Name: a rat model of ecohiv brain infection
Uploaded: 2021-01-21T14:30:00
Description: Watch this Scientific Journal Video about A Rat Model of EcoHIV Brain Infection at JoVE.com

Este trabalho foi financiado pelas bolsas NIH HD043680, MH106392, DA013137 e NS100624.

Todos os protocolos de animais foram revisados e aprovados pelo Comitê de Cuidados e Uso de Animais da Universidade da Carolina do Sul (número de garantia federal: D16-00028). Seis ratos F344/N machos adultos foram alojados em um ambiente controlado sob uma luz de 12/12: ciclo escuro com acesso ad libitum a alimentos e água. Todos os animais foram atendidos utilizando-se orientações estabelecidas pelos Institutos Nacionais de Saúde no Guia de Cuidado e Uso de Animais de Laboratório.
1. Em...

<ol>
	<li>Illenberger, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+Infection+and+Neurocognitive+Disorders+in+the+Context+of+Chronic+Drug+Abuse:+Evidence+for+Divergent+Findings+Dependent+upon+Prior+Drug+History.">HIV Infection and Neurocognitive Disorders in the Context of Chronic Drug Abuse: Evidence for Divergent Findings Dependent upon Prior Drug History.</a> Journal of Neuroimmune Pharmacology. 15 (4), 715-728 (2020).</li><li>Joseph, S. B., Swanstrom, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+HIV-1+entry+phenotypes+as+a+guide+to+changing+target+cells.">The evolution of HIV-1 entry phenotypes as a guide to changing target cells.</a> Journal of Leukocyte Biology. 103 (3), 421-431 (2018).</li><li>Potash, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+for+study+of+systemic+HIV-1+infection,+antiviral+immune+responses,+and+neuroinvasiveness.">A mouse model for study of systemic HIV-1 infection, antiviral immune responses, and neuroinvasiveness.</a> Proceedings of the National Academy of Sciences U S A. 102 (10), 3760-3765 (2005).</li><li>Albritton, L. M., Tseng, L., Scadden, D., Cunningham, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+putative+murine+ecotropic+retrovirus+receptor+gene+encodes+a+multiple+membrane-spanning+protein+and+confers+susceptibility+to+virus+infection.">A putative murine ecotropic retrovirus receptor gene encodes a multiple membrane-spanning protein and confers susceptibility to virus infection.</a> Cell. 57, 659-666 (1989).</li><li>Geraghty, P., Hadas, E., Kim, B. H., Dabo, A. J., Volsky, D. J., Foronjy, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+infection+model+of+chronic+obstructive+pulmonary+disease+in+mice.">HIV infection model of chronic obstructive pulmonary disease in mice.</a> American Journal of Physiology - Lung Cellular and Molecular Physiology. 312 (4), 500-509 (2017).</li><li>Gu, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EcoHIV+infection+of+mice+establishes+latent+viral+reservoirs+in+T+cells+and+active+viral+reservoirs+in+macrophages+that+are+sufficient+for+induction+of+neurocognitive+impairment.">EcoHIV infection of mice establishes latent viral reservoirs in T cells and active viral reservoirs in macrophages that are sufficient for induction of neurocognitive impairment.</a> PLoS Pathogens. 14 (6), 1007061 (2018).</li><li>Ellenbroek, B., Youn, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+in+neuroscience+research:+is+it+a+rat+race.">Rodent models in neuroscience research: is it a rat race.</a> Disease Models, Mechanisms. 9 (10), 1079-1087 (2016).</li><li>Feduccia, A. A., Duvauchelle, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+apparatus+and+method+for+drug+reinforcement.">Novel apparatus and method for drug reinforcement.</a> Journal of Visualized Experiments. (42), e1998 (2010).