The work was supported by grants from NIH/NINDS (NS054883), Craig H. Neilsen Foundation (280072), and the Veterans Administration and Canadian Spinal Research Organization. We thank the Rat Resource and Research Center, University of Missouri, Columbia, Missouri, for providing GFP rats.

Todos os protocolos de animais foram aprovados pelo cuidado e uso Comitê Institucional Animal (IACUC). Diretrizes do NIH para cuidados com animais de laboratório e segurança sejam rigorosamente seguidas. Os animais com procedimentos cirúrgicos foram tratadas adequadamente para minimizar a dor eo desconforto. 1. Medular Curgery e enxerto celular <ol><li> Instrumentos cirúrgicos autoclave antes de todas as cirurgias. Use Hot Bead Sterilizer (Ferramentas Ciência Belas) para remover patógenos e contaminantes microbianos de instrumentos entre os procedimentos em diferentes animais. Use luvas estéreis cirúrgicas, vestido e cortinas durante a cirurgia. Empregar técnica cirúrgica asséptica para cada procedimento cirúrgico. </li><li> Anestesiar ratos Fisher 344 fêmea com uma combinação (2 ml / kg) de cetamina (25 mg / ml), xilazina (1,3 mg / ml), e acepromazina (0,25 mg / ml) administrados através de injecção intraperitoneal (ip). </li><li> Raspar área nas costas e limpar a pele com Betadine e repetidamente etanol.</li><li> Cortar a pele usando uma lâmina # 10 e sem rodeios dissecar músculos camadas. Use um bisturi para isolar as vértebras, expor a vértebra T3, e realizar uma laminectomia dorsal utilizando uma ponta fina fórceps. </li><li> Faça uma incisão na dura longitudinalmente e transecto a medula espinhal no nível T4 usando uma combinação de Iridectomia tesoura e microaspirações para criar uma lacuna rostrocaudal aproximadamente 1 mm entre os cotos na medula espinhal. </li><li> Espere cerca de 1-2 minutos até que o sangramento totalmente interrompida, voltando músculos sutura com 3-0 Vycril e fechar a pele com grampos de sutura. </li><li> Injectar (5 ml) de Lactato de Ringer, buprenorfina (0,035 mg / kg), e ampicilina (33 mg / kg) por via subcutânea imediatamente após a cirurgia e manter os ratos em uma incubadora quente até acordado. </li><li> Injectar a solução de Ampicilina uma vez por dia até 10 dias, e buprenorfina, duas vezes por dia durante 3 dias, ou até que os sinais de dor e desconforto de Ringer e desaparecer. Não devolva um animal de companhia de outros animais até fully recuperado. </li><li> Esvaziar manualmente a bexiga duas vezes por dia por cerca de duas semanas, até o estabelecimento de bexiga reflexo esvaziamento, e, em seguida, esvaziar a bexiga, uma vez por dia durante toda a sobrevivência, se necessário. </li><li> Duas semanas mais tarde, voltar a anestesiar ratos SCI como descrito acima e re-expor o local da lesão da medula espinal. Mantenha a dura fechado para reter células implantadas no local da lesão. </li><li> Injectar 10 ml de solução de células (3,5 × 10 5 mL), coletados a partir de E14 GFP onipresente embriões de ratos transgênicos e incorporado em fibrinogênio e trombina 5,6, no epicentro da cavidade lesão ea interface rostral e caudal da lesão com injeção múltipla locais, utilizando uma micropipeta de vidro puxado com um diâmetro interno de 40 um, ligado a uma seringa Hamilton. </li><li> Suturar as camadas musculares e fechar a pele com agrafos. </li><li> Realize injeção pós-operatório e cuidados bexiga conforme descrito nas etapas 1,6-1,8. </li><li> Uma semana serfore perfusão, injetar 0,5% Fluorogold (FG, 0,4 ml em água destilada) por via intraperitoneal a etiqueta retrógrada simpáticas neurônios pré-ganglionares na medula espinhal 7. </li></ol> 2 Transmissor Implantação <ol><li> Soak transmittersin solução de glutaraldeído a 2% (4 ml de glutaraldeído a 50% em 96 ml de água destilada) por pelo menos 1 - 2 h (até 10 horas) à temperatura ambiente durante a esterilização. </li><li> Lavar cuidadosamente com transmissores estéril a 0,9% de solução salina 3 vezes e armazená-los em solução salina até à sua utilização (não mais do que 1 hora). </li><li> Oito semanas após o transplante de células E14 (10 semanas após a lesão), reanesthetize os ratos que foram submetidos a um SCI, com ou sem enxerto de células e ratos controle ingênuos. </li><li> Raspar a região abdominal e membros posteriores. Limpe a pele com Betadine. Coloque o rato na mesa cirúrgica em decúbito dorsal. </li><li> Incisar a