El presente estudio fue apoyado por becas del Consejo Nacional de Ciencia al Dr. Bai-Chuang Shyu (NSC 99-2320-B-001-016-MY3, NSC 100-2311-B-001-003-MY3, y NSC 102-2320- B-001-026-MY3). Este trabajo se llevó a cabo en el Instituto de Ciencias Biomédicas, que han recibido recursos de la Academia Sinica.

El protocolo en el presente estudio recibió la aprobación de la Academia Sinica Institucional Cuidado de Animales y el Utilización de Taiwán. 1. Preparativos Animal <ol><li> Obtener ratas macho Sprague-Dawley (aproximadamente 300 a 400 g). Mantener las ratas en una habitación con aire acondicionado (21 - 22 ° C, 50% de humedad) en virtud de un / 12 horas de luz / oscuridad ciclo de 12 horas (luces encendidas a las 8:00 AM) con acceso libre a comida y agua. </li></ol> 2. Procedimiento experimental <ol><li> Permitir todas las ratas para adaptarse al medio ambiente en sus jaulas durante 1 semana antes de los experimentos. Durante la adaptación, realizar las pruebas plantares von Frey y establecer líneas de base. </li><li> Prueba Frey von <ol><li> Coloque la rata en un recinto de acrílico (30 cm × 30 cm × 80 cm) durante 30 min para la habitación. </li><li> Obtener filamentos de von Frey (filamento no 11 -. 20) que tienen la misma longitud, pero variando los diámetros para proporcionar una gamade las fuerzas de 2-100 g. </li><li> Para evaluar el umbral de retirada de la pata en la pata trasera de la rata, usar filamentos de von Frey para estimular el centro de las patas traseras a través de un puerto en forma de red en la placa de acrílico en intervalos entre ensayos 5 min. Para registrar la presión máxima aplicada, utilizar los filamentos en orden ascendente, de abajo hacia arriba, hasta que se registró la presión máxima aplicada. 10 </li><li> Cuando las ratas exhiben una respuesta de retirada de la pata a la estimulación, registrar el número de filamentos. Determinar la respuesta de retirada en este ensayo de acuerdo con el estímulo más bajo. </li><li> Repetir el mismo procedimiento inmediato, para un total de tres veces consecutivas en la misma rata. Convertir el número de filamentos en la fuerza correspondiente (en gramos) y el promedio de los valores. </li></ol></li><li> Prueba Plantar <ol><li> Coloque la rata en una caja de plexiglás transparente (dividido en cuatro cuadros, 80 cm × 30 cm × 15 cm) durante 30 minutos para la habitación. </li><li> Use un infhaz rared para estimular el centro de la pata trasera a través de una placa de vidrio. Ajustar la intensidad de la luz infrarroja para obtener una latencia media de respuesta de retirada de la pata de aproximadamente 10 segundos. Llevar a cabo un ensayo oprimiendo una tecla que activa la fuente de luz infrarroja y se inicia un temporizador de estado sólido digital. Manipular la duración del haz de luz infrarroja. </li><li> Registrar la duración de la luz infrarroja cuando las ratas exhiben una respuesta de retirada de la pata. La duración más larga no debe exceder de 20 segundos en cada ensayo para evitar el daño tisular. Utilice un intervalo entre ensayos de al menos 5 min para evitar la estimulación sucesiva. </li><li> Repita la prueba con tres ensayos para las patas traseras izquierda y derecha, y calcular el promedio de cada pata trasera de cada rata. </li></ol></li><li> Cirugía Lesión colagenasa <ol><li> Anestesiar la rata con isoflurano al 4% hasta la pérdida de la respuesta del dedo del pie para los dedos y las respuestas somáticas que producen estímulos quirúrgicos. Mantener la anestesia con 1,5 - 2%, isoflurane para la duración de la cirugía. </li><li> Coloque la rata en un dispositivo estereotáxico con una simple resistencia de calentamiento para mantener la temperatura corporal a 36,5 - 37,5 ° C. Aplicar crema para los ojos en los ojos para evitar la sequedad mientras que bajo anestesia. </li><li> Afeitarse el pelo con una maquinilla eléctrica esterilizados, y hacer una incisión suave (aproximadamente 2-2,5 cm) con un bisturí a lo largo de la línea media del cuero cabelludo. Limpiar la piel y el cráneo con la alternancia de yodo providencia y la solución de alcohol al 75% para la desinfección. Durante la fase quirúrgica, utilice% de alcohol 75 para esterilizar todos los dispositivos, herramientas, y la mesa de trabajo para mantener las condiciones estériles. Antes de eso, todos los materiales quirúrgicos deben esterilizarse en un autoclave de vapor. </li><li> Utilice los guantes e instrumentos estériles, y perfore un agujero pequeño (3 mm de diámetro) en el cráneo con un taladro eléctrico sobre la VB (incluyendo ventral núcleo posteromedial del tálamo [VPM] y ventral núcleo talámico posterolateral [VPL]) del tálamo (3,0 -3,5 mm posterior, 3.0-3.5 mm lateralal bregma, y ​​5.5 a 5.8 mm de profundidad desde la superficie del cráneo). </li><li> Microinyectar 0,5 l de solución salina normal o 0.125 U de solución de colagenasa de tipo 4 en el grupo control y experimental, respectivamente. </li><li> Mantenga la aguja de inyección en lugar de una 5 minutos adicionales para permitir la difusión del fármaco. </li><li> Utilizar cemento dental para llenar el agujero en el cráneo, y la sutura de la incisión. Después de la sutura de la incisión, se aplican los anestésicos analgésicos locales (lidocaína) de ungüento en la herida, y devolverlos a sus jaulas. </li><li> Después de la cirugía, por separado albergar las ratas en jaulas de plástico hasta que mantienen decúbito esternal, y mantiene caliente hasta que se recupere de la anestesia. </li></ol></li><li> Las pruebas de comportamiento después de la recuperación quirúrgica <ol><li> Después de 7 días de recuperación de la cirugía, repita los procedimientos descritos en las secciones 2.2 y 2.3 para la fase de prueba. Llevar a cabo las pruebas de comportamiento