이 연구는 부분적으로 JH 장에 NIH 보조금 RO1NS053407에 의해 지원되었다. 우리는 그의 귀중한 공헌 씨 데이먼 Klebe 감사드립니다.

모든 절차는 실험실 동물의 관리 및 사용에 대한 NIH 가이드 준수 실시 Loma 린다 대학에서 동물 관리 및 사용위원회에 의해 승인되었습니다. 1. Presurgical 준비 무균 기술은 모든 수술 절차를하는 것이 좋습니다. stereotactic 장치를 소독하고 수술하기 전에 멸균 수술 용 도구를 준비합니다. 모든 동물 취급시 개인 보호 장비 (PPE)를 착용하십시오. 동물의 physiologic 체온을 유지하기 위해 수술하는 동안 가열 패드를 사용합니다. <ol><li> 트리플 빔 동물 척도를 이용하여 8-12주 기존 마우스를 무게. </li><li> 공동 주입 케타민 (100 밀리그램 / kg)과 xylazine (10 MG / kg) intraperitoneally는 마취에 대한 7-10 분 (충분한 진정 작용에 대한 모니터)을 적용 할 수 있습니다. </li><li> 열 담요에 마우스를 놓고 두피를 면도. </li><li> 두 눈에 안과 연고를 적용합니다. </li><li> 확보기도는에 의해 부드럽게 측면 혀를 이동하고, 조심스럽게 stereotactic 장치에 마우스의 머리를 보호합니다. 참고 : 머리가 stereotactic 프레임의 기초에 수평으로 고정해야합니다. </li><li> Betadine으로 수술 영역을 소독하고, 70 % 에탄올로 세정. 세 번 총 Betadine 및 70 % 에탄올의 응용 프로그램을 번갈아 가며 반복합니다. 면과 스쳐 applicators이 목적을 위해 사용할 수 있습니다. </li></ol> 2. 혈액 주입 모델 <ol><li> # 10 메스 블레이드와 두피의 1cm 긴 중간 선 절개를합니다. </li><li> 코로나 및 시상 봉합 (bregma)의 수직 교차점을 표시하기 위해 두개골을 덮고있는 부드러운 조직을 모두 삭제하려면 솜 스쳐 applicators을 사용합니다. </li><li> 분사 펌프에 해밀턴 주사기 (250 μl)를 탑재하고, stereotaxically bregma를 통해 바늘 (26 게이지)을 직접. </li><li> 다음, 바늘 0.2 mm의 위치에 stereotactic 속이는 팔을 조정 앞쪽 2측면 오른쪽 mm. 에서이 좌표가 1mm 드릴 비트와 다양한 속도의 드릴을 사용하여 작은 두개골 버 구멍을합니다. </li><li> 동물의 꼬리를 일시 중지하고 70 %의 에탄올과의 낮은 표면을 소독. </li><li> 펑크 중앙 꼬리 멸균 바늘 (예 : 26 게이지)와 동맥과 unheparinized 모세관 튜브에 동맥 혈액을 수집합니다. </li><li> 해밀턴 주사기의 유리 통에 모세관에서 신속하게 혈액을 전송 한 후 플런저를 삽입합니다. </li><li> 분사 펌프에 해밀턴 주사기를 포함하는 동맥 혈액의 지금 30 μl 이상을 다시 연결하고 베벨가 더 이상 표시 만 될 때까지 버 구멍을 통해서 바늘을 (시상 봉합을 향해 그 beveled 가장자리 포함) 삽입합니다. </li><li> 이 점 사전에서 바늘 3 ventrally mm 2 μl / 분의 속도로 autologous 혈액의 5 μl를 삽입. </li><li> 첫 번째 주입 미리 깊이 더 바늘 0.7 mm 완료 후. </li><li> 기다립니다5 분 후 오른쪽 striatum에 혈액의 25 μl를 삽입. </li><li> 두 번째 주입이 완료되면, 1 ㎜ / 분의 속도에서이 박탈되기 전에 추가 10 분에 적당한 위치에 바늘을 두십시오. </li><li> 뼈 왁스와 버 구멍을 밀봉하고 피부를 봉합 해. </li><li> 수술 후 진통제를 들어 미리 예열 유체 (정상 생리)에 피하 buprenorphine의 0.05 밀리그램 / kg를 주입. </li></ol> 3. Collagenase 주입 모델 <ol><li> 혈액 주입 모델에 설명 된대로 presurgical 준비 후, 반복 1-4 단계를 반복합니다. </li><li> 생리의 0.5 μl에 용해 세균 (clostridial) collagenase VII-S의 0.075U과 해밀턴 주사기 (10 μl)을 입력합니다. 