This paper is dedicated to my beloved wife Maren and our little Karl.

1. Preparação de Bovinos Olhos <ol><li> Obter bovina olhos imediatamente após o abate do animal. </li><li> Transportar os olhos protegidos em &quot;Sickel-solução&quot; uma forma especial, contendo NaCl 120 mM, KCl a 2 mM, 0,1 mM de MgCl2, 0,15 mM de CaCl2, 1,5 mM de NaH 2 PO 4, 13,5 mM de Na 2 HPO 4 e 5 mM de glucose no RT. </li><li> Realizar a preparação da retina em condições adaptadas escuro com uma luz vermelha ofuscante. </li><li> Retire a parte anterior do olho. Executar uma ca. incisão equatorial 4 milímetros posterior ao limbo. Posteriormente remover córnea, íris, corpo ciliar e lente em uma única peça. Mantenha as retinas em Sickel-solução. </li><li> Mecanicamente soltar os anexos vítreas para a superfície da retina e do vítreo remover da taça olho aberto. </li><li> Depois divida o olho em quatro quadrantes e perfurar rodada áreas de ca. 7 milímetros de diâmetro usando uma trefina. </li><li> Suavemente separar da retinado epitélio pigmentar e colocá-lo em um dispositivo de gravação dentro de uma caixa protegida da luz. O dispositivo de gravação é composto por uma manutenção de plástico com uma malha no meio; colocar a retina na malha e, em seguida, fixar com um anel de plástico directamente sobre os eléctrodos. NOTA: O mantenedor de plástico tem dois canais para permitir um fluxo constante do meio. </li></ol> 2. A gravação do eletrorretinograma (ERG) <ol><li> A fim de gravar o electrorretinograma, usar dois eléctrodos de prata / prata-cloreto de cada lado da retina e perfundir a retina a uma velocidade de perfusão constante de ca. 1 ml / min e temperatura constante de 37 ° C. Use a &quot;Sickel-solução&quot; saturada com oxigênio. </li><li> Antes de iniciar a medição, adaptar escuro da retina (proteger da luz durante todas as medições) e usar intervalos de estímulo de cinco min. Use um único flash xenon branco 1 Hz para a estimulação com uma intensidade definido para 6,3 mlx na superfície da retina. </li&gt; <li> Uso calibrado filtros de densidade neutra e um estímulo de luz de 10 ms controlado por um temporizador, de modo a apresentar respostas óptimas. </li><li> Para medir e processar os dados, filtrar o ERG e amplificá-lo (100 Hz filtro de alta freqüência, de 50 Hz filtro notch, 100.000 x amplificação), utilizando um RPS312RM Amplifier Grass. Tente filtrar frequências possíveis perturbadores que possam perturbar o sinal. A fim de processar os dados, use uma placa de aquisição analógico-digitais de dados em um computador desktop (PC compatível). </li><li> Após o período de adaptação ao escuro sob perfusão constante, medir a amplitude do sinal elétrico até estáveis ​​amplitudes b-ondas são gravadas. NOTA: As amplitudes são consideradas estáveis ​​se cinco medições individuais chegar a um valor médio e desviar-se menos de 10%. Um bom exemplo de medições individuais é dada na Figura 1. </li><li> Para iniciar o teste, substituir o oxigénio puro por qualquer azoto puro (número de experiências individuais, n = 5) para testar a hipóxiaou 250 um glutamato (n = 5). </li><li> Grave as respostas elétricas a cada 5 min para 45 min. </li><li> Após o período de testes, aspergir as retinas com padrão médio saturada com oxigênio por 75 min e olhar para as mudanças da amplitude da onda-b. Esta é a fase de lavagem. Medir a amplitude da onda-b da calha do uma onda para o pico da onda b. </li><li> Para investigar o efeito de hipóxia ou glutamato no potencial de fotorreceptores em condições escotópicas, suprimir a onda b pela adição de 1 mM para a solução nutriente. </li><li> Após a gravação de um potencial estável de fotorreceptores durante 30 min, realizar o procedimento como antes, expondo as retinas de 45 min para as diferentes soluções de irrigação com aspartato 1 mM. Use o mesmo período de washout (passo 2.8), como mencionado anteriormente. </li></ol> Análise 3. Dados <ol><li> A fim de avaliar estatisticamente os dados, garantir uma distribuição normal para todos os dados, por exemplo, utilizando o teste de Kolmogorov-Smirnovteste 15. </li><li> Calcula-se a redução dos a- e b-amplitudes de onda em percentagens após a fase de exposição, em comparação com a última medição antes da exposição. Comparar a redução das amplitudes-ERG após 45 min - no final do período de exposição - a ERG medido antes da aplicação. </li><li> Comparar a onda a- e b- no final da fase de lavagem para a amplitude correspondente antes de exposição a estudar a possibilidade de recuperação. </li><li> Para a análise estatística, use o software estatístico JMP software ou software SPSS. Calcular dados ao longo como a média ± desvio padrão. Estime significado pelo teste estatístico adequado. NOTA: Estes testes podem ser diferentes dependendo do escopo experimental. Neste cenário, use t-pareado de Student. </li></ol>

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The authors have nothing to disclose.

