우리는 건강의 국가 학회 및 자금 조달을 위한 신경 장애 및 치기의 국가 학회 (R01NS079691)를 인정하고 싶습니다.

<ol>
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저자는 TS가 MAP 하이드로겔을 상용화하고자하는 템포 치료제의 창시자라고 선언합니다.

여기서 우리는 쉽게 재현 할 수있는, 최소 침습, 영구 뇌졸중 모델을 설명하고 뇌졸중 후 5 일 동안 광원에 생체 물질을 주입하는 방법을 설명합니다. 포토thrombotic 염료 로즈 벵골과 스테레오 탁스 장치에 연결된 520 nm 콜라주 레이저의 사용은 우리에게 향상된 정밀도로 마우스의 모터 피질에 스트로크를 배치 할 수있는 기능을 제공합니다. 뇌졸중 후 5일 후, 경색의 위치는 조사의 중심에 눈으로 볼...

뇌졸중은 장애의 주요 원인이며 미국에서 다섯 번째로 주요 사망원인입니다 1. 모든 뇌졸중의 약 87%는 허혈성이며 나머지 13%의 대다수는 출혈2입니다. 허혈성 뇌졸중은 주변 조직에 동맥에서 혈류의 막힘으로 정의됩니다. 이 폐색은 수시로 살아남는 환자에 있는 영원한 무력으로 이끌어 내는 산소 부족 그리고 후속 괴사 귀착됩니다. 뇌졸중3의사망률이 감소했지만, 2030년까지 보급은340만명으로 증가할 것으로 예상된다. 장애인 생존자의 증가와 그에 따른 경제적 부담은 신경 수리 메커니즘에 초점을 맞춘 뇌졸중 연구를 위한 추진으로 이어졌습니다. 뇌졸중 다음에 는 괴사 영역이 팽창하는 것을 방지하는 흉터의 형성으로 이어지는 염증 성 기간이 있습니다. 괴사 코어를 둘러싼 지역은 "peri-farct"라고 하며 혈관 신생, 신경 발생 및 축산 발아증가를 포함하는 이 지역의 가소성이 동물 모델과 인간5에서관찰된 회복에 직접 연결된다는 강력한 증거가 있다. 뇌졸중 후 복잡한 상호 작용을 적절하게 복제 할 수있는 체외 모델이 없기 때문에 동물 모델은 뇌졸중 연구에 필수적입니다.
허혈성 스트로크를 생성하는 데 사용할 수있는 생체 내 모델이 몇 가지 있습니다. 마우스에 사용되는 가장 일반적인 모델 중 하나는 말단 또는 근위 (동맥 내 필라멘트를 통해) 폐색을 통해 중간 뇌동맥 폐색, 또는 MCAo입니다. 필라멘트 MCAo (fMCAo)라고도 하는 근위 모델은 일반적으로 대뇌 반구의 5 %에서 50 %까지 포괄하는 큰 허혈성 뇌졸중을 초래하며 요인6의수에 의존합니다. 이러한 모델에서 봉합사 또는 필라멘트는 내부 경동맥에서 중간 뇌동맥(MCA)의 기저부로 발전하여 특정 기간 동안 제자리에 보관된다. 임시 또는 영구로 만들 수 있는 폐색을 위한 이 방법은, 줄무늬에 중심이 되는 광원을 생성하고, 피질6을지나치게 관련시킬 수도 있고 관련되지 않을 수도 있다. 결과 뇌졸중 크기는 매우 가변적이며 레이저 도플러와 같은 이미징 기술은 각 마우스에서 절차의 효과를 확인하는 데 필요합니다. 30분 이상 지속되는 동맥 내 또는 발내 필라멘트 폐색은 크기 범위의 더 큰 끝에서 스트로크를 생성합니다. 일부 조사관은 상당한 실험 초점과 실험실 검증7을필요로하는 짧은 필라멘트 폐색 시간에 초점을 맞추고있다. 마우스에 있는 필라멘트 MCAo 모형은 인간 적인 치기 케이스에서 보인 것과 같이 세포 죽음, 허혈성 진행 및 peri-farct 영역의 형성의 유사한 단계를 따릅니다; 그러나, 더 큰 치기는 악성 뇌 경색의 질병 상태를 더 가깝게 닮은, 덜 일반적이다, 덜 치료 인간 뇌졸중6. 한편, 말단 MCA 폐색은 더 관련된 수술및 craniectomy를 요구합니다. 이 모델에서, 뇌의 표면을 따라 실행되는 MCA의 말단 부분은 봉합사 넥타이 또는 소작으로 직접 가려져 있습니다. 기술의 일부 변형에서, 경동맥은 일방적또는 일시적으로 양측으로 가려져 있다. 말단 MCAo의 장점은 필라멘트 모델보다 크기가 덜 가변적인 피질 기반 스트로크를 생성한다는 것입니다. 그러나, 해단 모델은 fMCAo6의관심사인 외부 경동맥(ECA)의 질부로 인해 더 열악한 행동 출력을 생성한다.