기포의 형성을 피하십시오. </li><li> 분사 펌프에 해밀턴 주사기를 다시 연결하고 베벨가 더 이상 표시 만 될 때까지 버 구멍을 통해 바늘 (26 게이지)를 삽입합니다. </li><li> ventrally 바늘 3.7 mm의 발전을하고 collagenase의 0.075U을 주입 전2 μl / 분의 속도로 n 인물은 바로 striatum. </li><li> 주입이 완료되면, 1 ㎜ / 분의 속도에서이 박탈되기 전에 추가 10 분에 적당한 위치에 바늘을 두십시오. </li><li> 뼈 왁스와 버 구멍을 밀봉하고 피부를 봉합 해. </li><li> 사전 예열 수술 유체의 피하 buprenorphine의 0.05 밀리그램 / kg를 주입. </li></ol> 4. 위장 운영 <ol><li> 혈액 주입 모델에 설명 된대로 presurgical 준비 후, 반복 1-4 단계를 반복합니다. </li><li> 버 구멍을 통해 ventrally 바늘 (26 게이지) 3.7 mm를 삽입합니다. 바늘은 1 ㎜ / 분의 속도로 인출되기 전에 10 분 동안 위치에 유지됩니다. </li><li> 뼈 왁스와 버 구멍을 밀봉하고 피부를 봉합 해. </li><li> 사전 예열 수술 유체의 피하 buprenorphine의 0.05 밀리그램 / kg를 주입. </li></ol> 5. 대표 결과 실험 intrastriatal 출혈은 형태학 세상의 시작과 끝설치류에서뿐만 아니라 행동 변화합니다. 이러한 변경은 프로 시저의 적절한 실행을 보장하기 위해, 또는 잠재적 치료의 효과를 조사하기 위해 평가 될 수있다. 타겟팅 된 뇌 영역 (예 : 기저 신경절)에 블리드를 생성하면 재현 접근 방식에 가장 필수적이며, 총 또는 histologically 뇌 스테인드 섹션 (예제 1-2)에 확인할 수 있습니다. 다양한 행동 평가를 통해 정량화 할 수 sensorimotor의 적자에서 기저 신경절 결과로 상해. 코너 회전 시험의 결과는 실험 마우스 오른쪽 단추로 양면 ICH 후, 마우스는 (그림 3 A) 사기는 수술 후 24, 72 시간에서 동물을 운영하지 않고 장애인 contralateral (왼쪽) 편에서 ipsilaterally와 거리에 훨씬 더 자주 바뀌을 보여 주었다. 또한, 적절히 vibrissae 자극 따라 표면에 장애 (왼쪽) forelimb을 배치 할 수있는 능력은 forelimb 배치 테스트를 통해 평가되었습니다. 24 72 시간의 수술 후, 마우스의 하위마우스 오른쪽 단추로 양면 ICH에 jected하는 것은 사기 작동하는 동물보다 훨씬 적은 위치를 보여 주었다. 뇌 부종의 측정은 자주 실험 ICH 후 뇌 손상의 정도를 수량화하기 위해 사용된다. autologous 혈액 (30 μl) 또는 박테리아 collagenase의 Intracerebral 주사 (0.075 U)는 중요한 24에서 ipsilateral 피질과 기저 신경절의 뇌 수분 함량의 증가 (그림 4)와 수술 후 72 시간 (그림 4 B) (으로 이어 ) 체하다에 비해. 