</li><li>Bertrand, S. J., Mactutus, C. F., Harrod, S. B., Moran, L. M., Booze, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+proteins+dysregulate+motivational+processes+and+dopamine+circuitry.">HIV-1 proteins dysregulate motivational processes and dopamine circuitry.</a> Scientific Reports. 8 (1), 7869 (2018).</li><li>McGaughy, J., Sarter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+vigilance+in+rats:+task+validation+and+effects+of+age,+amphetamine,+and+benzodiazepine+receptor+ligands.">Behavioral vigilance in rats: task validation and effects of age, amphetamine, and benzodiazepine receptor ligands.</a> Psychopharmacology. 117 (3), 340-357 (1995).</li><li>Li, H., Aksenova, M., Bertrand, S., Mactutus, C. F., Booze, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+filamentous+actin+(F-actin)+puncta+in+rat+cortical+neurons.">Quantification of filamentous actin (F-actin) puncta in rat cortical neurons.</a> Journal of Visualized Experiments. (108), (2016).</li><li>McLaurin, K. A., Li, H., Booze, R. M., Mactutus, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+Timing:+NeuroHIV+Progression+in+the+Post-cART+Era.">Disruption of Timing: NeuroHIV Progression in the Post-cART Era.</a> Scientific Reports. 9 (1), 827 (2019).</li><li>McLaurin, K. A., Moran, L. M., Li, H., Booze, R. M., Mactutus, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Power+of+Interstimulus+Interval+for+the+Assessment+of+Temporal+Processing+in+Rodents.">The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents.</a> Journal of Visualized Experiments. (146), e58659 (2019).</li><li>Li, H., McLaurin, K. A., Mactutus, C. F., Booze, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ballistic+Labeling+of+Pyramidal+Neurons+in+Brain+Slices+and+in+Primary+Cell+Culture.">Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture.</a> Journal of Visualized Experiments. (158), (2020).</li><li>Ko, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+but+not+Astrocytes+Harbor+HIV+DNA+in+the+Brains+of+HIV-1-Infected+Aviremic+Individuals+on+Suppressive+Antiretroviral+Therapy.">Macrophages but not Astrocytes Harbor HIV DNA in the Brains of HIV-1-Infected Aviremic Individuals on Suppressive Antiretroviral Therapy.</a> Journal of Neuroimmune Pharmacology. 14 (1), 110-119 (2019).</li><li>Sopper, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+simian+immunodeficiency+virus+infection+in+vitro+and+in+vivo+on+the+cytokine+production+of+isolated+microglia+and+peripheral+macrophages+from+rhesus+monkey.">The effect of simian immunodeficiency virus infection in vitro and in vivo on the cytokine production of isolated microglia and peripheral macrophages from rhesus monkey.</a> Virology. 220 (2), 320-329 (1996).</li><li>Llewellyn, G. N., Alvarez-Carbonell, D., Chateau, M., Karn, J., Cannon, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+infection+of+microglial+cells+in+a+reconstituted+humanized+mouse+model+and+identification+of+compounds+that+selectively+reverse+HIV+latency.">HIV-1 infection of microglial cells in a reconstituted humanized mouse model and identification of compounds that selectively reverse HIV latency.</a> Journal of NeuroVirology. 24 (2), 192-203 (2018).</li></ol>

Neste protocolo, estabelecemos um modelo de infecção por HIV induzida pelo EcoHIV em ratos. Especificamente, descrevemos uma injeção estereotaxa bilateral de EcoHIV no córtex que induziu com sucesso a infecção ativa do HIV no cérebro de rato 7 dias após a injeção. Futhermore, demonstramos que a infecção pelo EcoHIV em ratos pode ser um bom sistema biológico para estudar aspectos-chave da MÃO. Oito semanas após a infecção pelo EcoHIV, os ratos apresentaram prejuízos neurocognitivos significativos, que i...

