는 효과적으로 직접 내부 경동맥을 통해 뇌로 물질을 제공하는 마우스 미세 주입 기술을 설명한다. 중추 신경계에 대한 전달을 대상으로하는 것 외에도,이 기술은 정상적인 생리 장벽을 우회하므로 biologica에 매우 관련이없는뇌 내로 치료제 또는 병원체 통로 (L)에 관여 프로세스.

<table border="0" cellpadding="0" cellspacing="0" width="635">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Anesthesia instrument</td>
			<td style="width:149px;">Vetequip</td>
			<td align="right" style="width:119px;">901806</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Surgical scissors</td>
			<td style="width:149px;">Fine Science Tool</td>
			<td style="width:119px;">14558-09</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Surgical forceps straight tip</td>
			<td style="width:149px;">Fine Science Tool</td>
			<td style="width:119px;">00108-11</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Surgical forceps angled tip</td>
			<td style="width:149px;">Fine Science Tool</td>
			<td style="width:119px;">00109-11</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Spring scissors</td>
			<td style="width:149px;">Fine Science Tool</td>
			<td style="width:119px;">15000-08</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Nylon suture</td>
			<td style="width:149px;">Braintree Scientific</td>
			<td style="width:119px;">SUT-S 104</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Capillary tubing (Micro-Renathane 0.010&#8221; x 0.005&#8221; per ft.)&#160;</td>
			<td style="width:149px;">Braintree Scientific</td>
			<td style="width:119px;">MRE01050</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Closing suture</td>
			<td style="width:149px;">VWR</td>
			<td style="width:119px;">95057-036</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Isoflurane</td>
			<td style="width:149px;">Piramal</td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">2,3,5-Triphenyltetrazolium chloride</td>
			<td style="width:149px;">FisherScientific</td>
			<td style="width:119px;">50-121-8005</td>
		</tr>
	</tbody>
</table>

mouse microsurgery infusion technique for targeted substance delivery into the cns via the internal carotid artery

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous substances into the brain. These exogenous substances may induce injurious impact or constitute therapeutic strategy. The most common delivery methods of exogenous substances into the brain are based on systemic deliveries, such as subcutaneous or intravenous routes. Although commonly used, these approaches have several limitations, including low delivery efficacy into the brain. In contrast, surgical methods that locally deliver substances into the CNS are more specific and prevent the uptake of the exogenous substances by other organs. Several surgical methods for CNS delivery are available; however, they tend to be very traumatic. Here, we describe a mouse infusion microsurgery technique, which effectively delivers substances into the brain via the internal carotid artery, with minimal trauma and no interference with normal CNS functionality.

여기에 설명 된 마우스 미세 주입 기술은 매우 다목적 뇌 전이의 형성 (1) (12)의 대표 모델에서 종양 세포의 전달을 포함하여 직접 뇌에 다른 물질을 전달하기 위해 사용되어왔다. 이 기술은 또한 CNS 다른 병원균의 병리학 적 양상을 평가하기에 적합하다. HIV 감염의 마우스 모델에서, 주입은 수술 CCA 직접 바이러스 입자를 주입 하였다. 우리는 7 일 수술 후 CNS 실시간 PCR에 의해 HIV 양성 것으로 나타났습니다. 또한, 동측 반구 감염은 6-10 반대편 반구 (그림 2)보다 높은 주름이었다. 여기에 설명 된 주입 수술의 또 다른 적절한 방법은 제공하는 것입니다<html> 중추 신경계에 세포 외 소포 (ECVs), 주로 엑소 좀. 그림 3은 주입 후 마우스 24 시간의 동측 뇌의 녹색 형광 단백질 (GFP)으로 표시 CD63, 엑소 좀 마커의 존재를 보여줍니다. <img alt="그림 1" src="/files/ftp_upload/54804/54804fig1.jpg" /> 그림 1 : 내부 경동맥의 동맥을 통해 주입의 도식 표현. 삽입 위치 (a) 및 (b)는 각각 상부 및 하부 노트의 위치 파악을 도시한다; (c)는 모세관 튜브가 삽입 통과하는 ECA의 작은 컷 (회색 선)을 나타낸다. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54804/54804fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <html> 4804fig2.jpg &quot;/&gt; 내부 경동맥을 통해 바이러스 주입 후 실시간 PCR에 의해 HIV 검출 : 그림 2. 바 7 일 왼쪽 경동맥을 통해 주입을 게시 생쥐에서 수확 평균 HIV DNA 수준을 나타냅니다. 오차 막대는 표준 편차를 묘사. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54804/54804fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 3" src="/files/ftp_upload/54804/54804fig3.jpg" /> 그림 3 : 내부 경동맥을 통해 ECVs 주입 후 마우스 뇌 섹션 묘사 GFP - 태그 CD63, 엑소 좀 마커의 면역 형광. 대표적인 이미지는 CD63 발현 ECVs (녹색)에 연결된 마우스 뇌 미세 혈관을 나타낸다. 스케일 바 : 50 μm의.= &quot;_ 빈&quot;을 얻을&gt;이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.