행동 검사 (그림 3)과 뇌 부종 (그림 4)의 범위의 결과는 주어진 볼륨에서 혈액과 collagenase 주입 모델 사이에 차이점을 보여 주었다 없습니다. <img alt="그림 1" src="/files/ftp_upload/4289/4289fig1.jpg" /> 1 그림. 쥐 모델링 ICH. (A) bregma의 앞쪽 코로나 뇌 섹션 0.2 mm의 단순한 도식은 PR을 보여줍니다autologous 혈액 또는 collagenase 분사의 위치를​​ oposed. 측면 심실는 LV를 표시됩니다. CPU는 꼬리가있는-putamen, striatum의 일부를 의미하고, GP는 Globus는의 pallidus를 식별합니다. 모두 striatum뿐만 아니라 Globus는의 pallidus는 기저 신경절로 알려진 하위 대뇌 피질의 핵의 그룹에 속한다. autologous 전체 혈액의 intrastriatal 주입 후 24 시간에서 구할 bregma의 코로나 뇌 섹션 0.2mm의 앞의 (B) 대표 현미경 사진. <img alt="그림 2" src="/files/ftp_upload/4289/4289fig2.jpg" /> 그림 2. 혈종의 조직 학적 강령. 대표 hematoxylin 및 eosin (H &amp; E) 마우스 뇌의 스테인드 코로나 cryosection는 (10 μm), 세균 collagenase의 intrastriatal 주입 (0.075 U) 후 24 시간에 혈종의 크기를 보여주는. LH = 왼쪽 반구, RH = 오른쪽 뇌. &lt;<html> IMG ALT = &quot;그림 3&quot;SRC = &quot;/ files/ftp_upload/4289/4289fig3.jpg&quot;/&gt; 그림 3. 쥐 실험 ICH에 따라 Neurofunctional 평가는. autologous 혈액 (30 μl) 또는 박테리아 collagenase의 Intrastriatal 주입 (0.075 U)는 재현 neurofunctional 적자를 일으켰습니다. (A) 실험의 ICH 후 마우스는 수술 후 24, 72 시간에서 사기 작동하는 동물보다 훨씬 더 오른쪽으로 보여 주었다. 왼쪽 다리의 (B) 앞발 배치 용량은 수술 후 24, 72 시간에서 ICH 후 장애인을했다. 값은 ± SEM을 의미로 표현하고 학생 뉴먼-Keuls 방법 다음 순위에 분산 Kruskal-월리스 편도 분석과 분석 하였다. &lt;0.05의 AP 값이 통계적으로 의미있는 것으로 간주되었다, N = 6-12 당 그룹, * P 사기에 비해 &lt;0.05. <a href="http://www.jove.com/files/ftp_upload/4289/4289fig3large.jpg" target="_blank">큰 그림을 보려면 여기를 클릭하십시오</a> . igure 4 &quot;SRC =&quot;/ files/ftp_upload/4289/4289fig4.jpg &quot;/&gt; 4 그림. 쥐 실험 ICH에 따라 뇌 물 내용의 평가. autologous의 피 Intracerebral 주입 (30 μl) 또는 중요한 24 (A)에서 ipsilateral 피질과 기저 신경절의 뇌 수분 함량의 증가, 72 시간으로 이어 세균 collagenase (0.075 U) (B) ICH 유도 후. 값은 의미 ± SEM을로 표현하고 Tukey 게시물 임시 테​​스트에 이어 분산하는 방법 중 하나 분석과 분석 하였다. &lt;0.05의 AP 값이 통계적으로 의미있는 것으로 간주되었다, N = 6-10 그룹 별, * P 사기에 비해 &lt;0.05. <a href="http://www.jove.com/files/ftp_upload/4289/4289fig4large.jpg" target="_blank">큰 그림을 보려면 여기를 클릭하십시오</a> .