Os sistemas biológicos melhoraram nossa compreensão das doenças neurocognitivas associadas ao HIV-1 (HAND) e seus mecanismos neurais subjacentes2. Determinar qual sistema biológico é mais adequado para qualquer estudo, muitas vezes depende da questão do interesse2. A limitação da gama de modelos animais hospedeiros desafia os estudos sobre o desenvolvimento da doença do HIV-1. Para investigar a replicação viral e a patogênese do HIV-1, Potash et al.3 criaram um modelo de camundongo de infecção ativa pelo HIV-1, substituindo a região de codificação da glicoproteína de envelope de superfície do HIV, gp120, por gp 80 MLV ecotropic, que levou à replicação viral bem sucedida em camundongos4. Após injeções de veias na cauda em camundongos quióricos de HIV (EcoHIV), muitas características foram observadas semelhantes às de indivíduos soropositivos HIV-1 (por exemplo, linfócitos e macrófagos infectados, destinados a respostas imunes antivirais, e inflamação3,5,6).
Embora camundongos e ratos sejam ambos membros do Muridae, diferenças fundamentais de espécies podem influenciar sua adequação para questões experimentais específicas7. Portanto, a extensão do modelo de infecção do EcoHIV aos ratos (comumente utilizado em estudos de abuso de drogas e distúrbios neurocognitivos) seria vantajosa no estudo do neuroHIV. Por exemplo, seu tamanho maior torna a implantação do cateter jugular para procedimentos de autoadministração de medicamentos mais práticos8. Técnicas de autoadministração de medicamentos em ratos têm sidoutilizadas para avaliar a motivação no HIV-19 . Além disso, muitas tarefas neurocognitivas/comportamentais foram inicialmente projetadas para ratos10. Aqui, relatamos a utilização de injeções estereotribuíficas de EcoHIV em ratos para ampliar o modelo de infecção do EcoHIV e dar uma oportunidade fundamental para abordar novas questões relacionadas ao neuroHIV e à MÃO.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>293FT cells</td>
			<td>ThermoFisher Scientific</td>
			<td>R70007</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic solution</td>
			<td>Cellgro</td>
			<td>30004CI</td>
			<td>100X</td>
		</tr>
		<tr>
			<td>Corning BioCoatGelatin 75cm&#178; Rectangular Canted Neck Cell Culture Flask with Vented Cap</td>
			<td>Life Technologies</td>
			<td>354488</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning DMEM with L-Glutamine, 4.5 g/L Glucose and Sodium Pyruvate</td>
			<td>Life Technologies</td>
			<td>10013CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>VWR</td>
			<td>637-137</td>
			<td></td>
		</tr>
		<tr>
			<td>drill</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>World Precision Instruments</td>
			<td>14095</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #7 Forceps</td>
			<td>World Precision Instruments</td>
			<td>14097</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Snap-Cap Microcentrifuge Biopur Safe-Lock Tubes</td>
			<td>Life Technologies</td>
			<td>22600028</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethicon Vicryl Plus Antibacterial, 4-0 Polyglactin 910 Suture, 27in. FS-2</td>
			<td>Med Vet International</td>
			<td>VCP422H</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe</td>
			<td>Hamilton</td>
			<td>1701</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen Lipofectamine 3000 Transfection Reagent</td>
			<td>Life Technologies</td>
			<td>L3000015</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Forceps</td>
			<td>World Precision Instruments</td>
			<td>15914</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Scissors</td>
			<td>World Precision Instruments</td>
			<td>500216</td>
			<td></td>
		</tr>
		<tr>
			<td>Lentivirus-Associated p24 ELISA Kit</td>
			<td>Cell Biolabs, inc.</td>
			<td>VPK-107-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Lenti-X Concentrator</td>
			<td>Takara</td>
			<td>PT4421-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced Serum Medium</td>
			<td>Life Technologies</td>
			<td>11058021</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold</td>
			<td>Fisher Scientific</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>Sevoflurane</td>
			<td>Merritt Veterinary Supply</td>
			<td>347075</td>
			<td></td>
		</tr>
		<tr>
			<td>stereotaxic apparatus</td>
			<td>Kopf Instruments</td>
			<td>Model 900</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperFrost Plus Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-154%</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Scissors</td>
			<td>World Precision Instruments</td>
			<td>500086</td>
			<td></td>
		</tr>
	</tbody>
</table>