durante 4 semanas, mientras que el seguimiento de la salud y el estado de desarrollo de los animales. </li><li&gt; Controlar las condiciones sanitarias de los animales (por ejemplo, el peso corporal, la cantidad de la alimentación, y la libre circulación) entre los grupos control y experimentales para la fase posterior a la cirugía. </li></ol></li><li> Realizar la cánula de implantación con PE-50 Tubería <ol><li> Anestesiar la rata con isoflurano al 4%, y mantener la temperatura corporal a 36,5 - 37,5 ° C con una simple resistencia de calentamiento. </li><li> Con un escalpelo, cortar dos agujeros (2 cm de diámetro cada uno) en la línea media de la parte dorsal de las extremidades anteriores y la intersección de la parte ventral del hombro izquierdo y la cavidad torácica, respectivamente. </li><li> Disociar la piel y el músculo con un par de tijeras entre los dos orificios. </li><li> Conecte un extremo del tubo de PE 50 (20 cm de longitud) a la vena yugular externa a través del orificio ventral. Conectar el extremo terminal de la misma PE- 50 tubo en el orificio dorsal, y colocar sobre la piel. </li><li> Utilice una jeringa para inyectar solución salina en la vena yugular para verificar que el tubo PE- 50no está obstruido. </li><li> Suturar la incisión, e inyectar las ratas con 6 mg / kg de gentamicina (por vía intraperitoneal). </li><li> Enjuague el tubo cada dos días después de la cirugía con 0,3 ml de 0,9% de solución salina, seguido de 0,1 ml de solución salina con 20 U de heparina / ml. </li></ol></li><li> Prueba de comportamiento final <ol><li> Una semana después del paso 2.6, repita los pasos 2.2-2.3 para confirmar que el comportamiento es estable en comparación con el paso 2.5 después de la recuperación quirúrgica. </li></ol></li><li> La inyección del radiotrazador <ol><li> Coloque la rata en una jaula en reposo durante 5 - 10 minutos a la adaptación. </li><li> El uso de un divisor, conecte PE-50 tubos a dos jeringas de 1 ml. Llene una jeringa con solución salina normal y llenar la otra con [14 C] -IAP solución (125 Ci / kg en un volumen de 0,3 - 0,5 ml). </li><li> Inyectar el radiotrazador en la vena yugular externa, y reemplazar la jeringa con otra jeringa llena de cloruro de potasio 3 M. </li><li> Diez segundos después de que el radiotrazador injreflexión, inyectar cloruro de 3 M de potasio en virtud de una sobredosis de isoflurano, y el sacrificio de los animales de acuerdo a los métodos de eutanasia estándar (es decir, isoflurano de un vaporizador durante 50 min) sobre la base de las directrices de la Academia Sinica Institucional Cuidado de Animales y el Utilización de Taiwán. </li><li> Después de 1 minuto, exponer el cráneo, y recorte el músculo restante. Usando unas pinzas, retire la superficie dorsal del cráneo del cerebro. Recorte los lados del cráneo usando unas pinzas. A continuación, utilizando una espátula, cortar los bulbos olfatorios y las conexiones nerviosas a lo largo de la superficie ventral del cerebro, y quitar el cerebro. </li><li> Use un compuesto de temperatura óptima de corte (OCT) para congelar el cerebro en hielo seco y metilbutano (aproximadamente -55 ° C). Almacenar el tejido cerebral en un congelador. 11, 12 </li></ol></li><li> El rebanar el cerebro <ol><li> Oriente el tejido en un micrótomo, con el cerebro posterior hacia abajo. Use un criostato para cortar el cerebro en 20 y# 181; m de espesor secciones. </li><li> Coloque las rodajas de cerebro en portaobjetos de microscopio en un criostato a -20 ° C, con un intervalo de 240 m entre cada rebanada. </li><li> Colocar los portaobjetos de microscopio y cinco papeles de filtro estándar con radiactividad se nivelan en casetes de exposición durante 3 días a -20 ° C. De acuerdo con la secuencia de las rodajas de cerebro, disponer los portaobjetos de microscopio de arriba a abajo. Por último, colocar los papeles de filtro en la parte inferior de los cassettes. 12 </li><li> Retire la pantalla de fósforo de las gavetas de la exposición, y el uso de un generador de imágenes en modo variable para leer la pantalla de fósforo y generar imágenes para mostrar [14C] captación -IAP para los cortes de cerebro. </li></ol></li></ol> Análisis 3. Datos <ol><li> Después del paso 2.9.4, ajustar las imágenes usando Statistical Parametric Mapping (SPM) y el software ImageJ. Reconstruir todas las imágenes utilizando secciones coronales en serie. Suavizar y normalizar las imágenes de acuerdo con un modelo de cerebro de rata referencia.12, 13 </li><li> Para las evaluaciones cuantitativas, mida la región de interés (ROI) de las imágenes cerebrales utilizando el software ImageJ para determinar la intensidad de la señal de píxeles, y el uso de software estadístico para el análisis. 12, 13 </li><li> Para investigar las conexiones entre los diferentes núcleos del cerebro, usar software de análisis de correlación de MATLAB para visualizar relación de radiactividad en una matriz de correlación interregional, y visualizar las matrices como mapas de colores. Por último, utilizar software para el análisis de redes Pajek. 12, 13 </li><li> Utilice 2 × 5 bidireccional análisis mixto de la varianza (ANOVA), con el grupo y semanas como factores, para comparar la duración de la tolerancia al calor en la prueba plantar y fuerza mecánica en la prueba de von Frey en los grupos sham y CPSP al inicio del estudio y semana 1 - 5. uso de un 2 × 31 ANOVA de dos vías para medir la relación de radiactividad en función del grupo y área del cerebro. Cuando sea apropiado, llevar a cabo la diferencia honestamente significativa de Tukey (HSD) de las pruebas post hoc. Californiacorrelación de Pearson coeficientes cular para evaluar las correlaciones entre todas las áreas del cerebro seleccionados en la farsa y grupos CPSP. </li></ol>