덜 침습적 인 것으로 알려진 대체 뇌졸중 모델은 광혈전증 (PT) 모델입니다. PT 모델은 허혈의 잘 정의된 위치를 초래하고 높은 생존율8과연관된다. 이 기술은 빛 또는 레이저9로원하는 조직을 조사하기만 하면 인트라바스내 광 산화를 허용하는 인트라바스트(intraitone)에 주입된 감광성 염료에 의존한다. 자궁내막시, 내피 손상을 유발하는 산소 라디칼이 형성되어 조사부위8,9에서혈소판 응집 및 응고 형성을 활성화한다. 뇌졸중 크기와 위치를 엄격하게 제어할 뿐만 아니라 PT 모델의 높은 재현성은 생체 재료 연구에 이상적입니다. 정밀도는 레이저 및 스테레오탁스 좌표를 사용하여 가능하지만, 이 모델이 몇 가지 연구에 적합하지 않을 수 있다는 몇 가지 단점이 있습니다. fMCAo 모델과 달리 PT 스트로크 모델은 레퍼퍼프할 수 없습니다. 따라서, 재퍼퓨전 또는 레퍼퓨전 다음 메커니즘에 따른 손상에 특정한 신경보호제 조사를 위한 물질은 여기에서 유용하지 않을 것이다8. 또한 PT 모델의 미세 혈관 모욕으로 인해 상대적으로 작은 허혈성 음벌브라가 보입니다. 대신, 국소 혈관부종은 인간 뇌졸중에 특이하지 않은 것으로 발생하며, 이 모델은 peri-farct 영역6,8에초점을 맞춘 전임상 약물 연구에 바람직하지 않다.
뇌졸중의 생물 물질 전략의 전반적인 목표는 생리 활성 에이전트를 제공하거나 뇌 조직 성장을위한 대리 세포 외 매트릭스 역할을하는 것입니다. 우리가 우리의 방법을 사용하여 탐구할 한 가지 전략은 많은 현재 세포 치료가10전달되는peri-infarct 조직과 는 반대로, 스트로크 코어로 직접 하이드로겔을 전달하는 것입니다. 이 접근법에 대한 근거는 코어에서 발견되는 괴사 조직으로의 전달이 주변의 건강하거나 회복 조직을 방해하지 않을 것이라는 것입니다. 우리는 생물 물질 에 포함 된 임의의 활성 제의 확산은 코어에서 peri-farct에 도달 할 수있을 것입니다 가정, 우리는 하이드로 겔 생체 재료의 전달이 신경교 흉터(11)의두께를 감소 발견하기 때문에 특히. 이것은 peri-farct 지역이 치기 후에 신경 가소성을 전시하기 위하여 표시되었기 때문에 중요합니다, 매력적인 표적으로 만드는. 더욱이, 뇌졸중 코어에 대리 매트릭스의 전달은 새로운 조직의 형성을 유도하기 위해 혈관신생12 또는신경유발성 13인자를 로드할 수 있으며, 전달을 위한 세포(14). 세포 전달은 전달 중에 존재하는 가혹한 주입력 및 현지 환경으로부터 세포를 보호하고 분화 및이식(15)을장려하기 때문에 매트릭스를 사용하여 크게 향상된다.
이러한 주 사용 치료 생체 재료는 뇌졸중 응용 프로그램에서 임상 관련성이 있다, 현재 뇌졸중 후 신경 복구를 자극 하는 의료 치료. 복구에 관여하는 근본적인 신경 회로는 뇌졸중 코어16에인접한 뇌 조직에 있으며 뇌졸중 코어 자체는 실행 가능한 신경 조직이 없습니다. 괴사 뇌졸중 코어에 생체 물질을 전달하는 것은 이전에 언급 된 여러 메커니즘을 통해 인접 조직을 재생 프로세스로 자극 할 수있는 잠재력을 가지고 있을 것으로 예상됩니다( 성장 인자13의창고 방출, 조직 성장 의 자극 및 뇌 조직 개발의 촉진11,12,면역 반응의 변경 17, 줄기 세포 유래 치료제의 전달14, 18. 그러나 이러한 응용 프로그램의 가능성을 효과적으로 연구하기 위해서는 뇌졸중을 유도하고 생체 재료를 주입하는 일관되고 재현 가능한 방법이 필요합니다. PT 스트로크 모델은 스트로크의 방향과 위치를 정밀하게 제어하는 기술을 사용합니다. 스테레오탁틱 장치에 부착된 레이저는 방향을 안내하고, 스테레오택시 장치에 부착된 펌프는 추가 형태의 이미징 없이 재료의 사출 속도를 제어합니다. 따라서, 우리는 마우스의 모터 피질에서 PT 뇌졸중을 수행하고 뇌졸중 코어에 생체 물질을 주입하는 방법을 설명하기로 결정했습니다. 여기서, 우리는 추가된 세포 또는 성장 인자가 없는 주입을 위한 생물재료로 미세다공성 안구 입자(MAP) 하이드로겔을 사용합니다. 또한, 우리는 성공적으로 그대로 생물 재료로 뇌를 검색하는 방법을 설명하고, 우리는 생체 재료의 주입없이 뇌졸중 결과를 분석하는 데 사용되는 면역 화학 분석에 대해 논의.