pele do abdómen ventral coxa e interior, no lado direito, usando uma lâmina de # 15. </li><li> Corte através dos stecidos conjuntivos ubcutaneous para expor o conjunto de vasos femorais e nervo usando uma pequena tesoura. </li><li> Separe a artéria femoral da veia eo nervo usando uma pinça fina com ponta curva. </li><li> Coloque três suturas de seda por baixo da artéria e fazer um nó frouxo em cada sutura. </li><li> Aplicar 0,1 ml de lidocaína (2%) para a superfície da artéria para provocar vasodilatação para cateterismo subsequente. </li><li> Fixe o vaso distal com um nó de seda permanente e bloqueio temporário proximal por esticão um fio de seda frouxa. </li><li> Perfurar a artéria utilizando uma agulha curva de calibre 20 e inserir a ponta do cateter telémetro (8 cm de comprimento) utilizando uma ferramenta de inserção do cateter. </li><li> Insira o cateter rostral até 4 centímetros colocando assim a ponta na aorta torácica. </li><li> Ancorar o cateter no interior do vaso, amarrando três suturas de seda em torno da artéria femoral. </li><li> Adicione uma bolsa subcutânea ao longo do flanco entre a extremidade caudal do tórax e tele extensão mais cranial da gama de joelho com uma tesoura sem corte. </li><li> Inserir o corpo do transmissor para o bolso e sutura ao tecido conjuntivo em torno do transmissor a fim de evitar o movimento excessivo. </li><li> Sutura da pele com fio de seda n º 6. </li><li> Injectar (5 ml) de Lactato de Ringer, buprenorfina (0,035 mg / kg), e ampicilina (33 mg / kg) por via subcutânea imediatamente após a cirurgia e manter os ratos em uma incubadora quente até acordado. </li></ol> 3. Basal Pressão Arterial (MAPA) e freqüência cardíaca (FC) de gravação <ol><li> Quanto mais cedo um dia após a implantação transmissor, coloque um único animal no bloco receptor e ligue o transmissor. Aguarde cerca de 10-15 min para habituar o animal e estabilizar os parâmetros cardiovasculares. </li><li> Ficha de repouso e MAP HR, que são derivados a partir da pressão de pulso arterial com um sistema computadorizado de aquisição de dados para, pelo menos, 1 hora. Coletar dados a cada 5 segundos. </li><li> Monitorar a umanimais produtores de forma contínua e remover pontos de dados durante a ocorrência de espasmos visíveis. Para cada animal, pontos de dados médios obter valores médios. </li></ol> 4. Colorretal Distensão induzida Disreflexia Autonômica <ol><li> Contenha ratos NSC-enxertados ou controle SCI em uma toalha fornecidos com grãos de ração dentro do receptor transmissor. Normalmente, os ratos podem ficar cooperativamente durante o procedimento. </li><li> Inserir um cateter com ponta de balão de látex no reto por cerca de dois centímetros e prenda-o para a cauda com fita 8. </li><li> Ligue o transmissor e esperar por 10-15 min permitindo que a pressão arterial para voltar à pré-inserção da linha de base. </li><li> Induzir a distensão colorectal por inflação do balão, lentamente ao longo de 10 segundos com 1,4 mL de ar para 1 min, para gerar uma pressão de aproximadamente 30 mmHg. </li><li> MAP Record e HR 1 min antes, durante 1 min e 1 min após distensão colorretal; dados de amostra a cada 3 s durante o procedimento de 3 min. </li><li>Execute 2-3 ensaios por animal, com pelo menos 15 min de intervalo de recuperação entre os dois ensaios. </li><li> Animais Overdose (ip) com combinação de anestesia dose dupla descrita acima, se mais nenhuma avaliação. Perfundir animais com soro fisiológico seguido por paraformaldeído a 4%. </li><li> Para cada animal, a média dos valores antes e durante a distensão colorectal, respectivamente; calcular a diferença entre os valores iniciais e PAM e FC mudanças induzidas pela distensão para cada ensaio; média dos 2-3 ensaios obter valores médios. </li></ol> 5. Transmissor Limpeza <ol><li> Retire o transmissor do corpo do animal após a anestesia, mas antes da perfusão. Soak imediatamente num copo cheio com água destilada até que a limpeza; evitar a secagem do dispositivo telêmetro. </li><li> Transferir o telémetro de 1% de solução de limpeza enzimática Terg-A-Zyme (10 g / L de água) durante 24 horas à temperatura ambiente. </li><li> Regel a ponta do cateter de transmissor com uma agulha de calibre 30 rombaligado à seringa regel. </li><li> Secar cuidadosamente o transmissor usando um tecido macio dobrado e armazená-lo na bandeja de plástico original. </li></ol>