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En las pruebas de comportamiento, el grupo CPSP exhibió una reducción del umbral de retirada de la pata en la prueba de dolor térmico y la fuerza mecánica en la prueba de von Frey al inicio del estudio y las semanas 1 -. 5. Los resultados fueron consistentes con un estudio previo 14 El [14 C] -IAP método se basa en la intensidad de los píxeles de imágenes del cerebro para el análisis cuantitativo de los diferentes cortes de cerebro. Para evaluar los datos de las...

Hemorragia Stroke se ha demostrado que se producen en más de 8% de los pacientes que sufren de dolor neuropático, que se refiere al dolor posterior al accidente cerebrovascular como central (CPSP). 1-3 CPSP puede resultar de disfunción somatosensorial, induciendo de ese modo la hipersensibilidad y la alodinia. 4 Sin embargo , los mecanismos fisiopatológicos de la disfunción somatosensorial en CPSP siguen siendo inciertas. Por ejemplo, la pérdida de las sensaciones somáticas resulta de desaferenciación neuronal en la zona hemorrágica cerebral. La hiperalgesia puede ser causada por la hiperexcitabilidad de las neuronas nociceptivas centrales o desinhibición centro, 5, 6, pero los sustratos neurales que están implicados en los síntomas CPSP siguen siendo desconocidos. Algunos estudios han sugerido que la corteza prefrontal dorsolateral (CPFD), corteza cingulada anterior rostral (ACC), la amígdala, el hipocampo, gris periacueductal (PAG), la médula rostral ventromedial, y sus conexiones entre sí median el procesamiento nociceptivo. 7 adicionalLy, corteza prefrontal (córtex prefrontal medial) -amygdala circuitos, se mostró a estar implicado en la percepción relacionada con el dolor. 8 Los datos sobre los mecanismos fisiopatológicos de CPSP son diversos, y la activación de los sustratos neurales en CPSP necesita un examen más detallado. [14 C] -Iodoantipyrine (IAP) absorción se utiliza para observar indirectamente regional del flujo sanguíneo cerebral (rCBF), suponiendo una relación entre la actividad cerebral y CBF. A pesar de [14C] -IAP no puede evaluar la actividad cerebral en tiempo real, como por ejemplo con imágenes de resonancia magnética funcional (fMRI), que tiene varias ventajas. Por ejemplo, [14 C] -IAP es adecuado para medir de forma espontánea se producen eventos cerebrales durante los estados patológicos. 9 Por otra parte, [14 C] captación -IAP se mide sin anestesia. También cuesta menos que otros métodos de imagen, incluyendo fMRI y tomografía por emisión de positrones (PET). El [14 C] -IAP método ha sido sugerido para ser apropiado para measuring dolor espontáneo (por ejemplo, CPSP) que es inducido por lesiones del núcleo basal ventral (VB) del tálamo. 9 El presente protocolo describe cómo realizar la [14 C] -IAP método para evaluar la participación de los sustratos neurales de CPSP que es inducida por lesiones de la VB del tálamo en un modelo animal. La técnica ofrece una manera de determinar los mecanismos fisiopatológicos que subyacen a los síntomas CPSP en los niveles de comportamiento y neuronales.