<table>
	<tbody>
		<tr>
			<td>10% Normal Goat Serum</td>
			<td>VWR</td>
			<td>100504-028</td>
			<td>For blocking buffer</td>
		</tr>
		<tr>
			<td>2-ply alcohol pre pad, Sterile, Medium</td>
			<td>Medline</td>
			<td>MDS090735</td>
			<td></td>
		</tr>
		<tr>
			<td>25uL Hamilton Syringe 702RN, no needle</td>
			<td>Fishcer Scientific</td>
			<td>14824663</td>
			<td>Syringes used to inject biomaterials</td>
		</tr>
		<tr>
			<td>25uL Positive displacement pipette</td>
			<td>Gilson</td>
			<td>M-25</td>
			<td></td>
		</tr>
		<tr>
			<td>2x Beam Expander, 400-650nm</td>
			<td>Thorlabs</td>
			<td>GBE02-A</td>
			<td>Laser beam expander</td>
		</tr>
		<tr>
			<td>Adjustable Stage Platform</td>
			<td>Kopf Instruments</td>
			<td>901</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Glucose Transporter GLUT1 antibody, rabbit</td>
			<td>Abcam</td>
			<td>ab113435</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Iba1 Antibody, goat</td>
			<td>abcam</td>
			<td>ab5076</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Vacutainer Safety-Lok Blood Collection Sets. 25G, 12&#34;</td>
			<td>Medsupply</td>
			<td>367294</td>
			<td>For perfusions</td>
		</tr>
		<tr>
			<td>BKF12- Matte Black Aluminum Foil</td>
			<td>Thorlabs</td>
			<td>BKF12</td>
			<td>To cover anything that is reflective when using laser.</td>
		</tr>
		<tr>
			<td>Bone Iris Mini Scissors - 3-1/2&#34;</td>
			<td>Sklar surgical instruments</td>
			<td>64-2035</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 Mice</td>
			<td>Jackson Laboratory</td>
			<td>000664</td>
			<td>8-12 weeks of age</td>
		</tr>
		<tr>
			<td>Cage Assembly Rob</td>
			<td>Thorlabs</td>
			<td>ER3-P4</td>
			<td>3&#34; Long, diameter 6mm, 4 pack - for attaching laser to sterotax</td>
		</tr>
		<tr>
			<td>Carbon Steel Burrs -0.5mm Diameter</td>
			<td>Fine Science Tools</td>
			<td>19007-05</td>
			<td>For creating burr hole</td>
		</tr>
		<tr>
			<td>Chromium(III) potassium sulfate dodecahydrate</td>
			<td>VWR</td>
			<td>EM1.01036.0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Compact Controller for pigtailed lasers</td>
			<td>Thorlabs</td>
			<td>CLD1010LP</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton Swabs</td>
			<td>VWR</td>
			<td>89031-288</td>
			<td></td>
		</tr>
		<tr>
			<td>CP 25 pipette tips</td>
			<td>Gilson</td>
			<td>F148012</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-goat IgG H&#38;L (488)</td>
			<td>abcam</td>
			<td>ab150129</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Anti-rabbit IgG H&#38;L (647)</td>
			<td>abcam</td>
			<td>ab150075</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Anti-rat IgG H&#38;L (555)</td>
			<td>abcam</td>
			<td>ab150154</td>
			<td></td>
		</tr>
		<tr>
			<td>EMS DPX Mountant</td>
			<td>Elecron Microscopy Sciences</td>
			<td>13512</td>
			<td>Mounting solution for slides</td>
		</tr>
		<tr>
			<td>EMS Gelatin Powder Type A 300 Bloom</td>
			<td>Electron Microscopy Sciences</td>
			<td>16564</td>
			<td>For gelatin coating slides</td>
		</tr>
		<tr>
			<td>EMS Paraformaldehyde, Granular</td>
			<td>VWR</td>
			<td>100504-162</td>
			<td>For making 45 PFA</td>
		</tr>
		<tr>
			<td>ESD Worstation kit</td>
			<td>Elmstat</td>
			<td>WSKK5324SB</td>
			<td>Need for setting up the laser</td>
		</tr>
		<tr>
			<td>Fiber Bench Wall Plate, unthreaded</td>
			<td>Thorlabs</td>
			<td>HCA3</td>
			<td>Need for connecting laser to Kopf shaft</td>
		</tr>
		<tr>
			<td>FiberPort</td>
			<td>Thorlabs</td>
			<td>PAF-X-5-A</td>
			<td>FC/APC&#38; APC, f=4.6mm, 350-700nm, diameter 0.75mm</td>
		</tr>
		<tr>
			<td>Fine Scissors - straight/sharp-blunt/10cm</td>
			<td>Fine Science Tools</td>
			<td>14028-10</td>
			<td></td>
		</tr>
		<tr>
			<td>GFAP Antibody, rat</td>
			<td>Thermo Fisher Scientific</td>
			<td>13-0300</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating Plate</td>
			<td>Kopf Instruments</td>
			<td>HP-4M</td>
			<td></td>
		</tr>
		<tr>
			<td>ImmEdge Hydrophobic Barrier Pen</td>
			<td>Vector Laboratories</td>
			<td>H-4000</td>
			<td>For staining slides</td>
		</tr>
		<tr>
			<td>IMPAC 6-Integrated Multi Patient Anesthesia Center</td>
			<td>VetEquip</td>
			<td>901808</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodine Prep Pads</td>
			<td>Medx Supple</td>
			<td>MED MDS093917H</td>
			<td></td>
		</tr>
		<tr>
			<td>Jewlers Forceps #5</td>
			<td>GFS chemicals</td>
			<td>46085</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Safety Glasses</td>