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(Maywood). 230, 777-784 (2005).</li><li>Fujiwara, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+normobaric+oxygen+therapy+in+a+rat+model+of+intracerebral+hemorrhage.">Effect of normobaric oxygen therapy in a rat model of intracerebral hemorrhage.</a> Stroke. 42, 1469-1472 (2011).</li><li>Khatibi, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoflurane+posttreatment+reduces+brain+injury+after+an+intracerebral+hemorrhagic+stroke+in+mice.">Isoflurane posttreatment reduces brain injury after an intracerebral hemorrhagic stroke in mice.</a> Anesth Analg. 113, 343-348 (2011).</li></ol>

관심 없음 충돌이 선언 없습니다.

intracerebral 출혈 (ICH)의 동물 모델은 질병의 pathophysiology의 고급 이해에 크게 기여하고, 일반적으로 잠복기 분위기의 소설 치료 전략을 개발하고 평가하는 데 사용됩니다. autologous 혈액이나 세균 collagenase의 Intraparenchymal 주사는 설치류에서 ICH를 생성하는 잘 알려진 방법입니다. 두 방법은 처음 쥐에서 개발하지만, 유전자 변형과 한방 긴장의 급속하게 증가 가용성으로 인해, 쥐 더 출혈성 뇌 손상 <su...

<table border="1"><tbody><tr><td> 자료 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 논평 </td></tr><tr><td> Stereotactic 헤드 프레임 </td><td> Stoelting (주) </td><td> 51,600 </td><td></td></tr><tr><td> Nanomite의 주사기 펌프 </td><td> 하버드 장치 </td><td> PY2 70-2217 </td><td></td></tr><tr><td> 해밀턴 주사기 </td><td> 해밀턴 회사 </td><td> 1725RN (250 μl) 1701 RN (10 μl) </td><td> 250 μl 10 μl 주사기에 26 게이지 바늘. </td></tr><tr><td> Microdrill </td><td> 정밀 과학 도구 </td><td> 18000-17 </td><td></td></tr><tr><td> Microdrill 버 </td><td> 정밀 과학 도구 </td><td> 19007-09 </td><td> 0.9 mm 직경 </td></tr><tr><td> Collagenase 유형 VII-S </td><td> 시그마 - 알드리치 </td><td> C2399 </td><td></td></tr><tr><td> 모세관 튜브를 Microhematocrit </td><td> 피셔 과학 </td><td> 22-362-574 </td><td> unheparinized </td></tr><tr><td> 뼈 왁스 </td><td> Ethicon </td><td> W31 </td><td></td></tr><tr><td> 상처의 봉합 </td><td> Ethicon </td><td> 1676G </td><td></td></tr><tr><td> 케타민 </td><td> JHP 제약 </td><td> 42023-115-10 </td><td> Ketalar </td></tr><tr><td> Xylazine </td><td> 로이드 연구소 </td><td> 139-236 </td><td> AnaSed </td></tr></tbody></table>

modeling intracerebral hemorrhage in mice: injection of autologous blood or bacterial collagenase

자연 intracerebral 출혈 (ICH)는 모든 뇌졸중 관련 입원의 10-15%와 더 효과적인 치료법은 1,2 데이트를 할 수 없습니다있는 계정 잠재적으로 생명을 위협하는 신경 질병을 정의합니다. 때문에 인간의 ICH의 이질성의 다양한 잠복기 모델은 철저하게 3 예비 치료 전략을 탐색 할 필요합니다. 실험의 ICH는 일반적으로 autologous 혈액이나 세균 collagenase 4 중의 intraparenchymal 주입에 의해 동물에서 유도됩니다. 해당 모델은 출혈 유도와 부상의 진행의 pathophysiology에 따라 선택됩니다. 혈액 주입 모델은 빠른 속도로 진행 출혈을 모방 한 것이 었지요. 또한, 세균 collagenase는 효소 일반적으로 몇 시간 동안 5 발전 적극적으로 출혈의 원인, 뇌 모세 혈관의 기저 얇은 판을 방해. 결과 perihematomal 부종과 neurofunctional 적자는 이리저리 정량화 할 수m 모델 모두. 본 연구에서는, 우리는 설명과 autologous 전체 혈액 6 수정 이중 사출 모델뿐만 아니라 세균 collagenase 7 ICH 분사 모델, 둘 다 대상의 남성 CD-1 마우스의 기저 신경절 (코퍼스 striatum).을 평가 우리는 ICH 유도 한 후 neurofunctional 결손과 24에서 뇌 부종과 72 시간을 평가합니다. autologous 혈액 (30 μl) 또는 박테리아 collagenase (0.075U)의 Intrastriatal 주입 쥐 재현 neurofunctional 결손 및 수술 후 크게 증가 뇌 24의 부종과 72 시간 (P &lt;0.05)을 일으켰습니다. 결론적으로 두 모델은 일관성 출혈성 infarcts를 얻을 수 있으며 잠복기 ICH 연구에 대한 기본적인 방법을 나타냅니다.