a rat model of ecohiv brain infection

Tem sido bem estudado que o modelo de camundongo infectado pelo EcoHIV é de utilidade significativa na investigação de complicações neurológicas associadas ao HIV. O estabelecimento do modelo de ratos infectados pelo EcoHIV para estudos de abuso de drogas e distúrbios neurocognitivos seria benéfico no estudo de distúrbios neurocognitivos associados ao neurocognitivo neurocognitivo neurocognitivo (HAND) neurohiv e HIV-1. No presente estudo, demonstramos a criação bem-sucedida de um modelo de rato de infecção ativa pelo HIV usando HIV quimérico (EcoHIV). Primeiro, a construção lentiviral do EcoHIV foi embalada em células cultivadas de 293 FT por 48 horas. Em seguida, o meio condicional se concentrou e se concentrou. Em seguida, realizamos injeções estereóxicas bilaterais do EcoHIV-EGFP no tecido cerebral de rato F344/N. Uma semana após a infecção, sinais de fluorescência EGFP foram detectados no tecido cerebral infectado, indicando que o EcoHIV induz com sucesso uma infecção ativa pelo HIV em ratos. Além disso, foi realizada a imunostaining para o marcador de células microgliais, Iba1. Os resultados indicaram que a microglia era o tipo de célula predominante que abrigava o EcoHIV. Além disso, os ratos EcoHIV apresentaram alterações no processamento temporal, um potencial mecanismo neuroabportavioral subjacente da MÃO, bem como disfunção sináptica oito semanas após a infecção. Coletivamente, o presente estudo estende o modelo EcoHIV de infecção pelo HIV-1 ao rato oferecendo um valioso sistema biológico para estudar reservatórios virais do HIV-1 no cérebro, bem como comorbidades associadas à MÃO e comorbidades associadas, como o abuso de drogas.

O meio condicionado foi coletado a partir de células infectadas pelo EcoHIV-EGFP de 293FT. Em seguida, foi concentrado e titulado, em seguida, estereotaxicamente injetado no cérebro (região cortical) de ratos F344/N. Sete dias após a injeção, os ratos foram sacrificados e as imagens foram tiradas de fatias cerebrais coronais que variavam de bregma de 5,64 mm a bregma -4,68 mm. Na Figura 1A,há sinais ecohiv-egfp significativos em todo o cérebro, especialmente no córtex e no giro dent...

Watch this Scientific Journal Video about A Rat Model of EcoHIV Brain Infection at JoVE.com

A Rat Model of EcoHIV Brain Infection

Aqui, apresentamos um protocolo para estabelecer um novo modelo de infecção ativa pelo HIV usando HIV quimérico (EcoHIV), que é fundamental para melhorar nossa compreensão dos reservatórios virais do HIV-1 no cérebro e oferecer um sistema para estudar distúrbios neurocognitivos associados ao HIV e comorbidades associadas (ou seja, abuso de drogas).

Um modelo de rato de infecção cerebral ecohiv

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. ...