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Tradicionalmente, um fluido de preenchimento de uma cânula é inserida na artéria e ligada a um transdutor de pressão para registar os parâmetros cardiovasculares como um instantâneo de terminal em cada animal 11. Para monitorar continuamente o desempenho cardiovascular por um longo tempo, os sistemas de rádio-telemetria são empregados em muitos laboratórios. Esta ferramenta mais refinada pode gravar a pressão arterial em animais conscientes e movendo-se livremente. Em comparação com cateteres chei...

Disfunção cardiovascular ocorre após lesão da medula espinhal (SCI) em níveis elevados. Ela se manifesta na pressão desordenada arterial e freqüência cardíaca em repouso, hipotensão ortostática, hipotensão induzida por exercício, e Disreflexia autônomo, caracterizadas por episódios de hipertensão e bradicardia mediada pelo barorreflexo em resposta a estímulos sensoriais abaixo do nível da lesão 1,2. Estes sintomas interferem com a vida diária de pacientes feridos medula espinhal. Assim, é importante estabelecer instrumentos eficazes para a investigação das alterações cardiovasculares em animais com lesão medular e tratamentos experimentais. Para investigar a função cardiovascular em animais, várias técnicas têm sido usadas para monitorar a pressão arterial e freqüência cardíaca. Parâmetros cardiovasculares centrais podem ser registados através de inserção de cânula e de telemetria, ao passo que não invasivos cauda-punhos pode ser empregue para medir a pressão arterial periférica 3. Em comparação com outros métodos, teletria tem como principal vantagem que permite a gravação contínua em animais que se movem livremente e acompanhamento a longo prazo da função cardiovascular 4. Em modelos animais, SCI, as mudanças na pressão do sangue periférico após estimulação experimental pode não ser suficientemente grande para ser detectada. Assim, a técnica de monitoramento cardíaco adequado deve ser seleccionado para animais com SCI. No presente estudo, um sistema de rádio-telemetria foi introduzido para monitorar a função cardiovascular em ratos adultos após a transecção completa da medula espinhal. Os ratos receberam enxertos de rato singênico dia embrionário 14 (E14) células-tronco neurais derivadas de tronco cerebral (BS-NSC) no local da lesão. Ratos com lesões e sem transplante e ingênuos, ratos lesionados serviram como controle. O procedimento inclui a esterilização de telemetria transmissor e o implante (Figura 1), a gravação de parâmetros cardiovasculares basal, as respostas induzidas por distensão colorectal, e a limpeza e transmissorarmazenamento.