Neste estudo, um impacto significativo na amplitude da onda b após 45 minutos de hipoxia foi encontrado. Esta redução foi ainda significativo após a fase de washout. Um efeito semelhante sobre o potencial de fotorreceptores pode ser observada. Os resultados são compatíveis com outros dados publicados 16 e nos dão a oportunidade de estudar possíveis efeitos neuroprotetores após hipóxia. Depois de 45 minutos de exposição de 250 uM de glutamat...

Neuroprotection tem sido um forte campo de investigação na investigação oftalmológica nas últimas décadas. A retina é uma rede neuronal altamente sensível que depende significativamente a oxigenação e é fortemente influenciado pelo metabolismo das suas células vizinhas. As principais patologias oculares relacionadas com a lesão das células nervosas são oclusões vasculares retinianas, glaucoma e descolamento da retina. A oclusão da artéria retinal, como um exemplo para a oclusão vascular da retina, leva a uma perda súbita de visão devido a hipoxia da retina interna 1. Ela é freqüentemente associada com patologias vasculares gerais 2 e leva a uma perda visual persistente 1, com apenas 8% dos pacientes em recuperação da acuidade visual significativamente 1. Apesar de a fibrinólise arterial tem sido sugerida como uma opção de tratamento, o benefício não poderia ser mostrado em um ensaio clínico randomizado 3. Glaucoma e descolamento de retinaambos têm um aumento na concentração de glutamato 4-6. O glutamato sob condições fisiológicas é encontrado como um transmissor excitatório ao longo de todo o sistema nervoso central e retina interna a 7,8. Elevados níveis de glutamato foram encontradas não só em glaucoma e descolamento de retina 5,6, mas também na retinopatia diabética proliferativa 9. Um aumento em glutamato conduz possivelmente a excitotoxicidade e, portanto, danos das células nervosas 10. Na maioria dos casos de descolamento da retina e em alguns casos de cirurgia de retinopatia diabética proliferativa na retina (vitrectomia via pars plana) são necessários. Durante a manipulação mecânica vitrectomia, luz brilhante da fibra óptica ou tensão de cisalhamento exercida por altas taxas de fluxo de soluções de irrigação durante as operações longas exercer uma pressão adicional sobre a retina 11,12. Todas as doenças mencionadas têm em comum que a patologia está localizada ao retinuma só e representam a comunidade oftalmológica com o desafio de encontrar formas de proteger a retina como um sistema neurossensorial. O electrorretinograma (ERG) é o método padrão para a avaliação in vivo da função de fotorreceptores (uma onda) e a função da retina interna (b-ondas). O ERG é medido por eléctrodos de prata-introduzidos na córnea e os olhos estão a ser estimulado por um aumento do nível de luz para detectar defeitos em bastonetes ou cones ou na retina interna. Diferentes defeitos na retina pode ser detectada por alterações na amplitude (a força da resposta) ou a latência (o intervalo de tempo de resposta-para) do ERG. Diferentes protocolos e métodos de medição ERG (padrão-ERG, multifocal ERG-campo ou brilhante ERG) permitem uma maior diferenciação de defeitos. A técnica da retina isolado foi introduzida recentemente, tornando-se possível avaliar os efeitos sobre a retina, sem interferências de um animal, por exemplo o estudo dereações gerais 13,14. Foi o propósito deste estudo para avaliar e introduzir um modelo de estresse definido e padronizado para hipóxia e estresse glutamato na retina superfused isolado. Assim, nós estamos esperando para lançar as bases para futuros estudos sobre efeitos neuroprotetores de determinados agentes ou soluções de irrigação intra-oculares.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">120 mM NaCl&#160;</td>
			<td style="width:71px;">Merck Pharma, Germany</td>
			<td align="right" style="width:119px;">1,064,041,000</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">2 mM KCl,&#160;&#160;</td>
			<td style="width:71px;">Merck Pharma, Germany</td>
			<td align="right" style="width:119px;">1,050,010,250</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">0.1 mM MgCl2,&#160;</td>
			<td style="width:71px;">Merck Pharma, Germany</td>
			<td align="right" style="width:119px;">58,330,250</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">0.15 mM CaCl2</td>
			<td style="width:71px;">Merck Pharma, Germany</td>
			<td style="width:119px;">111 TA106282</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">1.5 mM NaH2PO4/13.5 mM Na2HPO4&#160;&#160;</td>
			<td style="width:71px;">Merck Pharma, Germany</td>
			<td align="right" style="width:119px;">1,065,860,500</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">5 mM glucose</td>
			<td style="width:71px;">Merck Pharma, Germany</td>
			<td align="right" style="width:119px;">40,741,000</td>
			<td></td>
		</tr>
	</tbody>
</table>