<table><tbody><tr><td>&lt;strong&gt;Anesthetic:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Halocarbon Products Corporation&amp;nbsp;</td><td>NDC 12164-002-25</td><td>4%</td></tr><tr><td>&lt;strong&gt;Surgery&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>homeothermic blanket system</td><td>Harvard Apparatus</td><td>Model 50&amp;ndash;7079</td><td>body temperature were maintained at 36.5 - 37.5 &amp;deg;C.</td></tr><tr><td>10 &amp;micro;l micro syringe</td><td>Hamilton</td><td>80008, Model 1701SN</td><td>injected with collagenase</td></tr><tr><td>polyethylene-50 tubing</td><td>Becton, Dickinson and Company</td><td>427411</td><td>catheterized into external jugular vein</td></tr><tr><td>1 c.c syringe</td><td>Terumo Medical Products</td><td>SS-01T</td><td>injected with &lt;sup&gt;14&lt;/sup&gt;C-IAP and saline.</td></tr><tr><td>saline (Sodium Chloride 0.9 gm)</td><td>Taiwan Biotech Co., LTD.</td><td>100-130-0201</td><td>To flush the tube</td></tr><tr><td>&lt;strong&gt;Drugs&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>type 4 collagenase</td><td>Sigma</td><td>C5138-500MG</td><td>0.125 U</td></tr><tr><td>Gentamicin</td><td>Sigma</td><td>G1264-250MG</td><td>6 mg/kg</td></tr><tr><td>Heparin</td><td>Sigma</td><td>H9399</td><td>20 U/ml; 0.1 ml/day</td></tr><tr><td>&lt;sup&gt;14&lt;/sup&gt;C-iodoantipyrine (IAP)</td><td>PerkinElmer</td><td>NEC712</td><td>125 mCi/kg in 300 ml of 0.9% saline</td></tr><tr><td>Potassium chloride</td><td>Merck</td><td>1.04936.1000</td><td>3 M</td></tr><tr><td>&lt;strong&gt;Behavior system:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>von Frey esthesiometer</td><td>Fabrication Enterprises, Inc.</td><td>Baseline Tactile Monofilaments 12-1666</td><td>mechanical hyperalgesia was assessed by measuring the withdrawal response to a mechanical stimulus</td></tr><tr><td>plantar test apparatus</td><td>IITC Life Science</td><td>IITC 390G Plantar Test</td><td>Thermal hyperalgesia was assessed by measuring the hind paw withdrawal latency in response to radiant heat.</td></tr><tr><td>&lt;strong&gt;Brain slice:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Optimal Cutting Temperature compound</td><td>Sakura Fintek Inc</td><td>4583</td><td>embedded the brain</td></tr><tr><td>dry ice</td><td></td><td></td><td>frozen in dry ice/methylbutane (approximately &amp;minus;55 &amp;deg;C)</td></tr><tr><td>methylbutane</td><td>Sigma</td><td>M32631-1L</td><td>frozen in dry ice/methylbutane (approximately &amp;minus;55 &amp;deg;C)</td></tr><tr><td>Cryostat</td><td>&amp;nbsp;Leica Biosystems Nussloch GmbH, Germany</td><td>Leica CM1850</td><td>Coronal brain slice were sectioned on this machine.</td></tr><tr><td>&lt;strong&gt;Data analyze&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>exposure cassettes with a phosphor screen</td><td>Amersham Biosciences&amp;nbsp;</td><td>20 cm x 25 cm</td><td>The slices were dried on glass slides and placed alongside five standard filter papers with graded radioactivity. All of the slides were exposed to the cassettes at &amp;minus;20 &amp;deg;C.</td></tr><tr><td>&amp;gamma;-counter</td><td>Beckman Coulter</td><td>Beckman LS 6500 Liquid Scintillation Counter</td><td>To measure the radioactivity count of the filter papers.</td></tr><tr><td>Typhoon 9410 Variable Mode Imager</td><td>&amp;nbsp;GMI, Inc.</td><td>WS-S9410</td><td>To read&amp;nbsp; phosphor screen which was exposed by brain slice</td></tr><tr><td>Statistical Parametric Mapping (SPM)</td><td>Wellcome Centre for Neuroimaging</td><td>version 8</td><td>all of the brains were averaged to create the final brain template. To determine significant differences between the images in these two groups, the images were derived by subtracting the sham group from the CPSP group.</td></tr><tr><td>ImageJ</td><td>http://imagej.nih.gov/ij</td><td>version 1.46</td><td>Adjacent sections were aligned both manually and using Stack- Reg, an automated pixel-based registration algorithm in ImageJ software. All of the original three-dimensionally reconstructed brains were smoothed and normalized to the reference rat brain model.</td></tr><tr><td>Matlab</td><td>MathWorks</td><td>version 2009b</td><td>used Pearson correlation coefficients to examine the relationships between the CPSP and sham groups. An inter-regional correlation matrix was calculated across animals from each group.</td></tr><tr><td>Pajek</td><td>http://Pajek.imfm.si/</td><td>version 3.06</td><td>Graphical theoretical analysis was performed on networks defined by the above correlation matrices using Pajek software.</td></tr></tbody></table>

autoradiographic measurements of [14c]-iodoantipyrine in rat brain following central post-stroke pain