			<td>Thorlabs</td>
			<td>LG10B</td>
			<td>Amber Lenses, 35% Visible light (googles versions available too)</td>
		</tr>
		<tr>
			<td>M27-1084 Powerful LED Dual Goose-neck</td>
			<td>United Scope</td>
			<td>LED-11C</td>
			<td></td>
		</tr>
		<tr>
			<td>Medical USP Grade Oxygen</td>
			<td>Airgas</td>
			<td>OX USP250</td>
			<td></td>
		</tr>
		<tr>
			<td>Miltex Adson Dressing Forceps, Disecting-grade</td>
			<td>Intefra Miltex</td>
			<td>V96-118</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Cord/Cordless Small Animal Trimmer</td>
			<td>Harvard aparatus</td>
			<td>72-6110</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-pump variable flow</td>
			<td>Thomas Scientific</td>
			<td>70730-064</td>
			<td>Pump for perfusions</td>
		</tr>
		<tr>
			<td>Mouse Brain Matrices, Coronal Slices, 1mm</td>
			<td>Kent Scientific</td>
			<td>RBMA-200c</td>
			<td>For TTC slices</td>
		</tr>
		<tr>
			<td>Mouse Gas Anethesia Head Holder</td>
			<td>Kopf Instruments</td>
			<td>923-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanojet Control Box</td>
			<td>Chemyx</td>
			<td>10050</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanojet pump header</td>
			<td>Chemxy</td>
			<td>10051</td>
			<td>Attach to stereotaxic device for injecting biomaterial</td>
		</tr>
		<tr>
			<td>Needle RN 30G PT STY 3, 0.5 inch</td>
			<td>Fishcer Scientific</td>
			<td>NC9459562</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-rupture ear bars 60&#186;</td>
			<td>Kopf Instruments</td>
			<td>922</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS buffer pH 7.4</td>
			<td>VWR</td>
			<td>97062 338</td>
			<td></td>
		</tr>
		<tr>
			<td>Pigtaled laser 520 nm, 100mW, 5G Pin</td>
			<td>Thorlabs</td>
			<td>LP520-MF100</td>
			<td></td>
		</tr>
		<tr>
			<td>Positive charge glass slides</td>
			<td>Hareta</td>
			<td>AHS90-WH</td>
			<td></td>
		</tr>
		<tr>
			<td>Power engergy meter</td>
			<td>Thorlabs</td>
			<td>PM100D</td>
			<td>Used to measure your mW laser output</td>
		</tr>
		<tr>
			<td>Puralube Vet Ointment</td>
			<td>Dr. Foster Smith</td>
			<td>9N-76855</td>
			<td></td>
		</tr>
		<tr>
			<td>Rectal Probe Mouse</td>
			<td>kopf Instruments</td>
			<td>Ret-3-ISO</td>
			<td></td>
		</tr>
		<tr>
			<td>Rose Bengal Dye 95%</td>
			<td>Sigma-Aldrich</td>
			<td>330000-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaft Modified 8-32 threaded hole 1/2&#34; depth</td>
			<td>Kopf Instruments</td>
			<td>1770-02</td>
			<td>For connecting laser to sterotaxic device</td>
		</tr>
		<tr>
			<td>Slim photodiode power sensor</td>
			<td>Thorlabs</td>
			<td>S130VC</td>
			<td>Used with power energy meter</td>
		</tr>
		<tr>
			<td>SM1-Threaed 30 mm Cage Plate 0.35&#34; thick 2 Retaining</td>
			<td>Thorlabs</td>
			<td>CP02</td>
			<td>For connecting laser expander</td>
		</tr>
		<tr>
			<td>Sol-M U-100 Insuline syringe with 1/2 unit markings 0.5 mL</td>
			<td>VWR</td>
			<td>10002-726</td>
			<td>To inject rose bengal</td>
		</tr>
		<tr>
			<td>StainTray Slide Staining System</td>
			<td>Simport Scientific</td>
			<td>M920-2</td>
			<td>For staining slides</td>
		</tr>
		<tr>
			<td>Sterotaxic device</td>
			<td>Kopf Instruments</td>
			<td>940</td>
			<td>Small Animal Stereotaxic Instrument</td>
		</tr>
		<tr>
			<td>Student Adson Forceps -1x2 teeth</td>
			<td>Fine Science Tools</td>
			<td>91127-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Student Fine Forceps - straight/broad Shanks</td>
			<td>Fine Science Tools</td>
			<td>91113-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature Controller</td>
			<td>Kopf Instruments</td>
			<td>TCAT-2LV</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek OCT compound</td>
			<td>VWR</td>
			<td>25608-930</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>VWR</td>
			<td>97063-864</td>
			<td></td>
		</tr>
		<tr>
			<td>Upper Bracket Clamp</td>
			<td>Kopf Instruments</td>
			<td>1770-c</td>
			<td>For connecting laser to sterotaxic device</td>
		</tr>
		<tr>
			<td>Vetbond Tissue Adhessive 3mL</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-361931</td>
			<td></td>
		</tr>
		<tr>
			<td>Vogue Professional My Manicurist</td>
			<td>Bargin Source</td>
			<td>6400</td>
			<td>For Burrs</td>
		</tr>
		<tr>
			<td>VWR Bead Sterilizers</td>
			<td>VWR</td>
			<td>75999-328</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Tek OCT compound</td>
			<td>Sakura</td>
			<td>4583</td>
			<td>For tissue embeding</td>
		</tr>
	</tbody>
</table>