Watch this Scientific Journal Video about Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery at JoVE.com

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

The present protocol describes a mouse microsurgery infusion technique, which effectively delivers substances directly into the brain via the internal carotid artery.

중추 신경계에 대상 물질 전달을위한 마우스 미세 주입 기술<i&gt;를 통해</i&gt; 내부 경동맥

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous ...

mouse-microsurgery-infusion-technique-for-targeted-substance-delivery

Nanyang Technological University

Research

JoVE Journal

Neuroscience

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

10.8K 조회수.  University of Miami. The present protocol describes a mouse microsurgery infusion technique, which effectively delivers substances directly into the brain via the internal carotid artery.

비디오: Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. However, because the uterine wall is opaque during early embryogenesis, the ability to target specific parts of the embryo for microinjection is greatly limited. Fortunately, high-frequency ultrasound imaging permits the generation of images that can be used in real time to guide a microinjection needle into the embryonic region of interest. Here we describe the use of such imaging to guide the injection of retroviral vectors into the ventricular system of the mouse forebrain at embryonic day (E) 9.5. This method uses a laparotomy to permit access to the uterine horns, and a specially designed plate that permits host embryos to be bathed in saline while they are imaged and injected. Successful surgeries often result in most or all of the injected embryos surviving to any subsequent time point of interest (embryonically or postnatally). The principles described here can be used with slight modifications to perform injections into the amnionic fluid of E8.5 embryos (thereby permitting infection along the anterior posterior extent of the neural tube, which has not yet closed), or into the ventricular system of the brain at E10.5/11.5. Furthermore, at mid-neurogenic ages (~E13.5), ultrasound imaging can be used direct injection into specific brain regions for viral infection or cell transplantation. The use of ultrasound imaging to guide in utero injections in mice is a very powerful technique that permits the molecular and cellular manipulation of mouse embryos in ways that would otherwise be exceptionally difficult if not impossible.

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. Here we describe the use of high-frequency ultrasound imaging to guide the injection of retroviral vectors into the mouse brain at embryonic day (E) 9.5.

마우스 Forebrain에 초음파 유도 Microinjection Utero에서 E9.5에서

In utero survival surgery in mice permits the molecular manipulation of gene expression during development.  However, because the uterine wall is opaque ...

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Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals through the vitreous fluid ensures that they act upon the entire RGC population. Viral vectors, plasmids or short interfering RNAs (siRNAs) can also be delivered to the vitreous chamber in order to infect or transfect retinal cells 5-12. The high tropism of Adeno-Associated Virus (AAV) vectors is beneficial to target RGCs, with an infection rate approaching 90% of cells near the injection site 6, 7, 13-15. Moreover, RGCs can be selectively transfected by applying siRNAs, plasmids, or viral vectors to the cut end of the optic nerve 16-19 or injecting vectors into their target the superior colliculus 10. This allows researchers to study apoptotic mechanisms in the injured neuronal population without confounding effects on other bystander neurons or surrounding glia. RGC apoptosis has a characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a window for experimental manipulations directed against pathways involved in apoptosis. Manipulations that directly target RGCs from the transected optic nerve stump are performed at the time of axotomy, immediately after cutting the nerve. In contrast, when substances are delivered via an intraocular route, they can be injected prior to surgery or within the first 3 days after surgery, preceding the initiation of apoptosis in axotomized RGCs. In the present article, we demonstrate several methods for experimental manipulations after optic nerve transection.

Optic Nerve transection is a widely used model of adult CNS injury. This model is ideal for performing a number of experimental manipulations that target the retina globally or directly target the injured neuronal population of retinal ganglion cells.

포유류의 CNS에서 시신경의 절개 후 실험에 대한 노는 방법

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be ...

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos1,2 that we have successfully adapted3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick5. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described6. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.
To date, in utero electroporation has been most widely used for the analysis of neocortical development1,2,3,4. More recent studies have targeted the embryonic retina7,8,9 and thalamus10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.

사용하여 여러 개의 CNS 준주로 효율적인 유전자 전달 Utero에서 Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

글루타민산 염과 GABA의 빠른 마이크로 이온 도입 : 시냅틱 통합을 조사하는 유용한 도구

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

마우스 망막을 사용하여 중추 신경계의 혈관 재생의 평가

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and genetic mechanisms that contribute to hearing loss. Introduction of vectors into the inner ear must be done in a way that widely distributes the agent throughout the cochlea while minimizing injury to the existing structures. This manuscript describes a post-auricular surgical approach that can be used for mouse cochlear therapy using molecular, pharmacologic, and viral delivery to mice postnatal day 10 and older via the round window membrane (RWM). This surgical approach enables rapid and direct delivery into the scala tympani while minimizing blood loss and avoiding animal mortality. This technique involves negligible or no damage to essential structures of the inner and middle ear as well as neck muscles while wholly preserving hearing. To demonstrate the efficacy of this surgical technique, the vesicular glutamate transporter 3 knockout (VGLUT3 KO) mice will be used as an example of a mouse model of congenital deafness that recovers hearing after delivery of VGLUT3 to the inner ear using an adeno-associated virus (AAV-1).

The described post-auricular surgical approach allows rapid and direct delivery into the mouse cochlear scala tympani while minimizing blood loss and animal mortality. This method can be used for cochlear therapy using molecular, pharmacologic and viral delivery to postnatal mice through the round window membrane.

라운드 창 멤브레인을 통해 마우스 내부 귀에 바이러스로 매개 유전자 전달을위한 수술 방법

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

마우스 해마에 올리고머 아밀로이드 - 베타의 정위 주입

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Autonomous and Rechargeable Microneurostimulator Endoscopically Implantable into the Submucosa

Gastric dysmotility can be a sign of common diseases such as longstanding diabetes mellitus. It is known that the application of high-frequency low-energetic stimulation can help to effectively moderate and alleviate the symptoms of gastric dysmotility. The goal of the research was the development of a miniature, endoscopically implantable device to a submucosal pocket. The implantable device is a fully customized electronic package which was specifically designed for the purpose of experiments in the submucosa. The device is equipped with a lithium-ion battery which can be recharged wirelessly by receiving an incident magnetic field from the charging/transmitting coil. The uplink communication is achieved in a MedRadio band at 432 MHz. The device was endoscopically inserted into the submucosal pocket of a live domestic pig used as an in vivo model, specifically in the stomach antrum. The experiment confirmed that the designed device can be implanted into the submucosa and is capable of bidirectional communication. The device can perform bipolar stimulation of muscle tissue.

The application of high-frequency low-energetic stimulation can alleviate the symptoms of gastric dysmotility. In this research, a miniature, endoscopically implantable and wirelessly rechargeable device which is implanted into a submucosal pocket is presented. Successful both-way communication and stimulation control were achieved during an experiment on live pig.

자율적이 고 충전식 Microneurostimulator 라고는 Submucosa에 이식

Gastric dysmotility can be a sign of common diseases such as longstanding diabetes mellitus. It is known that the application of high-frequency ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

화학유전학적으로 조작된 피질 인터뉴런 선조를 출생 후 마우스 뇌로 이식

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

Delivery of Antibodies into the Murine Brain via Convection-enhanced Delivery

Convection-enhanced delivery (CED) is a neurosurgical technique enabling effective perfusion of large brain volumes using a catheter system. Such an approach provides a safe delivery method by-passing the blood brain barrier (BBB), thus allowing treatment with therapeutics with poor BBB-permeability or those for which systemic exposure is not desired, e.g., due to toxicity. CED requires optimization of the catheter design, injection protocol, and properties of the infusate. With this protocol we describe how to perform CED of a solution containing up to 20 &#181;g of an antibody into the caudate putamen of mice. It describes preparation of step catheters, testing them in vitro and performing the CED in mice using a ramping injection program. The protocol can be readily adjusted for other infusion volumes and can be used for injecting various tracers or pharmacologically active or inactive substances, including chemotherapeutics, cytokines, viral particles, and liposomes.

Convection-enhanced delivery (CED) is a method enabling effective delivery of therapeutics into the brain by direct perfusion of large tissue volumes. The procedure requires the use of catheters and an optimized injection procedure. This protocol describes a methodology for CED of an antibody into a mouse brain.

대류 강화 전달을 통해 뮤린 뇌로 항체 전달

Convection-enhanced delivery (CED) is a neurosurgical technique enabling effective perfusion of large brain volumes using a catheter system. Such an ...