Watch this Scientific Journal Video about Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase at JoVE.com

Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase

intracerebral 출혈 (ICH)의 임상 관련 동물 모델은 출혈성 뇌졸중에 대한 지식을 확장하고 새로운 치료 전략을 검토 필요합니다. 본 연구에서는, 우리는 설명과 쥐의 기저 신경절 (코퍼스 striatum)에 autologous 전체 혈액이나 세균 collagenase 하나의 일방적 주사를 구현하는 두 ICH 모델을 평가합니다.

마우스의 모델링 Intracerebral 출혈 : Autologous 혈액이나 세균 Collagenase의 주입

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related ...

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24.0K 조회수.  Loma Linda University School of Medicine. intracerebral 출혈 (ICH)의 임상 관련 동물 모델은 출혈성 뇌졸중에 대한 지식을 확장하고 새로운 치료 전략을 검토 필요합니다. 본 연구에서는, 우리는 설명과 쥐의 기저 신경절 (코퍼스 striatum)에 autologous 전체 혈액이나 세균 collagenase 하나의 일방적 주사를 구현하는 두 ICH 모델을 평가합니다.

비디오: Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

마우스의 모델링 스트로크 - 필라멘트 모델 중간 대뇌 동맥 폐색

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

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Sequencing of Bacterial Microflora in Peripheral Blood: our Experience with HIV-infected Patients

The healthy gastrointestinal tract is physiologically colonized by a large variety of commensal microbes that influence the development 
	of the humoral and cellular mucosal immune system1,2. 
 Microbiota is shielded from the immune system via a strong mucosal barrier. Infections and antibiotics are known to alter both the normal 
	gastrointestinal tract barrier and the composition of resident bacteria, which may result in possible immune abnormalities3.
HIV causes a breach in the gastrointestinal barrier with progressive failure of mucosal immunity and leakage into the systemic circulation of bacterial bioproducts, such as lipopolysaccharide and 
	bacterial DNA fragments, which contribute to systemic immune activation4-7. Microbial translocation is implicated in HIV/AIDS immunopathogenesis and response to therapy 4,8.
We aimed to characterise the composition of bacteria translocating in peripheral blood of HIV-infected patients. To pursue our aim we set up a PCR reaction for the panbacteric 16S ribosomial gene 
	followed by a sequencing analysis.
Briefly, whole blood from both HIV-infected and healthy subjects is used. Given that healthy individuals present normal intestinal homeostasis no translocation of microflora is expected in 
	these patients. Following whole blood collection by venipuncture and plasma separation, DNA is extracted from plasma and used to perform a broad range PCR reaction for the panbacteric 
	16S ribosomial gene9. Following PCR product purification, cloning and sequencing analyses are performed.

Our experiment will show how to perform a sequencing analysis of bacterial species translocating in peripheral blood of HIV positive patients.

주변 혈액에 세균 Microflora의 시퀀싱 : HIV에 감염된 환자 경험

The healthy gastrointestinal tract is physiologically colonized by a large variety of commensal microbes that influence the development 
	of the humoral ...

Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most often results in failure of potential therapeutics designed for neurodegenerative disorders 1,2. Neurodegenerative diseases such as Spinal Muscular Atrophy (SMA), in which the lower motor neurons are affected, can benefit greatly from introducing the therapeutic agents into the CNS. The purpose of this video is to demonstrate two different injection paradigms to deliver therapeutic materials into neonatal mice soon after birth. One of these methods is injecting directly into cerebral lateral ventricles (Intracerebroventricular) which results in delivery of materials into the CNS through the cerebrospinal fluid 3,4. The second method is a temporal vein injection (intravenous) that can introduce different therapeutics into the circulatory system, leading to systemic delivery including the CNS 5. Widespread transduction of the CNS is achievable if an appropriate viral vector and viral serotype is utilized. Visualization and utilization of the temporal vein for injection is feasible up to postnatal day 6. However, if the delivered material is intended to reach the CNS, these injections should take place while the blood brain barrier is more permeable due to its immature status, preferably prior to postnatal day 2. The fully developed blood brain barrier greatly limits the effectiveness of intravenous delivery. Both delivery systems are simple and effective once the surgical aptitude is achieved. They do not require any extensive surgical devices and can be performed by a single person. However, these techniques are not without challenges. The small size of postnatal day 2 pups and the subsequent small target areas can make the injections difficult to perform and initially challenging to replicate.