<table border="0" cellpadding="0" cellspacing="0" width="580">
	<tbody>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:315px;">Table of Specific Materials/Equipment:</td>
			<td style="width:103px;"></td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="20">
			<td height="41" rowspan="2" style="height:41px;width:120px;">Reagents</td>
			<td rowspan="2" style="width:195px;">Company</td>
			<td rowspan="2" style="width:103px;">Catalogue</td>
			<td rowspan="2" style="width:164px;">Comment</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="62">
			<td height="83" rowspan="2" style="height:83px;width:120px;">Fibrinogen (rat)</td>
			<td rowspan="2" style="width:195px;">Sigma</td>
			<td rowspan="2" style="width:103px;">F6755-25MG</td>
			<td rowspan="2" style="width:164px;">2 hr at 37oC to dissovle</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="59">
			<td height="80" rowspan="2" style="height:80px;width:120px;">Thrombin (rat)</td>
			<td rowspan="2" style="width:195px;">Sigma</td>
			<td rowspan="2" style="width:103px;">T5772-100UN</td>
			<td rowspan="2" style="width:164px;">Dissovle in 10 mM CaCl2</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:120px;">1% Terg-A-Zyme</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:103px;">Z273287</td>
			<td style="width:164px;">Enzymatic solution for telemeter cleaning</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:120px;">Fluorogold</td>
			<td style="width:195px;">Fluorochrome</td>
			<td style="width:103px;"></td>
			<td style="width:164px;">Dissovle in distilled water and avoid light</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:120px;">Telemeter&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; (PA-C40)</td>
			<td style="width:195px;">Data Sciences International</td>
			<td style="width:103px;"></td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:120px;">Telementric recording and analysis system</td>
			<td style="width:195px;">Data Sciences International</td>
			<td style="width:103px;"></td>
			<td style="width:164px;">Signal stimulator, Data Exchange Matrix, receivers, Ambient pressure reference monitor</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:120px;">Balloon-tipped catheter</td>
			<td style="width:195px;">Edward Lifesciences</td>
			<td style="width:103px;">111F7-P</td>
			<td style="width:164px;">For colorectal distension</td>
		</tr>
	</tbody>
</table>

a radio-telemetric system to monitor cardiovascular function in rats with spinal cord transection and embryonic neural stem cell grafts

Alta lesão medular torácica ou cervical (SCI) pode levar à disfunção cardiovascular. Para monitorar os parâmetros cardiovasculares, que implantou um cateter ligado a um transmissor de rádio na artéria femoral de ratos que foram submetidos a um T4 espinhal transection cabo com ou sem enxerto de células-tronco embrionárias neural derivado do tronco cerebral que expressam a proteína fluorescente verde. Em comparação com outros métodos, como a inserção de uma cânula ou tail-cuff, a telemetria é vantajoso para monitorar continuamente a pressão arterial e freqüência cardíaca nos animais que se movem livremente. Ela também é capaz de longa duração de dados de aquisições múltiplas. Na medula espinhal feridos ratos, os dados cardiovasculares basais sob condição desenfreada e Disreflexia autonômico em resposta à distensão colorretal foram gravadas com sucesso. Além disso, os parâmetros cardiovasculares antes e depois da SCI pode ser comparada no mesmo rato, se um transmissor é implantado antes uma transecção da medula espinal. Uma limitação do Telemet descritory procedimento é que a implantação na artéria femoral podem influenciar o suprimento de sangue para o membro posterior ipsilateral.

Usando a técnica de telemetria acima descrito, registramos com sucesso parâmetros cardiovasculares em medula espinhal de animais feridos. Em animais com só SCI, MAP foi significativamente reduzida enquanto HR aumentou em relação a animais sem tratamento prévio, de acordo com relatórios anteriores 9. Em animais com BS-NSC enxertia, PAM e FC aproximou níveis medidos em animais ingênuos (Figura 2). Durante a distensão colorectal, um rato foi considerado como dysreflexic se o estímulo ...