Approximately 8% of stroke patients present symptoms of central post-stroke pain (CPSP). CPSP is associated with allodynia and hypersensitivity to nociceptive stimuli. Although some studies have shown that neuropathic pain may involve the dorsolateral prefrontal cortex, rostral anterior cingulate cortex, amygdala, hippocampus, periaqueductal gray, rostral ventromedial medulla, and medial thalamus, the neural substrates and their connections that mediate CPSP remain unclear. [14C]-Iodoantipyrine (IAP) uptake can be measured to evaluate spontaneously active pain. It can be used to assess the activation of neural substrates that may be involved in CPSP in an animal model. The [14C]-IAP method in rats is less expensive to perform compared with other brain mapping techniques. The present [14C]-IAP protocol is used to measure the activation of neural substrates that are involved in CPSP that is induced by lesions of the ventral basal nucleus (VB) of the thalamus in a rodent model.

La figura 1A representa la línea de tiempo experimental. Las ratas fueron asignadas a la farsa y grupos CPSP para las pruebas de comportamiento (es decir, von Frey prueba y plantar test). El primer día del experimento sirvió como línea de base, y las pruebas se repitieron en las semanas 1 - 5. PE-50 cateterismo se realizó en la vena yugular externa en la semana 4. Se inyectó heparina (20 U / ml, 0,1 ml / día) durante las semanas 4 y 5. Cinco minutos despu...

Watch this Scientific Journal Video about Autoradiographic Measurements of [14C]-Iodoantipyrine in Rat Brain Following Central Post-Stroke Pain at JoVE.com

Autoradiographic Measurements of [14C]-Iodoantipyrine in Rat Brain Following Central Post-Stroke Pain

We present a protocol to measure [14C]-iodoantipyrine (IAP) uptake and assess the activation of neural substrates that are involved in central post-stroke pain (CPSP) in a rodent model.

Las mediciones de autorradiografía de [<sup&gt; 14</sup&gt; C] -Iodoantipyrine en cerebro de rata Siguiendo central posterior al accidente cerebrovascular Dolor

Approximately 8% of stroke patients present symptoms of central post-stroke pain (CPSP). CPSP is associated with allodynia and hypersensitivity to ...

autoradiographic-measurements-14c-iodoantipyrine-rat-brain-following

Research

JoVE Journal

Medicine

Autoradiographic Measurements of [14C]-Iodoantipyrine in Rat Brain Following Central Post-Stroke Pain

8.7K vistas.  Fo Guang University. We present a protocol to measure [14C]-iodoantipyrine (IAP) uptake and assess the activation of neural substrates that are involved in central post-stroke pain (CPSP) in a rodent model.

Video: Autoradiographic Measurements of [14C]-Iodoantipyrine in Rat Brain Following Central Post-Stroke Pain

2-Vessel Occlusion/Hypotension: A Rat Model of Global Brain Ischemia

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ischemia produces damage to specific brain regions shown to be highly sensitive to ischemia 1. Hippocampal neurons have higher sensitivity to ischemic insults compared to other cell populations, and specifically, the CA1 region of the hippocampus is particularly vulnerable to ischemia/reperfusion 2.
The design of therapeutic interventions, or study of mechanisms involved in cerebral damage, requires a model that produces damage similar to the clinical condition and in a reproducible manner. Bilateral carotid vessel occlusion with hypotension (2VOH) is a model that produces reversible forebrain ischemia, emulating the cerebral events that can occur during cardiac arrest and resuscitation. We describe a model modified from Smith et al. (1984) 2, as first presented in its current form in Sanderson, et al. (2008) 3, which produces reproducible injury to selectively vulnerable brain regions 3-6. The reliability of this model is dictated by precise control of systemic blood pressure during applied hypotension, the duration of ischemia, close temperature control, a specific anesthesia regimen, and diligent post-operative care. An 8-minute ischemic insult produces cell death of CA1 hippocampal neurons that progresses over the course of 6 to 24 hr of reperfusion, while less vulnerable brain regions are spared. This progressive cell death is easily quantified after 7-14 days of reperfusion, as a near complete loss of CA1 neurons is evident at this time.
In addition to this brain injury model, we present a method for CA1 damage quantification using a simple, yet thorough, methodology. Importantly, quantification can be accomplished using a simple camera-mounted microscope, and a free ImageJ (NIH) software plugin, obviating the need for cost-prohibitive stereology software programs and a motorized microscopic stage for damage assessment.