Usando ratos, este protocolo pode ser usado para estabelecer um novo modelo de infecção ativa pelo HIV usando HIV quimérico. com ratos, o estabelecimento do modelo de infecção do EcoHIV para estudos de abuso de drogas e distúrbios neurocognitivos pode ser benéfico para o estudo de distúrbios neurológicos e neurocognitivos associados ao HIV-1. O procedimento de demonstração será Hailong Li, um pesquisador associado do meu laboratório.Para a embalagem do vírus em 293 células T, semente cinco vezes 10 para a quinta 293 células T por mililitro de DMEM complementado com 10% FBS em cada um dos dois frascos de 75 centímetros quadrados revestidos de gelatina e incubar as células a 37 graus Celsius até que as culturas atinjam 50% de confluência. No dia da transfecção, diluir 22,5 microliters de reagente de transfecção em 750 microliters de frasco médio por frasco em um tubo de microcentrífuga de 1,5 mililitro e vórtice da solução por três segundos. Diluir 20 microgramas do DNA plasmídeo quimérico EcoHIV em 750 microlitres de meio em um segundo tubo de microcentrífuga de 1,5 mililitro por frasco com mistura completa.Combine o DNA diluído com o reagente de transfecção diluído com mistura suave e incubar a solução resultante por 15 minutos à temperatura ambiente. No final da incubação, adicione cada suspensão do vírus a 10 mililitros de meio DMEM pré-guerra e adicione cada solução suplementada por vírus a um frasco de 293 células T. Após dois dias de cultura celular, acumule o supernacido dos frascos em um único tubo cônico de 50 mililitros para centrifugação e transfira o supernante esclarecido para um novo tubo de 50 mililitros.Adicione oito mililitros de Concentredor Lenti-X ao supernanato esclarecido e misture com inversão suave. Após uma incubação de dois dias a quatro graus Celsius, centrifugar a mistura e remover cuidadosamente o supernascer, em seguida, resuspenque suavemente a pelota com 100 microliters de 100 milmolarES PBS e use um kit P24 ELISA para titer a concentração do vírus. Para a injeção EcoHIV-EGFP, após confirmar a falta de resposta ao reflexo do pedal, raspe os cabelos da região cerebral e esterilize a pele exposta duas vezes com 70% de etanol e um esfoliante à base de clorexidina.Fixar o rato em uma posição propensa em um aparelho estereotaxic e fazer uma incisão de cinco a seis centímetros através da pele ao longo da linha média do couro cabeludo. Marque uma posição de perfuração a 0,8 milímetros lateral e uma rostral de 1,2 milímetros para bregma e fure um buraco de 0,4 milímetros de diâmetro em cada posição do crânio. Carregue 1,04 vezes 10 para a sexta unidade de transdução por mililitro da solução de lentivírus EcoHIV em uma seringa de injeção de 10 microliteres e fixe a seringa no aparelho estereotaxico.Mova a agulha perto da superfície de um orifício de perfuração e insira a agulha 2,5 mililitros no orifício. Infundir um microliter de solução de vírus a uma taxa de 0,2 microliters de vírus por minuto. Quando todo o vírus tiver sido injetado, deixe a agulha dentro da área de injeção por cinco minutos antes de retirar lentamente a agulha até que ela esteja fora do crânio do rato.Feche a incisão da pele com uma sutura de fio de seda 4-0 e esterilize a solução com 70% de etanol, em seguida, coloque o rato em uma câmara de recuperação com uma almofada de aquecimento com monitoramento até a recumbência. Uma a oito semanas após a infusão viral, depois de confirmar a falta de resposta ao reflexo do pedal, fixar o rato em uma posição supina dentro de um capô de fumaça e incisar a pele ao longo da linha média torácica. Corte o diafragma para abrir a cavidade torácica e insira uma agulha de 20 por 25 milímetros no ventrículo esquerdo.Abra imediatamente o átrio direito com tesouras e perfunda 50 mililitros de PBS de 100 mililitros pré-refrigerados a uma taxa de fluxo de cinco mililitros por minuto. Quando todo o PBS for entregue, perfuse com 100 mililitros de frio 4%paraformaldeído. Quando todo o fixador tiver sido entregue, remova o cérebro do crânio e fixe o tecido durante a noite em um fresco 4% de desidida.Na manhã seguinte, coloque a amostra em 40 mililitros de 30% de sacarose em 100 mililitros pbs em um tubo de 50 mililitros por cerca de três dias até que o cérebro se instale no fundo do tubo antes de estalar o tecido cerebral em butanol metil por dois minutos a menos 80 graus Celsius. Após o congelamento, use um criostat de menos 20 graus Celsius e um pincel fino para adquirir seções coronais de 50 mícrons de espessura. Quando todas as seções tiverem sido obtidas, cubra as amostras com 300 microliters de meio anti-fade e monte-as com 22 tampas de 50 mililitros.Quando o meio secar, imagem as seções por microscopia confocal. Sete dias após a injeção de Lentivírus, imagens de fatias cerebrais coronal de ratos revelam uma presença significativa de sinais EcoHIV-EGFP em todo o tecido cerebral, especialmente dentro do córtex e do giro dentonato hipocampal. A rotulagem dupla com e as sondas Iba1 e EcoHIV-EGFP fornecem fortes evidências de que a microglia é o tipo de célula predominante que abriga a expressão EcoHIV no cérebro.Oito semanas após a infecção, os animais injetados pelo EcoHIV apresentam uma relativa insensibilidade à manipulação do intervalo interestimuloso, como evidenciado por uma função de intervalo relativamente mais plana em comparação com os controles salinos. Além disso, os ratos injetados pelo EcoHIV apresentam alterações profundas em sua morfologia dendrítica da coluna vertebral, como evidenciado por uma frequência relativa aumentada de espinhos dendráticos mais curtos com aumento dos diâmetros da cabeça e pescoço em relação aos animais de controle. A concentração e titulação do meio da condição antes da injeção estereotaxica é fundamental para garantir os resultados consistentes e replicáveis em experimentos.Esta injeção estereotaxa regional específica do EcoHIV fornece uma infecção eficiente pelo HIV dentro do cérebro e produz déficits de processamento temporal em ratos. A utilização de injeções estereotaxas de HIV em ratos para estender o modelo de infecção pelo EcoHIV oferece uma oportunidade fundamental para abordar novas questões relacionadas ao neuro HIV em questão.