injection of hydrogel biomaterial scaffolds to the brain after stroke

뇌졸중은 장애의 주요 원인과 미국에서 다섯 번째 주요 사망 원인입니다. 모든 뇌졸중의 약 87%는 허혈성 뇌졸중이며 뇌에 혈액을 공급하는 혈관의 갑작스런 막힘으로 정의됩니다. 막힘의 분 안에, 세포는 정지하기 시작하고 돌이킬 수 없는 조직 손상을 초래합니다. 현재 치료 치료는 혈전 제거 또는 용해에 초점을 맞추어 재관류를 허용하고 더 심각한 뇌 손상을 예방합니다. 일시적인 뇌 가소성은 시간이 지남에 따라 손상된 조직의 일부를 구출 할 수 있지만, 환자의 상당한 분수는 결코 해결되지 않을 신경 적자로 남아 있습니다. 뇌졸중으로 인한 신경 학적 적자를 치료하는 치료 옵션의 부족이있다, 이 증가하는 환자 인구를 치료하는 새로운 전략을 개발하는 필요성을 강조. 주사용 생체 재료는 현재 뇌 가소성을 향상시키고 활성 제또는 줄기 세포의 전달을 통해 내인성 수리를 개선하도록 설계되고 있습니다. 이러한 접근법을 테스트하는 한 가지 방법은 설치류 스트로크 모델을 활용하고, 생체 물질을 스트로크 코어에 주입하고, 수리를 평가하는 것입니다. 뇌졸중 코어의 정확한 위치를 아는 것은 뇌졸중 후 정확한 치료에 필수적이므로 예측 가능한 뇌졸중 위치를 초래하는 뇌졸중 모델은 주입 전에 이미징의 필요성을 피하는 것이 바람직하다. 다음 프로토콜은 광혈전 뇌졸중을 유도하는 방법, 제어되고 정확한 방식으로 하이드로겔을 주입하는 방법, 생물 물질을 손상시키면서 뇌를 추출하고 저온절하는 방법을 다룰 것입니다. 또한, 이러한 동일한 하이드로겔 물질이 줄기 세포의 공동 전달에 어떻게 사용될 수 있는지 강조할 것입니다. 이 프로토콜은 뇌졸중 코어내로 다른 주사용 생체 물질을 사용하는 것으로 일반화될 수 있다.