Bilateral carotid occlusion coupled with systemic hypotension produces global brain ischemia in the rat, resulting in damage to the hippocampus with reproducible severity. Animal subjects are impaired with predictable patterns of brain damage, they recover expediently, and mortality rates are comparatively low.

2 vasos Oclusión / Hipotensión: un modelo de rata de isquemia cerebral global

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ...

Investigación

Medicina

A Middle Cerebral Artery Occlusion Technique for Inducing Post-stroke Depression in Rats

Post-stroke depression (PSD) is the most recurrent of all psychiatric complications resulting from an ischemic stroke. A greater majority (about 60%) of all ischemic stroke patients suffer from PSD, a disorder considered to be an ischemic stroke-related precursor for increased death and degradation in health. The pathophysiology of PSD is still obscure. To study the mechanism of development and occurrence of PSD further, and to find out a therapy, we attempted to develop a new protocol that requires occluding the middle cerebral artery (MCA) via the internal carotid artery (ICA) in rats. This protocol describes a model of PSD induced in rats through the middle cerebral artery occlusion (MCAO). Also used in the experiment are the Porsolt forced swim test and the sucrose preference test to confirm and evaluate the depressive mood of the rats under investigation. Rather than inserting the catheter through the external carotid artery (ECA), as stipulated for the original procedure, this MCAO technique has the monofilament passing directly through the ICA. This MCAO technique was developed a few years ago and leads to a reduction in mortality and variability. It is generally accepted that the criteria used are preferred in the selection of biological models. The data obtained with this protocol show that this model of MCAO could be a way of inducing PSD in rats and could potentially lead to the understanding of the pathophysiology and the future development of new drugs and other neuroprotective agents.

Here we present a protocol to induce post-stroke depression in rats by occluding the middle cerebral artery via the internal carotid artery. We use the Porsolt forced swim test and the sucrose preference test to confirm and evaluate induced depressive moods.

Una técnica de oclusión de la arteria cerebral media para inducir la depresión post-accidente cerebrovascular en ratas

Post-stroke depression (PSD) is the most recurrent of all psychiatric complications resulting from an ischemic stroke. A greater majority (about 60%) of ...

Comportamiento

A Dual Tracer PET-MRI Protocol for the Quantitative Measure of Regional Brain Energy Substrates Uptake in the Rat

We present a method for comparing the uptake of the brain&#39;s two key energy substrates: glucose and ketones (acetoacetate [AcAc] in this case) in the rat. The developed method is a small-animal positron emission tomography (PET) protocol, in which&#160;11C-AcAc and 18F-fluorodeoxyglucose (18F-FDG) are injected sequentially in each animal. This dual tracer PET acquisition is possible because of the short half-life of 11C (20.4 min). The rats also undergo a magnetic resonance imaging (MRI) acquisition seven days before the PET protocol. Prior to image analysis, PET and MRI images are coregistered to allow the measurement of regional cerebral uptake (cortex, hippocampus, striatum, and cerebellum). A quantitative measure of 11C-AcAc and 18F-FDG brain uptake (cerebral metabolic rate; &#956;mol/100 g/min) is determined by kinetic modeling using the image-derived input function (IDIF) method. Our new dual tracer PET protocol is robust and flexible; the two tracers used can be replaced by different radiotracers to evaluate other processes in the brain. Moreover, our protocol is applicable to the study of brain fuel supply in multiple conditions such as normal aging and neurodegenerative pathologies such as Alzheimer&#39;s and Parkinson&#39;s diseases.

Small-animal positron emission tomography enables the assessment of the brain&#39;s two main energy substrates:&#160;glucose and ketones. In the present method, 11C-acetoacetate and 18F-fluorodeoxyglucose are injected sequentially in each animal, and their uptake is measured quantitatively in specific brain regions determined from the magnetic resonance images.

Un Protocolo de PET-MRI Dual trazador para la medición cuantitativa de la Regional del cerebro Energía Sustratos captación en la rata

We present a method for comparing the uptake of the brain&#39;s two key energy substrates: glucose and ketones (acetoacetate [AcAc] in this case) in the ...

Neurociencias

Measurement of Glutamate Uptake using Radiolabeled L-[3H]-Glutamate in Acute Transverse Slices Obtained from Rodent Resected Hippocampus