Research

JoVE Journal

Neuroscience

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

Entrega de genes ao cérebro pós-natal do rato por Non-ventricular Injection sHRNA e Eletroporação

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

Neurociência

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

Cirurgia estereotáxica para lesão excitotóxica de áreas específicas do cérebro do rato adulto

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies of this phenomenon have been performed in either models with severe intrauterine ischemia or models with gradient degree of intrauterine hypoperfusion. No study has been performed in a model on uniform mild intrauterine hypoperfusion (MIUH). Two models have been used for studies of MIUH: a model based on suture ligation of either side of the arterial arcade formed with the uterine and ovarian arteries, and a transient model based on clipping the bilateral ovarian arteries and aorta having patency. Those two rodent models of MIUH have some limitations, e.g., not all fetuses are subjected to MIUH, depending on their position in the uterine horn. In our MIUH model, all fetuses are subjected to a comparable level of intrauterine hypoperfusion. MIUH was achieved by mild stenosis of all four arteries feeding the uterus, i.e., the bilateral uterine and ovarian arteries.
Arterial stenosis was induced by metal microcoils wrapped around the feeding arteries. Producing arterial stenosis with microcoils allowed us to control, optimize, and reproduce decreased blood flow with very little inter-animal variability and a low mortality rate, thus enabling accurate evaluation. When microcoils with an inner diameter of 0.24 mm were used, the blood flow in both the placenta and fetus was mildly decreased (approximately 30% from the pre-stenosis level in the placenta). The offspring of our MIUH model clearly demonstrates long-lasting alterations in neurological, neuroanatomical and behavioral test results.

Mild intrauterine hypoperfusion was produced by artery stenosis with metal microcoils wrapped around the uterine and ovarian arteries in rats at embryonic day 17. This procedure produced prenatal hypoperfusion and intrauterine growth restriction.

Um modelo do rato de leve hipoperfusão intra-uterino com estenose Microcoil

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies ...