이 방법의 목적은 뇌졸중 후 뇌에 생체 물질을 주입하는 방법을 시연하는 것이었습니다. 장미 벵골과 520 nm 레이저를 가진 광혈전 모델은 크기와 위치 모두에서 뇌졸중 병변의 제어 방향에 사용되었다. 뇌졸중 후 5일 동안 경색은수술(도 1B)및 TTC 및 이미징 IHC 염색 슬라이드(도2)에의해 시각화될 수 있다. 2x 렌즈를 가진 레이저 직경의 증가는 레이저만으...

Watch this Scientific Journal Video about Injection of Hydrogel Biomaterial Scaffolds to The Brain After Stroke at JoVE.com

Injection of Hydrogel Biomaterial Scaffolds to The Brain After Stroke

뇌졸중은 최소한의 치료 옵션과 잃어버린 뇌 조직을 재생하기위한 현재 임상 치료와 글로벌 문제입니다. 여기에서 우리는 설치류의 모터 피질에 정확한 광혈전 뇌졸중을 만들고 뇌졸중 후 조직 재생에 미치는 영향을 연구하기 위해 하이드로 겔 생체 재료의 후속 주입을 만드는 방법을 설명합니다.

하이드로겔 생체 재료 비계의 주입 뇌졸중 후 뇌에

Stroke is the leading cause of disability and the fifth-leading cause of death in the United States. Approximately 87% of all strokes are ischemic strokes ...

injection-of-hydrogel-biomaterial-scaffolds-to-the-brain-after-stroke

Research

JoVE Journal

Biology

4.9K 조회수.  Duke University. 뇌졸중은 최소한의 치료 옵션과 잃어버린 뇌 조직을 재생하기위한 현재 임상 치료와 글로벌 문제입니다. 여기에서 우리는 설치류의 모터 피질에 정확한 광혈전 뇌졸중을 만들고 뇌졸중 후 조직 재생에 미치는 영향을 연구하기 위해 하이드로 겔 생체 재료의 후속 주입을 만드는 방법을 설명합니다.

비디오: Injection of Hydrogel Biomaterial Scaffolds to The Brain After Stroke

Methods for Patch Clamp Capacitance Recordings from the Calyx

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. Electrical recordings from the presynaptic terminal allow the measurement of action potentials, calcium channel currents, vesicle fusion (exocytosis) and subsequent membrane uptake (endocytosis). The fusion of vesicles containing neurotransmitter causes the vesicle membrane to be added to the cell membrane of the calyx. This increase in the amount of cell membrane is measured as an increase in capacitance. The subsequent reduction in capacitance indicates endocytosis, the process of membrane uptake or removal from the calyx membrane. Endocytosis, is necessary to maintain the structure of the calyx and it is also necessary to form vesicles that will be filled with neurotransmitter for future exocytosis events. Capacitance recordings at the calyx of Held have made it possible to directly and rapidly measure vesicular release and subsequent endocytosis in a mammalian CNS nerve terminal. In addition, the corresponding postsynaptic activity can be simultaneously measured by using paired recordings. Thus a complete picture of the presynaptic and postsynaptic electrical activity at a central nervous system synapse is achievable using this preparation. Here, the methods for slice preparation, morphological features for identification of calyces of Held, basic patch clamping techniques, and examples of capacitance recordings to measure exocytosis and endocytosis are presented.

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal.

칼리에서 패치 클램프 커패시턴스 레코딩을위한 방법

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. ...

연구

생물학

Intravitreous Injection for Establishing Ocular Diseases Model

Intravitreous injection is a widely used technique in visual sciences research. It can be used to establish animal models with ocular diseases or as direct application of local treatment. This video introduces how to use simple and inexpensive tools to finish the intravitreous injection procedure. Use of a 1 ml syringe, instead of a hemilton syringe, is used. Practical tips for how to make appropriate injection needles using glass pipettes with perfect tips, and how to easily connect the syringe needle with the glass pipette tightly together, are given.
To conduct a good intravitreous injection, there are three aspects to be observed: 1) injection site should not disrupt retina structure; 2) bleeding should be avoided to reduce the risk of infection; 3) lens should be untouched to avoid traumatic cataract. In brief, the most important point is to reduce the interruption of normal ocular structure. To avoid interruption of retina, the superior nasal region of rat eye was chosen. Also, the puncture point of the needle was at the par planar, which was about 1.5 mm from the limbal region of the rat eye. A small amount of vitreous is gently pushed out through the puncture hole to reduce the intraocular pressure before injection. With the 45&deg; injection angle, it is less likely to cause traumatic cataract in the rat eye, thus avoiding related complications and influence from lenticular factors. In this operation, there was no cutting of the conjunctiva and ocular muscle, no bleeding. With quick and minor injury, a successful intravitreous injection can be done in minutes. 
The injection set outlined in this particular protocol is specific for intravitreous injection. However, the methods and materials presented here can also be used for other injection procedures in drug delivery to the brain, spinal cord or other organs in small mammals.