Delivery of Therapeutic Agents Through Intracerebroventricular ICV and Intravenous IV Injection in Mice

This article demonstrates two very different methods of injection: 1) into the brain (intracerebroventricular) and 2) systemic (intravenous) to introduce the therapeutic agents into the central nervous system of neonatal mice.

마우스에 Intracerebroventricular을 통해 치료 대리점 납품 (ICV)과 정맥 (IV) 사출

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most ...

Assessing Changes in Volatile General Anesthetic Sensitivity of Mice after Local or Systemic Pharmacological Intervention

One desirable endpoint of general anesthesia is the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is less straightforward than it is in human patients. A widely used behavioral surrogate for hypnosis in rodents is the loss of righting reflex (LORR), or the point at which the animal no longer responds to their innate instinct to avoid the vulnerability of dorsal recumbency. We have developed a system to assess LORR in 24 mice simultaneously while carefully controlling for potential confounds, including temperature fluctuations and varying gas flows. These chambers permit reliable assessment of anesthetic sensitivity as measured by latency to return of the righting reflex (RORR) following a fixed anesthetic exposure. Alternatively, using stepwise increases (or decreases) in anesthetic concentration, the chambers also enable determination of a population's sensitivity to induction (or emergence) as measured by EC50 and Hill slope. Finally, the controlled environmental chambers described here can be adapted for a variety of alternative uses, including inhaled delivery of other drugs, toxicology studies, and simultaneous real-time monitoring of vital signs.

Loss of the righting reflex has long served as a standard behavioral surrogate for unconsciousness, also called hypnosis, in laboratory animals. Alterations in volatile anesthetic sensitivity caused by pharmacological interventions can be detected with a carefully controlled high-throughput assessment system, which may be adapted for delivery of any inhaled therapeutic.

One desirable endpoint of general anesthesia is  the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is  less ...

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

Intrastriatal Injection of Autologous Blood or Clostridial Collagenase as Murine Models of Intracerebral Hemorrhage

Intracerebral hemorrhage (ICH) is a common form of cerebrovascular disease and is associated with significant morbidity and mortality. Lack of effective treatment and failure of large clinical trials aimed at hemostasis and clot removal demonstrate the need for further mechanism-driven investigation of ICH. This research may be performed through the framework provided by preclinical models. Two murine models in popular use include intrastriatal (basal ganglia) injection of either autologous whole blood or clostridial collagenase. Since, each model represents distinctly different pathophysiological features related to ICH, use of a particular model may be selected based on what aspect of the disease is to be studied. For example, autologous blood injection most accurately represents the brain&#39;s response to the presence of intraparenchymal blood, and may most closely replicate lobar hemorrhage. Clostridial collagenase injection most accurately represents the small vessel rupture and hematoma evolution characteristic of deep hemorrhages. Thus, each model results in different hematoma formation, neuroinflammatory response, cerebral edema development, and neurobehavioral outcomes. Robustness of a purported therapeutic intervention can be best assessed using both models. In this protocol, induction of ICH using both models, immediate post-operative demonstration of injury, and early post-operative care techniques are demonstrated. Both models result in reproducible injuries, hematoma volumes, and neurobehavioral deficits. Because of the heterogeneity of human ICH, multiple preclinical models are needed to thoroughly explore pathophysiologic mechanisms and test potential therapeutic strategies.

Preclinical models of intracerebral hemorrhage are utilized to mimic certain aspects of clinical disease. Thus, mechanisms of injury and potential therapeutic strategies may be explored. In this protocol, two models of intracerebral hemorrhage are described, intrastriatal (basal ganglia) injections of autologous blood or collagenase.

뇌출혈의 쥐과 모델로자가 혈액이나 클로스 트리 디움 콜라게나 제의 줄무늬 체 주입

Intracerebral hemorrhage (ICH) is a common form of cerebrovascular disease and is associated with significant morbidity and mortality. Lack of effective ...