Watch this Scientific Journal Video about A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts at JoVE.com

A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts

Apresenta-se um protocolo para a utilização de um sistema de rádio-telemétrico para registar os parâmetros cardiovasculares em ratos T4 medular transeccionados oito semanas após o tronco cerebral embrionária das células estaminais neurais de enxerto no local da lesão. A telemetria é uma técnica avançada para avaliar com precisão a função cardiovascular em que se movem livremente na medula espinhal ratos lesionados conscientes.

Um sistema de rádio-telemetria para monitorar a função cardiovascular em ratos com Medula Espinhal transecção e Embryonic Stem Neural enxertos de células-

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter ...

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10.4K visualizações.  Drexel University College of Medicine.  Apresenta-se um protocolo para a utilização de um sistema de rádio-telemétrico para registar os parâmetros cardiovasculares em ratos T4 medular transeccionados oito semanas após o tronco cerebral embrionária das células estaminais neurais de enxerto no local da lesão. A telemetria é uma técnica avançada para avaliar com precisão a função cardiovascular em que se movem livremente na medula espinhal ratos lesionados conscientes. 

Vídeo: A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts

Assessing Endothelial Vasodilator Function with the Endo-PAT 2000

The endothelium is a delicate monolayer of cells that lines all blood vessels, and which comprises the systemic and lymphatic capillaries. By virtue of the panoply of paracrine factors that it secretes, the endothelium regulates the contractile and proliferative state of the underlying vascular smooth muscle, as well as the interaction of the vessel wall with circulating blood elements. Because of its central role in mediating vessel tone and growth, its position as gateway to circulating immune cells, and its local regulation of hemostasis and coagulation, the the properly functioning endothelium is the key to cardiovascular health. Conversely, the earliest disorder in most vascular diseases is endothelial dysfunction.
In the arterial circulation, the healthy endothelium generally exerts a vasodilator influence on the vascular smooth muscle. There are a number of methods to assess endothelial vasodilator function. The Endo-PAT 2000 is a new device that is used to assess endothelial vasodilator function in a rapid and non-invasive fashion. Unlike the commonly used technique of duplex ultra-sonography to assess flow-mediated vasodilation, it is totally non-operator-dependent, and the equipment is an order of magnitude less expensive. The device records endothelium-mediated changes in the digital pulse waveform known as the PAT ( peripheral Arterial Tone) signal, measured with a pair of novel modified plethysmographic probes situated on the finger index of each hand. Endothelium-mediated changes in the PAT signal are elicited by creating a downstream hyperemic response. Hyperemia is induced by occluding blood flow through the brachial artery for 5 minutes using an inflatable cuff on one hand. The response to reactive hyperemia is calculated automatically by the system. A PAT ratio is created using the post and pre occlusion values. These values are normalized to measurements from the contra-lateral arm, which serves as control for non-endothelial dependent systemic effects. Most notably, this normalization controls for fluctuations in sympathetic nerve outflow that may induce changes in peripheral arterial tone that are superimposed on the hyperemic response.
In this video we demonstrate how to use the Endo-PAT 2000 to perform a clinically relevant assessment of endothelial vasodilator function.

A noninvasive procedure to assess endothelial function is demonstrated using the Endo-PAT 2000.

Avaliação da função endotelial com a Vasodilatador 2000 Endo-PAT

The endothelium is a delicate monolayer of cells that lines all blood vessels, and which comprises the systemic and lymphatic capillaries. By virtue of ...

Medicina

Acute and Chronic Tactile Sensory Testing after Spinal Cord Injury in Rats

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia3. Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI4-5. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically5. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery4-5. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.

We describe two tactile sensory testing methods for acute or chronic periods of spinal cord injury in rats. These validated procedures can detect the development and maintenance of allodynia-like sensations.

Aguda e crônica teste sensorial tátil após a lesão medular em ratos

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory ...