Glutamate removal from the extracellular space by high-affinity Na+-dependent transporters is essential to ensure that the brain&#39;s intrinsic connectivity mechanisms work properly and homeostasis is maintained. The hippocampus is a unique brain structure that manages higher cognitive functions, and is the subject of several studies regarding neurologic diseases. The investigation of physiological and pathological mechanisms in rodent models can benefit from acute hippocampal slice (AHS) preparations. AHS has the advantage of providing reliable information on cell function since the cytoarchitecture and synaptic circuits are preserved. Although AHS preparations are commonly used in neurochemistry laboratories, it is possible to find some methodological differences in the literature. Considering that distinctive slice preparation protocols might change the hippocampal regions analyzed, this current protocol proposes a standard technique for obtaining transverse AHS from resected hippocampus. This simple-to-perform protocol may be used in mice and rats&#39; experimental models and allow several ex vivo approaches investigating neurochemical dynamics (in dorsal, intermediate and ventral hippocampus) in different backgrounds (e.g., transgenic manipulations) or after in vivo manipulations (e.g., pharmacological treatments or suitable rodent models to study clinical disorders). After dissecting the hippocampus from the rodent brain, transverse slices along the septo-temporal axis (300 &#181;m thick) were obtained. These AHS contain distinct parts of the hippocampus and were subjected to an individual neurochemical investigation (as an example: neurotransmitter transporters using their respective substrates). As the hippocampus presents a high density of excitatory synapses, and glutamate is the most important neurotransmitter in the brain, the glutamatergic system is an interesting target for in vivo observed phenomena. Thus, the current protocol provides detailed steps to explore glutamate uptake in ex vivo AHS using L-[3H]-Glutamate. Using this protocol to investigate hippocampal function may help to better understand the influence of glutamate metabolism on mechanisms of neuroprotection or neurotoxicity.

Measurement of Glutamate Uptake using Radiolabeled L-[3H]-Glutamate in Acute Transverse Slices Obtained from Rodent Resected Hippocampus

This article describes a reliable and simple way to obtain ex vivo acute hippocampal transverse slices from mice and rats using a tissue chopper. Slices obtained from resected hippocampi can be submitted for functional glutamate uptake analysis to investigate glutamatergic system homeostasis.

Glutamate removal from the extracellular space by high-affinity Na+-dependent transporters is essential to ensure that the brain&#39;s intrinsic ...

Bioquímica

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to return to normal in a large majority of patients2, which is mainly due to current lack of clinical treatment for acute stroke. This necessitates understanding the physiological effects of cerebral ischemia on brain tissue over time and is a major area of active research. Towards this end, experimental progress has been made using rats as a preclinical model for stroke, particularly, using non-invasive methods such as 18F-fluorodeoxyglucose (FDG) coupled with Positron Emission Tomography (PET) imaging3,10,17. Here we present a strategy for inducing cerebral ischemia in rats by middle cerebral artery occlusion (MCAO) that mimics focal cerebral ischemia in humans, and imaging its effects over 24 hr using FDG-PET coupled with X-ray computed tomography (CT) with an Albira PET-CT instrument. A VOI template atlas was subsequently fused to the cerebral rat data to enable a unbiased analysis of the brain and its sub-regions4. In addition, a method for 3D visualization of the FDG-PET-CT time course is presented. In summary, we present a detailed protocol for initiating, quantifying, and visualizing an induced ischemic stroke event in a living Sprague-Dawley rat in three dimensions using FDG-PET.

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Brain damage resulting from cerebral ischemia may be non-invasively imaged and studied in rats using pre-clinical positron emission tomography coupled with the injectable radioactive probe, 18F-fluorodeoxyglucose. Further, the use of modern software tools that include volume of interest (VOI) brain templates dramatically increase the quantitative information gleaned from these studies.

De imagen no invasivas y Análisis de la isquemia cerebral en ratas vivas utilizando Tomografía por emisión de positrones con<sup&gt; 18</sup&gt; F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to ...

Intracranial Pharmacotherapy and Pain Assays in Rodents

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the development of effective therapeutics. Intracranial pharmacology presents an important tool for understanding the molecular and cellular mechanisms of pain in the brain, as well as for novel treatments. Here we present a protocol that integrates intracranial pharmacology with pain behavior testing. Specifically, we show how to infuse analgesic drugs into a select brain region, which may be responsible for pain modulation. Furthermore, to determine the effect of the candidate drug in the central nerve system, pain assays are performed after intracranial treatment. Our results demonstrate that intracranial administration of analgesic drugs in a targeted region can provide relief of pain in rodents. Thus, our protocol successfully demonstrates that intracranial pharmacology, combined with pain behavior testing, can be a powerful tool for the study of pain mechanisms in the brain.

Here we present a protocol to perform intracranial pharmacological experiments followed by pain behavior assays in rodents. This protocol allows researchers to deliver molecular and cellular targets in the brain, for pharmacologic agents in the treatment of pain.

Análisis de dolor en roedores y farmacoterapia intracraneal

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the ...

Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it is thought that dysregulation of protein synthesis in the nervous system may be a core phenotype in some developmental disorders. Accurate measurement of rates of cerebral protein synthesis in animal models is important for understanding these disorders. The method that we have developed was designed to be applied to the study of awake, behaving animals. It is a quantitative autoradiographic method, so it can yield rates in all regions of the brain simultaneously. The method is based on the use of a tracer amino acid, L-[1-14C]-leucine, and a kinetic model of the behavior of L-leucine in the brain. We chose L-[1-14C]-leucine as the tracer because it does not lead to extraneous labeled metabolic products. It is either incorporated into protein or rapidly metabolized to yield 14CO2 which is diluted in a large pool of unlabeled CO2 in the brain. The method and the model also allow for the contribution of unlabeled leucine derived from tissue proteolysis to the tissue precursor pool for protein synthesis. The method has the spatial resolution to determine protein synthesis rates in cell and neuropil layers, as well as hypothalamic and cranial nerve nuclei. To obtain reliable and reproducible quantitative data, it is important to adhere to procedural details. Here we present the detailed procedures of the quantitative autoradiographic L-[1-14C]-leucine method for the determination of regional rates of protein synthesis in vivo.