Modeling Spontaneous Metastatic Renal Cell Carcinoma (mRCC) in Mice Following Nephrectomy

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully recapitulate early- and late-stage metastatic disease progression. Typical tumor implantation models utilize ectopic or orthotopic primary tumor implantation, but few include systemic spontaneous metastatic disease that mimics the clinical setting. This protocol describes the key steps to develop RCC disease progression stages similar to patients. First, it uses a highly metastatic mouse tumor cell line in a syngeneic model to show orthotopic tumor cell implantation. Methods include superficial and internal implantation into the sub-capsular space with cells combined with matrigel to prevent leakage and early spread. Next it describes the procedures for excision of tumor-bearing kidney (nephrectomy), with critical pre- and post- surgical mouse care. Finally, it outlines the steps necessary to monitor and assess micro-and macro-metastatic disease progression, including bioluminescent imaging as well provides a detailed visual necropsy guide to score systemic disease distribution. The goal of this protocol description is to facilitate the widespread use of clinically relevant metastatic RCC models to improve the predictive value of future therapeutic testing.&#160;

Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy

Models of spontaneous metastatic renal cell carcinoma (RCC) disease progression can be used for evaluating treatments in a clinically relevant setting. This protocol demonstrates different procedures for orthotopic kidney tumor cell implantation, proper nephrectomy, and finally outlines a necropsy guide for visual and bioluminescent scoring of metastatic burden and localization.

마우스의 자발적인 전이성 신장 세포 암 (mRCC)를 모델링에 따라 신장 절제술

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully ...

Ultrasound-guided Transthoracic Intramyocardial Injection in Mice

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. Experiments involving myocardial injection are currently performed by direct surgical access through a thoracotomy. While convenient when performed at the time of another experimental manipulation such as coronary artery ligation, the need for an invasive procedure for intramyocardial delivery limits potential experimental designs. With ever improving ultrasound resolution and advanced noninvasive imaging modalities, it is now feasible to routinely perform ultrasound-guided, percutaneous intramyocardial injection. This modality efficiently and reliably delivers agents to a targeted region of myocardium. Advantages of this technique include the avoidance of surgical morbidity, the facility to target regions of myocardium selectively under ultrasound guidance, and the opportunity to deliver injectate to the myocardium at multiple, predetermined time intervals. With practiced technique, complications from intramyocardial injection are rare, and mice quickly return to normal activity on recovery from anesthetic. Following the steps outlined in this protocol, the operator with basic echocardiography experience can quickly become competent in this versatile, minimally invasive technique.

Echocardiography-guided percutaneous intramyocardial injection represents an efficient, reliable, and targetable modality for the delivery of gene transfer agents or cells into the murine heart. Following the steps outlined in this protocol, the operator can quickly become competent in this versatile, minimally invasive technique.

마우스의 초음파 유도 경 흉부 심근 내 주입

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

마우스의 모델링 스트로크 : 말초 중간 대뇌 동맥의 영구 응고

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

The Rabbit Blood-shunt Model for the Study of Acute and Late Sequelae of Subarachnoid Hemorrhage: Technical Aspects

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and controllable animal models that simulate both conditions are presently uncommon. Therefore, new models are needed in order to mimic human pathophysiological conditions resulting from SAH.
This report describes the technical nuances of a rabbit blood-shunt SAH model that enables control of intracerebral pressure (ICP). An extracorporeal shunt is placed between the arterial system and the subarachnoid space, which enables examiner-independent SAH in a closed cranium. Step-by-step procedural instructions and necessary equipment are described, as well as technical considerations to produce the model with minimal mortality and morbidity. Important details required for successful surgical creation of this robust, simple and consistent ICP-controlled SAH rabbit model are described.

The experimental intracranial pressure-controlled blood shunt subarachnoid hemorrhage (SAH) model in the rabbit combines the standard procedures &#8212; subclavian artery cannulation and transcutaneous cisterna magna puncture, which enables close mimicking of human pathophysiological conditions after SAH. We present step-by-step instructions and discuss key surgical points for successful experimental SAH creation.