A Contusion Model of Severe Spinal Cord Injury in Rats

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is described to obtain a consistent model of severe SCI. Use of a stereotactic frame and computer controlled impactor allows for creation of reproducible injury. Hypothermia and urinary tract infection pose significant challenges in the post-operative period. Careful monitoring of animals with daily weight recording and bladder expression allows for early detection of post-operative complications. The functional results of this contusion model are equivalent to transection models. The contusion model can be utilized to evaluate the efficacy of both neuroprotective and neuroregenerative approaches.

A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent model.

Um modelo de contusão grave de lesão medular em ratos

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is ...

Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. Clinically, more than 50% of SCI affect the cervical spine1. As a consequence of the primary injury, a cascade of secondary mechanisms including inflammation, apoptosis, and demyelination occur finally leading to tissue scarring and development of intramedullary cavities2,3. Both represent physical and chemical barriers to cell transplantation, integration, and regeneration. Therefore, shaping the inhibitory environment and bridging cavities to create a supportive milieu for cell transplantation and regeneration is a promising therapeutic target4. Here, a contusion/compression model of cervical SCI using an aneurysm clip is described. This model is more clinically relevant than other experimental models, since complete transection or ruptures of the cord are rare. Also in comparison to the weight drop model, which in particular damage the dorsum columns, circumferential compression of the spinal cord appears advantageous. Clip closing force and duration can be adjusted to achieve different injury severity. A ring spring facilitates precise calibration and constancy of clip force. Under physiological conditions, synthetic self-assembling peptides (SAP) self-assemble into nanofibers and thus, are appealing for application in SCI5. They can be injected directly into the lesion minimizing damage to the cord. SAPs are biocompatible structures erecting scaffolds to bridge intramedullary cavities and thus, equip the damaged cord for regenerative treatments. K2(QL)6K2 (QL6) is a novel SAP introduced by Dong et al.6 In comparison to other peptides, QL6 self-assembles into &#946;-sheets at neutral pH6.14 days after SCI, after the acute stage, SAPs are injected into the center of the lesion and neural precursor cells (NPC) are injected into adjacent dorsal columns. In order to support cell survival, transplantation is combined with continuous subdural administration of growth factors by osmotic micro pumps for 7 days.

Treating cervical spinal cord injury with both self-assembling peptides (SAP) and neural precursor cells (NPC), together with growth factors, is a promising approach to promote regeneration and recovery. A contusion/compression aneurysm clip rat model of cervical SCI and combined treatment involving SAP injection and NPC transplantation is established.

Use Synergetic de células neuronais precursoras e Peptídeos de auto-montagem em Experimental Cervical Spinal Cord Injury

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

Neural Stem Cell Transplantation em Experimental contusão Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) below the site of injury. The secondary ischemia that develops following the initial mechanical insult is a serious complication of the SCI and severely impairs the function and viability of surviving neuronal and non-neuronal cells in the SC. In addition, ischemia is also responsible for the growth of lesion during chronic phase of injury and interferes with the cellular repair and healing processes. Thus there is a need to develop a spinal cord ischemia model for studying the mechanisms of ischemia-induced pathology. Focal ischemia induced by photothrombosis (PT) is a minimally invasive and very well established procedure used to investigate the pathology of ischemia-induced cell death in the brain. Here, we describe the use of PT to induce an ischemic lesion in the spinal cord of mice. Following retro-orbital sinus injection of Rose Bengal, the posterior spinal vein and other capillaries on the dorsal surface of SC were irradiated with a green light resulting in the formation of a thrombus and thus ischemia in the affected region. Results from histology and immunochemistry studies show that PT-induced ischemia caused spinal cord infarction, loss of neurons and reactive gliosis. Using this technique a highly reproducible and relatively easy model of SCI in mice can be achieved that would serve the purpose of scientific investigations into the mechanisms of ischemia induced cell death as well as the efficacy of neuroprotective drugs. This model will also allow exploration of the pathological changes that occur following SCI in live mice like axonal degeneration and regeneration, neuronal and astrocytic Ca2+ signaling using two-photon microscopy.

Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice.