Intravitreous injection is a widely used technique in visual sciences research for ocular diseases or as direct application of local treatment. This video demonstrated a protocol for intravitreous injection using a 1ml syringe with glass pipette. Useful tips about avoiding massive bleeding and lens damage are given.

안구 질환 모델 구축을위한 Intravitreous 사출

Intravitreous injection is a widely used technique in visual sciences research. It can be used to establish animal models with ocular diseases or as ...

Zebrafish Brain Ventricle Injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid.  In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation.  In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue.  However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space.  Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy.  The brightfield and the fluorescent images are then processed and superimposed in Photoshop.  This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image.  Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages.  We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3).

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.

Zebrafish의 두뇌 뇌실 주입

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved ...

The Gateway to the Brain: Dissecting the Primate Eye

The visual system in humans is considered the gateway to the world and plays a principal role in the plethora of sensory, perceptual and cognitive processes. It is therefore not surprising that quality of vision is tied to quality of life . Despite widespread clinical and basic research surrounding the causes of visual disorders, many forms of visual impairments, such as retinitis pigmentosa and macular degeneration, lack effective treatments. Non-human primates have the closest general features of eye development to that of humans. Not only do they have a similar vascular anatomy, but amongst other mammals, primates have the unique characteristic of having a region in the temporal retina specialized for high visual acuity, the fovea1. Here we describe a general technique for dissecting the primate retina to provide tissue for retinal histology, immunohistochemistry, laser capture microdissection, as well as light and electron microscopy. With the extended use of the non-human primate as a translational model, our hope is that improved understanding of the retina will provide insights into effective approaches towards attenuating or reversing the negative impact of visual disorders on the quality of life of affected individuals.

The non-human primate is an important translational species for our understanding of development and aging. The anatomical organization of the primate retina may provide important insights into normal and pathological conditions in humans.

두뇌에 게이트웨이 : 프리 메이트 &#39;눈&#39;을 분석 해 봅시다

The visual system in humans is considered the  gateway  to the world and plays a principal role in the plethora of sensory, perceptual and cognitive ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

성인 Zebrafish에 Intraperitoneal 사출

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

Mapping the After-effects of Theta Burst Stimulation on the Human Auditory Cortex with Functional Imaging

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent progress, the functional properties and lateralization of the human auditory cortex are far from being fully understood. Transcranial Magnetic Stimulation (TMS) is a non-invasive technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses, and represents a unique method of exploring plasticity and connectivity. It has only recently begun to be applied to understand auditory cortical function 2. 
An important issue in using TMS is that the physiological consequences of the stimulation are difficult to establish. Although many TMS studies make the implicit assumption that the area targeted by the coil is the area affected, this need not be the case, particularly for complex cognitive functions which depend on interactions across many brain regions 3. One solution to this problem is to combine TMS with functional Magnetic resonance imaging (fMRI). The idea here is that fMRI will provide an index of changes in brain activity associated with TMS. Thus, fMRI would give an independent means of assessing which areas are affected by TMS and how they are modulated 4. In addition, fMRI allows the assessment of functional connectivity, which represents a measure of the temporal coupling between distant regions. It can thus be useful not only to measure the net activity modulation induced by TMS in given locations, but also the degree to which the network properties are affected by TMS, via any observed changes in functional connectivity.
Different approaches exist to combine TMS and functional imaging according to the temporal order of the methods. Functional MRI can be applied before, during, after, or both before and after TMS. Recently, some studies interleaved TMS and fMRI in order to provide online mapping of the functional changes induced by TMS 5-7. However, this online combination has many technical problems, including the static artifacts resulting from the presence of the TMS coil in the scanner room, or the effects of TMS pulses on the process of MR image formation. But more importantly, the loud acoustic noise induced by TMS (increased compared with standard use because of the resonance of the scanner bore) and the increased TMS coil vibrations (caused by the strong mechanical forces due to the static magnetic field of the MR scanner) constitute a crucial problem when studying auditory processing. 
This is one reason why fMRI was carried out before and after TMS in the present study. Similar approaches have been used to target the motor cortex 8,9, premotor cortex 10, primary somatosensory cortex 11,12 and language-related areas 13, but so far no combined TMS-fMRI study has investigated the auditory cortex. The purpose of this article is to provide details concerning the protocol and considerations necessary to successfully combine these two neuroscientific tools to investigate auditory processing. 
Previously we showed that repetitive TMS (rTMS) at high and low frequencies (resp. 10 Hz and 1 Hz) applied over the auditory cortex modulated response time (RT) in a melody discrimination task 2. We also showed that RT modulation was correlated with functional connectivity in the auditory network assessed using fMRI: the higher the functional connectivity between left and right auditory cortices during task performance, the higher the facilitatory effect (i.e. decreased RT) observed with rTMS. However those findings were mainly correlational, as fMRI was performed before rTMS. Here, fMRI was carried out before and immediately after TMS to provide direct measures of the functional organization of the auditory cortex, and more specifically of the plastic reorganization of the auditory neural network occurring after the neural intervention provided by TMS. 
Combined fMRI and TMS applied over the auditory cortex should enable a better understanding of brain mechanisms of auditory processing, providing physiological information about functional effects of TMS. This knowledge could be useful for many cognitive neuroscience applications, as well as for optimizing therapeutic applications of TMS, particularly in auditory-related disorders.