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier (BBB). Rats with induced ischemic stroke were established and used as in vivo models to investigate the functional integrity of the BBB. Spectrophotometric detection of Evans blue (EB) in the brain samples with ischemic injury could provide reliable justification for the research and development of novel therapeutic modalities. This method generates reproducible results, and is applicable in any laboratory without a need for special equipment. Here, we present a visualized and technical guideline on the detection of the extravasation of EB following induction of ischemic stroke in rats.

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

The overall goal of this procedure is to provide a highly reproducible technique for in vivo assessment of the blood-brain barrier disruption in rat models of ischemic stroke.

Uma avaliação In Vivo do rompimento da barreira hemato - encefálica em um modelo do rato de acidente vascular cerebral isquêmico

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier ...

A Rat Model of Central Fatigue Using a Modified Multiple Platform Method

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed as a water tank with narrow platforms on the bottom. The model rats were put into the tank and stood on the platforms for 14 h (18:00 - 8:00) per day for a consecutive 21 days, with a blank control group set for contrast. At the end of modeling, rats in the model group showed an obvious fatigued appearance. To assess the model, we performed several behavioral tests, including the open field test (OFT), the elevated plus maze (EPM) test, and the exhaustive swimming (ES) test. The results showed that anxiety, spatial cognition impairment, poor muscle performance, and declined voluntary activity presented in model rats confirm the diagnosis of&#160;central fatigue. Changes of the central neurotransmitters also verified the result. In conclusion, the model successfully simulated central fatigue, and future study with the model may help reveal the pathological mechanism of the disease.

Here, we present a protocol to introduce a rat model of central fatigue using the modified multiple platform method (MMPM).

Um rato do modelo de fadiga Central usando um modificado método de plataforma múltipla

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Uma abordagem alternativa para estudar eventos primários na neurodegeneração usando fatias de cérebro de rato Ex Vivo

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

A Benchtop Approach to the Location Specific Blood Brain Barrier Opening using Focused Ultrasound in a Rat Model

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic delivery including precise localization to a target brain region and reduction of off target side effects. However, stereotaxic surgery is highly invasive which limits its translational efficacy, requires long recovery times, and provides challenges when targeting multiple brain regions. Focused ultrasound (FUS) can be used in combination with circulating microbubbles to transiently open the blood brain barrier (BBB) in millimeter sized regions. This allows intracranial localization of systemically delivered agents that cannot normally cross the BBB. This technique provides a noninvasive alternative to stereotaxic surgery. However, to date this technique has yet to be widely adopted in neuroscience laboratories due to the limited access to equipment and standardized methods. The overall goal of this protocol is to provide a benchtop approach to FUS BBB opening (BBBO) that is affordable and reproducible and can therefore be easily adopted by any laboratory.

Focused ultrasound with microbubble agents can open the blood brain barrier focally and transiently. This technique has been used to deliver a wide range of agents across the blood brain barrier. This article provides a detailed protocol for the localized delivery to the rodent brain with or without MRI guidance.

Uma abordagem benchtop para a abertura da barreira cerebral de sangue específica do local usando ultrassom focado em um modelo de rato

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic ...

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered tissue samples. Traditional immunolabeling procedures require cutting the sample into thin sections, which restricts the ability to label and examine intact structures. However, if brain tissue can remain intact during processing, structures and circuits can remain intact for the analysis. Previously established clearing methods take significant time to completely clear the tissue, and the harsh chemicals can often damage sensitive antibodies. The iDISCO method quickly and completely clears tissue, is compatible with many antibodies, and requires no special lab equipment. This technique was initially validated for the use in mice tissue, but the current protocol adapts this method to image hemispheres of control and transgenic rat brains. In addition to this, the present protocol also makes several adjustments to preexisting protocol to provide clearer images with less background staining. Antibodies for Iba-1 and tyrosine hydroxylase were validated in the HIV-1 transgenic rat and in F344/N control rats using the present hydrophobic tissue clearing method. The brain is an interwoven network, where structures work together more often than separately of one another. Analyzing the brain as a whole system as opposed to a combination of individual pieces is the greatest benefit of this whole brain clearing method.