glutamate and hypoxia as a stress model for the isolated perfused vertebrate retina

Neuroprotection tem sido um forte campo de investigação na investigação oftalmológica nas últimas décadas e afeta doenças como glaucoma, oclusão vascular da retina, descolamento de retina, e retinopatia diabética. Era o objeto deste estudo a introdução de um modelo de estresse padronizado para futuro teste pré-clínico terapêutico. Retina bovina foram preparadas e perfundido com uma solução saturada de oxigénio padrão, e o ERG foi gravado. Depois de gravar b-ondas estáveis, hipoxia (N 2) puro ou estresse glutamato (250 um glutamato) foi exercida por 45 min. Para investigar os efeitos sobre sozinho função de fotorreceptores, 1 mM aspartato foi adicionada para obter a-ondas. ERG-recuperação foi monitorada por 75 min. Para hipoxia, uma diminuição na amplitude da onda-um de 87,0% foi observada (p &lt;0,01) após um tempo de exposição de 45 min (36,5% de redução após o final da lavagem p = 0,03). Além disso, um decr inicialfacilidade de amplitudes de ondas de b-87.23% foi registada, que atingiu significado estatístico (p &lt;0,01, diminuição de 25,5% no final da lavagem, p = 0,03). Para 250 um glutamato, uma redução inicial de 7,8% de um amplitudes de ondas (p&gt; 0,05), seguidas por uma redução de 1,9% (p&gt; 0,05). Uma redução de 83,7% da amplitude da onda b (p &lt;0,01) foi observado; após uma lavagem de 75 min, a redução foi de 2,3% (p = 0,62). Neste estudo, um modelo de tensão é apresentada normalizada que pode ser útil para identificar possíveis efeitos neuroprotectores no futuro.

Após 1 hora de perfusão dos preparativos da retina com solução padrão saturado de oxigênio (Figura 1A e B) ERG-amplitudes mostrou estabilização e menor variação de amplitudes entre as medidas individuais. pH, pressão osmótica, temperatura, e pO 2 (excepto para o teste de hipóxia) foram mantidas constantes em todos os testes. Para isolar o sinal a partir do sinal de fotorreceptores da retina interna, aspartato 1 mM foi adicionado para a solução padrão ...

Watch this Scientific Journal Video about Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina at JoVE.com

Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina

Com este estudo, apresentamos um modelo de estresse padronizado para a retina bovina superfundido isolado para o futuro teste pré-clínico terapêutico. O efeito de qualquer hipóxia (puro N 2) ou estresse glutamato (250 mM glutamato) em função da retina representado por a- e amplitudes da onda-b foi avaliado.

Glutamato e hipóxia como um modelo de estresse para o isolado perfundido Vertebrate Retina

Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades and affects diseases such as glaucoma, retinal ...

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7.8K visualizações.  University Eye Hospital Tübingen.  Com este estudo, apresentamos um modelo de estresse padronizado para a retina bovina superfundido isolado para o futuro teste pré-clínico terapêutico. O efeito de qualquer hipóxia (puro N 2) ou estresse glutamato (250 mM glutamato) em função da retina representado por a- e amplitudes da onda-b foi avaliado. 

Vídeo: Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson's Disease

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our understanding of the mechanisms underlying parkinsonian symptoms, since it recapitulates the changes in basal ganglia circuitry and pharmacology observed in parkinsonian patients1-4. However, the precise cellular and molecular changes occurring at cortico-striatal synapses of the output pathways within the striatum, which is the major input region of the basal ganglia remain elusive, and this is believed to be site where pathological abnormalities underlying parkinsonian symptoms arise3,5.
In PD, understanding the mechanisms underlying changes in basal ganglia circuitry following degeneration of the nigro-striatal pathway has been greatly advanced by the development of bacterial artificial chromosome (BAC) mice over-expressing green fluorescent proteins driven by promoters specific for the two striatal output pathways (direct pathway: eGFP-D1; indirect pathway: eGFP-D2 and eGFP-A2a)8, allowing them to be studied in isolation. For example, recent studies have suggested that there are pathological changes in synaptic plasticity in parkinsonian mice9,10. However, these studies utilised juvenile mice and acute models of parkinsonism. It is unclear whether the changes described in adult rats with stable 6-OHDA lesions also occur in these models. Other groups have attempted to generate a stable unilaterally-lesioned 6-OHDA adult mouse model of PD by lesioning the medial forebrain bundle (MFB), unfortunately, the mortality rate in this study was extremely high, with only 14% surviving the surgery for 21 days or longer11. More recent studies have generated intra-nigral lesions with both a low mortality rate &gt;80% loss of dopaminergic neurons, however expression of L-DOPA induced dyskinesia11,12,13,14 was variable in these studies. Another well established mouse model of PD is the MPTP-lesioned mouse15. Whilst this model has proven useful in the assessment of potential neuroprotective agents16, it is less suitable for understanding mechanisms underlying symptoms of PD, as this model often fails to induce motor deficits, and shows a wide variability in the extent of lesion17, 18.
Here we have developed a stable unilateral 6-OHDA-lesioned mouse model of PD by direct administration of 6-OHDA into the MFB, which consistently causes &gt;95% loss of striatal dopamine (as measured by HPLC), as well as producing the behavioural imbalances observed in the well characterised unilateral 6-OHDA-lesioned rat model of PD. This newly developed mouse model of PD will prove a valuable tool in understanding the mechanisms underlying generation of parkinsonian symptoms.

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson&#039;s Disease

A protocol for performing unilateral 6-OHDA lesions of the medial forebrain bundle in mice is described. This method has a low mortality rate (13.3 %) with 89% of the surviving animals showing &#62;95% loss of striatal dopamine and 90.63&#177;-4.02 % ipsiversive rotational bias towards the side of the lesion.

Desenvolvimento de um Unilateralmente-lesionadas modelo de rato 6-OHDA da Doença de Parkinson

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our ...

Medicina

The Trier Social Stress Test Protocol for Inducing Psychological Stress

This article demonstrates a psychological stress protocol for use in a laboratory setting. Protocols that allow researchers to study the biological pathways of the stress response in health and disease are fundamental to the progress of research in stress and anxiety.1 Although numerous protocols exist for inducing stress response in the laboratory, many neglect to provide a naturalistic context or to incorporate aspects of social and psychological stress. Of psychological stress protocols, meta-analysis suggests that the Trier Social Stress Test (TSST) is the most useful and appropriate standardized protocol for studies of stress hormone reactivity.2 In the original description of the TSST, researchers sought to design and evaluate a procedure capable of inducing a reliable stress response in the majority of healthy volunteers.3 These researchers found elevations in heart rate, blood pressure and several endocrine stress markers in response to the TSST (a psychological stressor) compared to a saline injection (a physical stressor).3 Although the TSST has been modified to meet the needs of various research groups, it generally consists of a waiting period upon arrival, anticipatory speech preparation, speech performance, and verbal arithmetic performance periods, followed by one or more recovery periods. The TSST requires participants to prepare and deliver a speech, and verbally respond to a challenging arithmetic problem in the presence of a socially evaluative audience.3 Social evaluation and uncontrollability have been identified as key components of stress induction by the TSST.4 In use for over a decade, the goal of the TSST is to systematically induce a stress response in order to measure differences in reactivity, anxiety and activation of the hypothalamic-pituitary-adrenal (HPA) or sympathetic-adrenal-medullary (SAM) axis during the task.1 Researchers generally assess changes in self-reported anxiety, physiological measures (e.g. heart rate), and/or neuroendocrine indices (e.g. the stress hormone cortisol) in response to the TSST. Many investigators have adopted salivary sampling for stress markers such as cortisol and alpha-amylase (a marker of autonomic nervous system activation) as an alternative to blood sampling to reduce the confounding stress of blood-collection techniques. In addition to changes experienced by an individual completing the TSST, researchers can compare changes between different treatment groups (e.g. clinical versus healthy control samples) or the effectiveness of stress-reducing interventions.1

This article describes a protocol for inducing psychological stress in participants, which enables researchers to measure psychological, physiological and neuroendocrine responses to stress within single participants or between groups.

O Trier Protocolo de teste de estresse social para induzir Estresse Psicológico

This article demonstrates a psychological stress protocol for use in a laboratory setting.  Protocols that allow researchers to study the biological ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Biomarcadores em um modelo animal para Revelando correlatos neurais, hematológica e Comportamental do TEPT

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

O<em&gt; Em ovo</em&gt; CAM-ensaio como um Xenograftmodel para o Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

Descolamento de Retina modelo em roedores por injeção sub-retiniana de hialuronato de sódio

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Excitotóxico Estimulação do Microslices cérebro como um<em&gt; In vitro</em&gt; Modelo de AVC

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Isolated Hepatic Perfusion as a Treatment for Liver Metastases of Uveal Melanoma

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic agent melphalan. Briefly, the procedure starts with the setup of a percutaneous veno-venous bypass from the femoral vein to the external jugular vein. Via a laparotomy, catheters are then inserted into the proper hepatic artery and the caval vein. The portal vein and the caval vein, both supra- and infrahepatically, are then clamped. The arterial and venous catheters are connected to a heart lung machine and the liver is perfused with melphalan (1 mg/kg body weight) for 60 min. This way it is possible to locally perfuse the liver with a high dose of a chemotherapeutic agent, without leakage to the systemic circulation.
In previous studies including patients with isolated liver metastases of uveal melanoma, an overall response rate of 33-100% and a median survival between 9 and 13 months, have been reported. The aim of this protocol is to give a clear description of how to perform the procedure and to discuss IHP as a treatment option for liver metastases of uveal melanoma.

Here, we present a protocol how to perform an isolated liver perfusion (IHP) with melphalan and we also discuss IHP as a treatment option for liver metastases of uveal melanoma.

Isolado hepática Perfusão como um tratamento para o fígado Metástases de Uveal Melanoma

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic ...

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA thrombolysis) rarely produces benefits in mechanical occlusion-based experimental models, which dominate preclinical stroke research. This split between the bench and bedside suggests the need to employ tPA-responsive models in preclinical stroke research. To this end, a simple and tPA-reactive thrombotic stroke model is invented and described here. This model consists of transient occlusion of the unilateral common carotid artery and delivery of 7.5% oxygen through a face mask in adult mice for 30 min, while maintaining the animal rectal temperature at 37.5 &#177; 0.5 &#176;C. Although reversible ligation of the unilateral carotid artery or hypoxia each suppressed cerebral blood flow only transiently, the combination of both insults caused lasting reperfusion deficits, fibrin and platelet deposition, and large infarct in the middle cerebral artery-supplied territory. Importantly, tail-vein injection of recombinant tPA at 0.5, 1, or 4 hr post-tHI (10 mg/kg) provided time-dependent reduction of the mortality rate and infarct size. This new stroke model is simple and can be standardized across laboratories to compare experimental results. Further, it induces thrombosis without craniectomy or introducing pre-formed emboli. Given these unique merits, the tHI model is a useful addition to the repertoire of preclinical stroke research.

Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients.

Um Modelo de acidente vascular cerebral trombótico Baseado em Transient isquemia-hipoxia cerebral

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA ...

A Model to Simulate Clinically Relevant Hypoxia in Humans

In case of apnea, arterial partial pressure of oxygen (pO2) decreases, while partial pressure of carbon dioxide (pCO2) increases. To avoid damage to hypoxia sensitive organs such as the brain, compensatory circulatory mechanisms help to maintain an adequate oxygen supply. This is mainly achieved by increased cerebral blood flow. Intermittent hypoxia is a commonly seen phenomenon in patients with obstructive sleep apnea. Acute airway obstruction can also result in hypoxia and hypercapnia. Until now, no adequate model has been established to simulate these dynamics in humans. Previous investigations focusing on human hypoxia used inhaled hypoxic gas mixtures. However, the resulting hypoxia was combined with hyperventilation and is therefore more representative of high altitude environments than of apnea. Furthermore, the transferability of previously performed animal experiments to humans is limited and the pathophysiological background of apnea induced physiological changes is poorly understood. In this study, healthy human apneic divers were utilized to mimic clinically relevant hypoxia and hypercapnia during apnea. Additionally, pulse-oximetry and Near Infrared Spectroscopy (NIRS) were used to evaluate changes in cerebral and peripheral oxygen saturation before, during, and after apnea.

Hypoxia simulation in humans has usually been performed by inhaling hypoxic gas mixtures. For this study, apneic divers were used to simulate dynamic hypoxia in humans. Additionally, physiological changes in desaturation and re-saturation kinetics were evaluated with non-invasive tools such as Near-Infrared-Spectroscopy (NIRS) and peripheral oxygenation saturation (SpO2).

Um modelo para simular hipóxia clinicamente relevantes em humanos

In case of apnea, arterial partial pressure of oxygen (pO2) decreases, while partial pressure of carbon dioxide (pCO2) increases. To avoid damage to ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Medição de contratilidade isométrica da artéria mesentérica Mouse usando fio miografia

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...