Protein synthesis is&#160;a critical biological process for cells. In brain, it is required for adaptive changes. Measurement of rates of protein synthesis in the intact brain requires careful methodological considerations. Here we present the L-[1-14C]-leucine quantitative autoradiographic method for determination of regional rates of cerebral protein synthesis in vivo.

Método autoradiográfico cuantitativo para la determinación de las tasas regionales de síntesis de proteínas cerebrales in Vivo

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it ...

PET Imaging of Neuroinflammation Using [11C]DPA-713 in a Mouse Model of Ischemic Stroke

Neuroinflammation is central to the pathological cascade following ischemic stroke. Non-invasive molecular imaging methods have the potential to provide critical insights into the temporal dynamics and role of certain neuroimmune interactions in stroke. Specifically, Positron Emission Tomography (PET) imaging of translocator protein 18 kDa (TSPO), a marker of activated microglia and peripheral myeloid-lineage cells, provides a means to detect and track neuroinflammation in vivo. Here, we present a method to accurately quantify neuroinflammation using [11C]N,N-Diethyl-2-[2-(4-methoxyphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl]acetamide ([11C]DPA-713), a promising second generation TSPO-PET radiotracer, in distal middle cerebral artery occlusion (dMCAO) compared to sham-operated mice. MRI was performed 2 days post-dMCAO surgery to confirm stroke and define the infarct location and volume. PET/Computed Tomography (CT) imaging was carried out 6 days post-dMCAO to capture the peak increase in TSPO levels following stroke. Quantitation of PET images was conducted to assess the uptake of [11C]DPA-713 in the brain and spleen of dMCAO and sham mice to assess central and peripheral levels of inflammation. In vivo [11C]DPA-713 brain uptake was confirmed using ex vivo autoradiography.

PET Imaging of Neuroinflammation Using [11C]DPA-713 in a Mouse Model of Ischemic Stroke

Positron Emission Tomography (PET) imaging of translocator protein 18 kDa (TSPO) provides a non-invasive means to visualize the dynamic role of neuroinflammation in the development and progression of brain diseases. This protocol describes TSPO-PET and ex vivo autoradiography to detect neuroinflammation in a mouse model of ischemic stroke.

Animal doméstico proyección de imagen de neuroinflamación usando [11C] DPA-713 en un modelo murino de ictus isquémico

Neuroinflammation is central to the pathological cascade following ischemic stroke. Non-invasive molecular imaging methods have the potential to provide ...

Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The method is based on the specific binding of a radioligand to its biological target. Therefore, frozen tissue sections are incubated with radioligand solution, and the binding to the target is subsequently localized by the detection of radioactive decay, for example, by using photosensitive film or phosphor imaging plates. Resulting digital autoradiograms display remarkable spatial resolution, which enables quantification and localization of radioligand binding in distinct anatomical structures. Moreover, quantification allows for the pharmacological characterization of ligand affinity by means of dissociation constants (Kd), inhibition constants (Ki) as well as the density of binding sites (Bmax) in selected tissues. Thus, the method provides information about both target localization and ligand selectivity. Here, the technique is exemplified with autoradiographic characterization of the high-affinity &#947;-hydroxybutyric acid (GHB) binding sites in mammalian brain tissue, with special emphasis on methodological considerations regarding the binding assay parameters, the choice of the radioligand and the detection method.

The method of autoradiography is routinely used to study binding of radioligands to tissue sections for determination of qualitative or quantitative pharmacology.

Autorradiografía como un método sencillo y potente para la visualización y caracterización de dianas farmacológicas

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The ...

Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke involves middle cerebral artery occlusion (MCAO). Infarct zone, brain edema and blood-brain barrier (BBB) breakdown are measured as parameters that reflect the extent of brain injury after MCAO. A significant limitation to this method is that these measurements are normally obtained in different rat brain samples, leading to ethical and financial burdens due to the large number of rats that need to be euthanized for an appropriate sample size. Here we present a method to accurately assess brain injury following MCAO by measuring infarct zone, brain edema and BBB permeability in the same set of rat brains. This novel technique provides a more efficient way to evaluate the pathophysiology of stroke.

This protocol describes a novel technique of measuring the three most important parameters of ischemic brain injury on the same set of rodent brain samples. Using only one brain sample is highly advantageous in terms of ethical and economic costs.

Medición del edema cerebral post-accidente cerebrovascular, zona infarta y descomposición de la barrera hematoencefálica en un solo conjunto de muestras cerebrales de roedores

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke ...

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