Fototrombose induzida por isquemia focal como um Modelo de Lesão Medular em Ratos

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

A Neonatal do rato Spinal Cord Injury Compression Modelo

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. Research models for bypass graft failure are essential for a better understanding of pathobiological and pathophysiological processes during graft patency loss. Large animal models, such as pigs or sheep, resemble human anatomical structures but require special facilities and equipment. This video describes a rat vein interposition model to investigate vein graft patency loss. Rats are inexpensive and easy to handle. Compared to mouse models, the convenient size of rats permits better operability and enables a sufficient amount of material to be obtained for further diverse analysis. In brief, the inferior epigastric vein of a donor rat is harvested and used to replace a segment of the femoral artery. Anastomosis is conducted via single stitches and sealed with fibrin glue. Graft patency can be monitored non-invasively using duplex sonography. Myointimal hyperplasia, which is the main cause for graft patency loss, develops progressively over time and can be calculated from histological cross sections.

This video demonstrates a model to study the development of myointimal hyperplasia after venous interposition surgery in rats.

Veia Interposição Modelo: Um modelo adequado para estudar Bypass Graft Perviedade

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. ...

Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues contributes to type 2 diabetes mellitus (T2DM) development and appetite regulation in the hypothalamus. Chemokine CCL5 and C-C chemokine receptor type 5 (CCR5) levels have been suggested to mediate arteriosclerosis and glucose intolerance in type 2 diabetes mellitus (T2DM). In addition, CCL5 plays a neuroendocrine role in the hypothalamus by regulating food intake and body temperature, thus, prompting us to investigate its function in hypothalamic insulin signaling and the regulation of peripheral glucose metabolism.
The micro-osmotic pump brain infusion system is a quick and precise way to manipulate CCL5 function and study its effect in the brain. It also provides a convenient alternative approach to generating a transgenic knockout animal. In this system, CCL5 signaling was blocked by intracerebroventricular (ICV) infusion of its antagonist, MetCCL5, using a micro-osmotic pump. The peripheral glucose metabolism and insulin responsiveness was detected by the Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT). Insulin signaling activity was then analyzed by protein blot from tissue samples derived from the animals.
After 7-14 days of MetCCL5 infusion, the glucose metabolism and insulin responsiveness was impaired in mice, as seen in the results of the OGTT and ITT. The IRS-1 serine302 phosphorylation was increased and the Akt activity was reduced in mice hypothalamic neurons following CCL5 inhibition. Altogether, our data suggest that blocking CCL5 in the mouse brain increases the phosphorylation of IRS-1 S302 and interrupts hypothalamic insulin signaling, leading to a decrease in insulin function in peripheral tissues as well as the impairment of glucose metabolism.

This protocol studies the role of chemokine (C-C motif) ligand 5 (CCL5) in the hypothalamus by delivering an antagonist, MetCCL5, into the mouse brain using a micro-osmotic pump brain infusion system. This transient inhibition of CCL5 activity interrupted hypothalamic insulin signaling, leading to glucose intolerance and peripheral systemic insulin sensitivity.

Estudando o sinal de insulina hipotalâmico a intolerância de glicose periférica com um sistema de infusão contínua de drogas dentro do cérebro de rato

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues ...

Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine balloon denudation model, which is comparable with established vessel injury models in large animals. The aorta denudation model with balloon catheters mimics the clinical setting and leads to comparable pathobiological and physiological changes. Briefly, after performing a horizontal incision in the aorta abdominalis, a balloon catheter will be inserted into the vessel, inflated, and introduced retrogradely. Inflation of the balloon will lead to intima injury and overdistension of the vessel. After removing the catheter, the aortic incision will be closed with single stiches. The model shown in this article is reproducible, easy to perform, and can be established quickly and reliably. It is especially suitable for evaluating expensive experimental therapeutic agents, which can be applied in an economical fashion. By using different knockout-mouse strains, the impact of different genes on MH development can be assessed.

This article demonstrates a murine model to study the development of myointimal hyperplasia (MH) after aortic balloon injury.

Lesão baseada no balão para induzir a hiperplasia Myointimal da Aorta Abdominal de Mouse

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine ...