Name: autoradiographic measurements of [14c]-iodoantipyrine in rat brain following central post-stroke pain
Uploaded: 2016-07-18T15:00:00
Description: Watch this Scientific Journal Video about Autoradiographic Measurements of [14C]-Iodoantipyrine in Rat Brain Following Central Post-Stroke Pain at JoVE.com

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><p class="jove_ti...

<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 border="0" cellpadding="0" cellspacing="0" width="785">
	<tbody>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:785px;">Anesthetic:</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isoflurane</td>
			<td style="width:229px;">Halocarbon Products Corporation&#160;</td>
			<td style="width:173px;">NDC 12164-002-25</td>
			<td style="width:167px;">4%</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:785px;">Surgery</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">homeothermic blanket system</td>
			<td style="width:229px;">Harvard Apparatus</td>
			<td style="width:173px;">Model 50&#8211;7079</td>
			<td style="width:167px;">body temperature were maintained at 36.5-37.5&#176;C.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">10&#181;l micro syringe</td>
			<td style="width:229px;">Hamilton</td>
			<td style="width:173px;">80008, Model 1701SN</td>
			<td style="width:167px;">injected with collagenase</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">polyethylene-50 tubing</td>
			<td style="width:229px;">Becton, Dickinson and Company</td>
			<td style="width:173px;">427411</td>
			<td style="width:167px;">catheterized into external jugular vein</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:216px;">1 c.c syringe</td>
			<td style="width:229px;">Terumo Medical Products</td>
			<td style="width:173px;">SS-01T</td>
			<td style="width:167px;">injected with 14C-IAP and saline.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">saline (Sodium Chloride 0.9gm)</td>
			<td style="width:229px;">Taiwan Biotech Co., LTD.</td>
			<td style="width:173px;">100-130-0201</td>
			<td style="width:167px;">To flush the tube</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:785px;">Drugs:</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">type 4 collagenase</td>
			<td style="width:229px;">Sigma</td>
			<td style="width:173px;">C5138-500MG</td>
			<td style="width:167px;">0.125 U</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gentamicin</td>
			<td style="width:229px;">Sigma</td>
			<td style="width:173px;">G1264-250MG</td>
			<td style="width:167px;">6 mg/kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Heparin</td>
			<td style="width:229px;">Sigma</td>
			<td style="width:173px;">H9399</td>
			<td style="width:167px;">20 U/ml; 0.1 ml/day</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">14C-iodoantipyrine (IAP)</td>
			<td style="width:229px;">PerkinElmer</td>
			<td style="width:173px;">NEC712</td>
			<td style="width:167px;">125 mCi/kg in 300 ml of 0.9% saline</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Potassium chloride</td>
			<td style="width:229px;">Merck</td>
			<td style="width:173px;">1.04936.1000</td>
			<td style="width:167px;">3 M</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:785px;">Behavior system:</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">von Frey esthesiometer</td>
			<td style="width:229px;">Fabrication Enterprises, Inc.</td>
			<td style="width:173px;">Baseline Tactile Monofilaments 12-1666</td>
			<td style="width:167px;">mechanical hyperalgesia was assessed by measuring the withdrawal response to a mechanical stimulus</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">plantar test apparatus</td>
			<td style="width:229px;">IITC Life Science</td>
			<td style="width:173px;">IITC 390G Plantar Test</td>
			<td style="width:167px;">Thermal hyperalgesia was assessed by measuring the hind paw withdrawal latency in response to radiant heat.</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:785px;">Brain slice:</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Optimal Cutting Temperature compound</td>
			<td style="width:229px;">Sakura Fintek Inc</td>
			<td style="width:173px;">4583</td>
			<td style="width:167px;">embedded the brain</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">dry ice</td>
			<td style="width:229px;"></td>
			<td style="width:173px;"></td>
			<td style="width:167px;">frozen in dry ice/methylbutane (approximately &#8722;55&#176;C)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">methylbutane</td>
			<td style="width:229px;">Sigma</td>
			<td style="width:173px;">M32631-1L</td>
			<td style="width:167px;">frozen in dry ice/methylbutane (approximately &#8722;55&#176;C)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Cryostat</td>
			<td style="width:229px;">&#160;Leica Biosystems Nussloch GmbH, Germany</td>
			<td style="width:173px;">Leica CM1850</td>
			<td style="width:167px;">Coronal brain slice were sectioned on this machine.</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:785px;">Data analyze</td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:216px;">exposure cassettes with a phosphor screen</td>
			<td style="width:229px;">Amersham Biosciences&#160;</td>
			<td style="width:173px;">20 cm x 25 cm</td>
			<td style="width:167px;">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 &#8722;20&#176;C.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">&#947;-counter</td>
			<td style="width:229px;">Beckman Coulter</td>
			<td style="width:173px;">Beckman LS 6500 Liquid Scintillation Counter</td>
			<td style="width:167px;">To measure the radioactivity count of the filter papers.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Typhoon 9410 Variable Mode Imager</td>
			<td style="width:229px;">&#160;GMI, Inc.</td>
			<td style="width:173px;">WS-S9410</td>
			<td style="width:167px;">To read&#160; phosphor screen which was exposed by brain slice</td>
		</tr>
		<tr height="231">
			<td height="231" style="height:231px;width:216px;">Statistical Parametric Mapping (SPM)</td>
			<td style="width:229px;">Wellcome Centre for Neuroimaging</td>
			<td style="width:173px;">version 8</td>
			<td style="width:167px;">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 height="294">
			<td height="294" style="height:294px;width:216px;">ImageJ</td>
			<td style="width:229px;">http://imagej.nih.gov/ij</td>
			<td style="width:173px;">version 1.46</td>
			<td style="width:167px;">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 height="210">
			<td height="210" style="height:210px;width:216px;">Matlab</td>
			<td style="width:229px;">MathWorks</td>
			<td style="width:173px;">version 2009b</td>
			<td style="width:167px;">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 height="126">
			<td height="126" style="height:126px;width:216px;">Pajek</td>
			<td style="width:229px;">http://Pajek.imfm.si/</td>
			<td style="width:173px;">version 3.06</td>
			<td style="width:167px;">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 ...

Research

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Medicine

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

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