Here we present a hydrophobic tissue clearing method that allows for the viewing of target molecules as part of intact brain structures. This technique has now been validated for F344/N control and HIV-1 transgenic rats of both sexes.

Um método de limpeza de tecido hidrofóbico para tecido cerebral de rato

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered ...

Induction and Assessment of Levodopa-induced Dyskinesias in a Rat Model of Parkinson's Disease

Levodopa (L-DOPA) remains the gold-standard therapy used to treat Parkinson&#39;s disease (PD) motor symptoms. However, unwanted involuntary movements known as L-DOPA-induced dyskinesias (LIDs) develop with prolonged use of this dopamine precursor.&#160;It is estimated that the incidence of LIDs escalates to approximately 90% of individuals with PD within 10&#8211;15 years of treatment. Understanding the mechanisms of this malady and developing both novel and effective anti-dyskinesia treatments requires consistent and accurate modeling for pre-clinical testing of therapeutic interventions. A detailed method for reliable induction and comprehensive rating of LIDs following 6-OHDA-induced nigral lesioning in a rat model of PD is presented here. Dependable LID assessment in rats provides a powerful tool that can be readily utilized across laboratories to test emerging therapies focused on reducing or eliminating this common treatment-induced burden for individuals with PD.

Induction and Assessment of Levodopa-induced Dyskinesias in a Rat Model of Parkinson&#039;s Disease

This article describes methods to induce and evaluate levodopa-induced dyskinesias in a rat model of Parkinson&#39;s disease. The protocol offers detailed information regarding the intensity and frequency of a range of dyskinetic behaviors, both dystonic and hyperkinetic, providing a reliable tool to test treatments targeting this unmet medical need.

Indução e Avaliação de Dyskinesias induzidas por Levodopa em um modelo de rato da doença de Parkinson

Levodopa (L-DOPA) remains the gold-standard therapy used to treat Parkinson&#39;s disease (PD) motor symptoms. However, unwanted involuntary movements ...

Validando a resposta a lesões e avaliações comportamentais em um modelo de rato mTBI

Rat Model of Closed-Head Mild Traumatic Injury and its Validation

Animal models are crucial for advancing our understanding of mild traumatic brain injury (mTBI) and guiding clinical research. To achieve meaningful insights, developing a stable and reproducible animal model is essential. In this study, we report a detailed description of a closed-head mTBI model and a representative validation method using Sprague-Dawley rats to verify the modeling effect. The model involves dropping a 550 g mass weight from a height of 100 cm directly onto the head of a rat on a destructible surface, followed by a 180-degree turn. To assess the injury, rats underwent a series of neurobehavioral assessments 10 min post-injury, including time of loss of consciousness, first seeking-behavior time, escape ability, and beam balance ability test. During the acute and subacute stages following the injury, behavioral tests were conducted to assess motor coordination ability (Beam task), anxiety (Open Field test), and learning and memory abilities (Morris Water Maze test). The closed-head mTBI model produced a consistent injury response with minimal mortality and replicated real-life situations. The validation method effectively verified the model development and ensured the stability and consistency of the model.

Autor em destaque: Avançando na pesquisa de lesões cerebrais traumáticas - um modelo de cabeça fechada para replicação precisa e avaliação rápida 

Here, we present a closed-head mild traumatic brain injury (mTBI) rat model and its validation exhibiting remarkable similarity to human mTBI concerning behavioral manifestations during the acute and subacute stages.

Modelo de Lesão Traumática Leve de Cabeça Fechada em Rato e sua Validação

Animal models are crucial for advancing our understanding of mild traumatic brain injury (mTBI) and guiding clinical research. To achieve meaningful ...