Auditory processing is the basis of speech and music-related processing. Transcranial Magnetic Stimulation (TMS) has been used successfully to study cognitive, sensory and motor systems but has rarely been applied to audition. Here we investigated TMS combined with functional Magnetic Resonance Imaging to understand the functional organization of auditory cortex.

쎄의 후유증을 매핑하는 기능 영상과 함께 인간 청각 컴퓨터에 접속 자극을 버스트

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent ...

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of genes within the mammary epithelium has been difficult to achieve due to the lack of appropriate targeting molecules that are independent of developmental stages such as pregnancy and lactation. Herein, we describe a technique for localized delivery of reagents to the mammary gland at any stage in adulthood via intraductal injection into the nipples of mice. The injections can be performed on live mice, under anesthesia, and allow for a non-invasive and localized drug delivery to the mammary gland. Furthermore, the injections can be repeated over several months without damaging the nipple. Vital dyes such as Evans Blue are very helpful to learn the technique. Upon intraductal injection of the blue dye, the entire ductal tree becomes visible to the eye. Furthermore, fluorescently labeled reagents also allow for visualization and distribution within the mammary gland. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents, and small molecules.

A protocol for the non-invasive intraductal delivery of aqueous reagents to the mouse mammary gland is described. The method takes advantage of localized injection into the nipples of mammary glands targeting mammary ducts specifically. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents and small molecules.

마우스 유선 화 된 약물 전달을위한 췌관 주입

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of ...

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

횡단 단백질의 재구성, 현미경 및 패치 클램프 연구 거대한 단일 층 소포에 전압 게이트 이온 채널, KvAP,

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

쥐의 측면 꼬리 정맥에서 혈액을 샘플링

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

A High Output Method to Isolate Cerebral Pericytes from Mouse

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood brain barrier formation and physiology is now demonstrated but its molecular basis remains largely unknown. As the pathophysiological role of cerebral pericytes in neurological disorders is intriguing and of great importance, the in vitro models are not only sufficiently appropriate but also able to incorporate different techniques for these studies. Several methods have been proposed as in vitro models for the extraction of cerebral pericytes, although an antibiotic-free protocol with high output is desirable. Most importantly, a method that has increased output per extraction reduces the usage of more animals.
Here, we propose a simple and efficient method for extracting cerebral pericytes with sufficiently high output. The mouse brain tissue homogenate is mixed with a BSA-dextran solution for the separation of the tissue debris and microvascular pellet. We propose a three-step separation followed by filtration to obtain a microvessel rich filtrate. With this method, the quantity of microvascular fragments obtained from 10 mice is sufficient to seed 9 wells (9.6 cm2 each) of a 6-well plate. Most interestingly with this protocol, the user can obtain 27 pericyte rich wells (9.6 cm2 each) in passage 2. The purity of the pericyte cultures are confirmed with the expression of classical pericyte markers: NG2, PDGFR-&#946; and CD146. This method demonstrates an efficient and feasible in vitro tool for physiological and pathophysiological studies on pericytes.

We present a protocol for the extraction of murine cerebral pericytes. Based on an antibiotic-free enrichment oriented pericyte extraction, this protocol is a valuable tool for in vitro studies providing high purity and high yield, thus decreasing the number of experimental animals used.

마우스에서 대뇌 골후혈구를 분리하는 고출력 방법

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood ...