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

1. Preparación de la especie bovina Ojos <ol><li> Obtener ojos bovinos directamente siguientes al sacrificio del animal. </li><li> Transportar los ojos protegidos en &quot;Sickel-solución&quot; un medio especial que contiene NaCl 120 mM, 2 mM KCl, 0,1 mM de MgCl 2, 0,15 mM CaCl 2, 1,5 mM NaH 2 PO 4 mM de glucosa, 13,5 mM Na 2 HPO 4 y 5 en RT. </li><li> Realizar la preparación de la retina en condiciones adaptados a la oscuridad con una luz roja tenue. </li><li> Retire la parte anterior del ojo. Realizar una incisión ecuatorial ca. 4 mm por detrás del limbo. A partir de entonces eliminar córnea, el iris, el cuerpo ciliar y la lente en una sola pieza. Mantenga las retinas en Sickel-solución. </li><li> Mecánicamente aflojar los archivos adjuntos vítreo a la superficie de la retina y quitar el vítreo de la copa ojo abierto. </li><li> Luego divida el ojo en cuatro cuadrantes y rompa ronda áreas de ca. 7 mm de diámetro utilizando un trépano. </li><li> Separar suavemente la retinadel epitelio pigmentario y colocarlo en un dispositivo de grabación dentro de una caja protegida de la luz. El dispositivo de grabación consta de un mantenedor de plástico con una malla en el centro; colocar la retina en la malla y luego fijar con un anillo de plástico directamente sobre los electrodos. NOTA: El mantenedor de plástico tiene dos canales para permitir un flujo constante del medio. </li></ol> 2. Grabación de la Electrorretinograma (ERG) <ol><li> Con el fin de registrar el electrorretinograma, utilizar dos electrodos de plata / cloruro de plata-a cada lado de la retina y perfundir la retina a una velocidad de perfusión constante de ca. 1 ml / min y temperatura constante de 37 ° C. Utilice la opción &quot;Sickel-solución&quot; saturada de oxígeno. </li><li> Antes de iniciar la medición, oscuro adaptar la retina (protegerlo de la luz durante todas las mediciones) y utilizar intervalos de estímulo de cinco minutos. Use un 1 Hz solo flash de xenón blanco para la estimulación con una intensidad ajustado a 6,3 mlx en la superficie de la retina. </li&gt; <li> Uso calibrado filtros de densidad neutra y un estímulo de luz de 10 microsegundos controlado por un temporizador con el fin de tener respuestas óptimas. </li><li> Para medir y procesar los datos, filtrado del ERG y amplificarlo (filtro de paso alto de 100 Hz, 50 Hz filtro de muesca, 100000 x amplificación) utilizando un RPS312RM Amplificador Hierba. Trate de filtrar las posibles frecuencias perturbadoras que puedan perturbar la señal. Para procesar los datos, utilice una tarjeta de adquisición analógico-digital de datos en un ordenador de sobremesa (PC compatible). </li><li> Después del periodo de adaptación a la oscuridad bajo perfusión constante, medir las amplitudes de la señal eléctrica hasta que se registran las amplitudes de la onda b estables. NOTA: Amplitudes se consideran estables si cinco mediciones individuales alcanzan un valor medio y se desvían menos de 10%. Un buen ejemplo de mediciones individuales se da en la Figura 1. </li><li> Para iniciar la prueba, reemplazar oxígeno puro ya sea por nitrógeno puro (número de experimentos individuales, n = 5) para la prueba de hipoxiao glutamato 250 m (n = 5). </li><li> Anote las respuestas eléctricas cada 5 min durante 45 min. </li><li> Después del período de prueba, perfundir las retinas con medio estándar saturada de oxígeno durante 75 minutos y ver los cambios de la amplitud de la onda b. Esta es la fase de lavado. Mida la amplitud de la onda b del punto más bajo de la onda a que el pico de la onda b. </li><li> Para investigar el efecto de la hipoxia o glutamato en el potencial de los fotorreceptores en condiciones escotópicas, suprimir la onda b mediante la adición de 1 mM a la solución de nutrientes. </li><li> Después de grabar un potencial fotorreceptor estable durante 30 minutos, llevar a cabo el procedimiento que antes, exponiendo las retinas de 45 minutos a las diferentes soluciones de riego con 1 mM aspartato. Utilice el mismo período de lavado (paso 2.8) como se mencionó anteriormente. </li></ol> Análisis 3. Datos <ol><li> A fin de evaluar estadísticamente los datos, garantizar una distribución normal para todos los datos, por ejemplo, utilizando la prueba de Kolmogorov-Smirnovprueba 15. </li><li> Calcular la reducción de las amplitudes A y B en porcentajes de onda después de la fase de exposición en comparación con la última medición antes de la exposición. Comparación de la reducción de los ERG-amplitudes después de 45 min - al final del período de exposición - a ERG medido antes de la aplicación. </li><li> Comparar la onda a- y b- al final de la fase de lavado a la amplitud correspondiente antes de la exposición para examinar una posible recuperación. </li><li> Para el análisis estadístico, utilice el software software estadístico JMP o software SPSS. Calcular datos en todo momento como la media ± desviación estándar. Estimar importancia por la prueba estadística apropiada. NOTA: Estas pruebas pueden ser diferentes según el ámbito experimental. En esta configuración, utilice la prueba t pareada de Student. </li></ol>

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The authors have nothing to disclose.

En este estudio, se encontró un impacto significativo en la amplitud de la onda b después de 45 min de la hipoxia. Esta reducción fue todavía significativa después de la fase de lavado. Un efecto similar sobre el potencial de los fotorreceptores se pudo observar. Los resultados son apoyados por otros datos publicados 16 y darnos la oportunidad de estudiar los posibles efectos neuroprotectores después de la hipoxia. Después de 45 min de exposici?...

Neuroprotección ha sido un fuerte campo de la investigación en la investigación oftalmológica en las últimas décadas. La retina es una red neuronal altamente sensible que depende significativamente de la oxigenación y está influenciada fuertemente por el metabolismo de sus células circundantes. Las principales patologías oculares relacionados con el daño de las células nerviosas son oclusiones vasculares de la retina, glaucoma y desprendimiento de retina. Oclusión de la arteria de la retina, como un ejemplo para la oclusión vascular retiniana, conduce a una pérdida repentina de la visión debido a la hipoxia de la retina interna 1. A menudo se asocia con patologías vasculares generales 2 y conduce a una pérdida de la visión persistente 1, con sólo el 8% de los pacientes que se recuperan de la agudeza visual significativamente 1. Aunque la fibrinólisis arterial se ha sugerido como una opción de tratamiento, el beneficio no se pudo demostrar en un ensayo clínico aleatorizado 3. El glaucoma y desprendimiento de retinaambos tienen un aumento en la concentración de glutamato 4-6. El glutamato en condiciones fisiológicas se encuentra como un transmisor excitador en todo el sistema nervioso central y el 7,8 retina interna. Niveles de glutamato elevados se han encontrado no sólo en el glaucoma y desprendimiento de retina 5,6, sino también en la retinopatía diabética proliferativa 9. Un aumento en el glutamato posiblemente conduce a la excitotoxicidad y, por lo tanto, el daño de la célula nerviosa 10. En la mayoría de los casos de desprendimiento de retina y, en algunos casos de retinopatía diabética proliferativa de la cirugía sobre la retina (vitrectomía pars plana) son necesarios. Durante la vitrectomía pars plana manipulación mecánica, la luz brillante de la fibra óptica o el estrés de cizalla ejercida por las altas tasas de flujo de las soluciones de riego durante las operaciones largas ejercen una presión adicional sobre la retina 11,12. Todas las enfermedades mencionadas tienen en común que la patología se localiza en el retinuna sola y plantean la comunidad oftalmológica con el desafío de encontrar formas de proteger la retina como un sistema neurosensorial. El electrorretinograma (ERG) es el método estándar para la evaluación de la función in vivo de los fotorreceptores (una onda) y la función de la retina interna (la onda b). El ERG se mide por plata-electrodos introducidos en la córnea y los ojos están siendo estimulado por un creciente nivel de luz para detectar defectos en varillas o conos o en la retina interna. Diferentes defectos de la retina pueden ser detectados por cambios en la amplitud (la fuerza de la respuesta) o la latencia (la respuesta de intervalo de tiempo-a-) de la ERG. Diferentes métodos de protocolo y medición ERG (patrón-ERG, multifocal-ERG o brillante campo ERG) permiten una mayor diferenciación de los defectos. La técnica de la retina aislada se ha introducido recientemente, por lo que es posible evaluar los efectos sobre la retina sin interferencias de por ejemplo un animal de estudioreacciones generales 13,14. Era el propósito de este estudio para evaluar e introducir un modelo de estrés definido y estandarizado para la hipoxia y el estrés de glutamato en la retina aislada superfused. Por lo tanto, tenemos la esperanza de sentar las bases para futuros estudios sobre los efectos neuroprotectores de determinados agentes o soluciones de irrigación intraoculares.

<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

Neuroprotección ha sido un fuerte campo de la investigación en la investigación oftalmológica en las últimas décadas y afecta a enfermedades como el glaucoma, la oclusión vascular retiniana, desprendimiento de retina y la retinopatía diabética. Era el objeto de este estudio para introducir un modelo de estrés estandarizado para futuras pruebas terapéutica preclínica. Retinas bovinas se prepararon y perfundidos con una solución estándar saturada de oxígeno, y el ERG se registró. Después de registrar ondas b estables, hipoxia (n pura 2) o el estrés glutamato (250 micras glutamato) se ejerció durante 45 min. Para investigar los efectos sobre la función de los fotorreceptores solo, se añadió aspartato 1 mM para obtener una de las ondas. ERG-recuperación se controló durante 75 min. Para la hipoxia, una disminución en una onda de amplitud de 87,0% se observó (p &lt;0,01) después de un tiempo de exposición de 45 min (disminución de 36,5% después del final de la lavado p = 0,03). Además, un decr inicialfacilidad de amplitudes b-onda de 87,23% se registró, que alcanzó significación estadística (p &lt;0,01, disminución de 25,5% al ​​final del lavado, p = 0,03). Por 250 micras glutamato, una reducción inicial del 7,8% de las amplitudes de una onda (p&gt; 0,05), seguido de una reducción del 1,9% (p&gt; 0,05). Una reducción del 83,7% de las amplitudes de la onda b (p &lt;0,01) fue observado; después de un lavado de 75 min, la reducción fue del 2,3% (p = 0,62). En este estudio, un modelo de estrés se presenta estandarizada que puede ser útil para identificar posibles efectos neuroprotectores en el futuro.

Después de 1 hora de la perfusión de los preparativos de la retina con solución estándar saturada de oxígeno (Figura 1A y B) ERG-amplitudes mostraron estabilización y una menor variación de amplitudes entre las mediciones individuales. pH, la presión osmótica, la temperatura, y pO 2 (excepto para las pruebas de hipoxia) se mantuvieron constantes para todas las pruebas. Para aislar la señal de los fotorreceptores de la señal de la retina interna, 1 mM aspar...

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

Con este estudio, presentamos un modelo de estrés estandarizado para la retina bovina superfundido aislada para futuras pruebas terapéutica preclínica. Se evaluó el efecto de cualquiera de hipoxia (N 2 puro) o estrés glutamato (250 glutamato mM) sobre la función retiniana representado por a- y amplitudes de la onda b.

El glutamato y la hipoxia como un Modelo de estrés para el perfundido aislado Vertebrados 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 vistas.  University Eye Hospital Tübingen.  Con este estudio, presentamos un modelo de estrés estandarizado para la retina bovina superfundido aislada para futuras pruebas terapéutica preclínica. Se evaluó el efecto de cualquiera de hipoxia (N 2 puro) o estrés glutamato (250 glutamato mM) sobre la función retiniana representado por a- y amplitudes de la onda b. 

Video: 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.

Desarrollo de una manera unilateral-lesionado 6-OHDA modelo de ratón de la enfermedad 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 ...

Investigación

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.

El estrés social Trier Protocolo de prueba para la inducción de estrés 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 en un modelo animal para Revelar correlatos neurales, hematológicas y conductuales de estrés postraumático

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.

La<em&gt; En ovo</em&gt; CAM-ensayo como un Xenograftmodel para el 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.

Desprendimiento de Retina modelo en roedores por inyección subretiniana de Hialuronato de Sodio

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.

Excitotóxica Estimulación del cerebro Microslices como<em&gt; In vitro</em&gt; Modelo de Carrera

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.

Aislado hepática de perfusión como un tratamiento para las metástasis hepáticas de melanoma uveal

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.

Un accidente cerebrovascular trombótico Basado En transitoria cerebral por hipoxia-isquemia Modelo

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).

Un modelo para simular la hipoxia clínicamente relevantes en seres 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.

Medición de la contractilidad isométrica de la arteria mesentérica de ratón con cable miografía

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