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

The present study was supported by National Science Council grants to Dr. Bai-Chuang Shyu (NSC 99-2320-B-001-016-MY3, NSC 100-2311-B-001-003-MY3, and NSC 102-2320-B-001-026-MY3). This work was conducted at the Institute of Biomedical Sciences, which received funding from Academia Sinica.

The protocol in the present study received approval from the Academia Sinica Institutional Animal Care and Utilization Committee in Taiwan.
1. Animal Preparations
<ol>
	<li>Obtain male Sprague-Dawley rats (approximately 300 - 400 g). Maintain the rats in an air-conditioned room (21 - 22 &#176;C, 50% humidity) under a 12 hr/12 hr light/dark cycle (lights on at 8:00 AM) with free access to food and water.</li>
</ol>
2. Experimental Procedure
<ol>
	<li>Allow all of the...

<ol>
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In the behavioral tests, the CPSP group exhibited reductions of the paw withdrawal threshold in the thermal pain test and mechanical force in the von Frey test at baseline and weeks 1 - 5. The findings were consistent with a previous study.14
The [14C]-IAP method relies on the pixel intensity of brain images for the quantitative analysis of different brain slices. To evaluate the data in the brain images, the pixel signal intensity was defined. In the present study, the e...

Stroke hemorrhage has been shown to occur in more than 8% of patients who suffer from neuropathic pain, referred to as central post-stroke pain (CPSP).1-3 CPSP can result from somatosensory dysfunction, thereby inducing hypersensitivity and allodynia.4 However, the pathophysiological mechanisms of somatosensory dysfunction in CPSP remain uncertain. For example, the loss of somatic sensations results from neuronal deafferentation in the hemorrhagic brain area. Hyperalgesia may be caused by the hyperexcitability of central nociceptive neurons or central disinhibition,5, 6 but the neural substrates that are involved in CPSP symptoms remain unknown. Some studies have suggested that the dorsolateral prefrontal cortex (dPFC), rostral anterior cingulate cortex (ACC), amygdala, hippocampus, periaqueductal gray (PAG), rostral ventromedial medulla, and their connections with each other mediate nociceptive processing.7 Additionally, medial prefrontal cortex (mPFC)-amygdala circuits were shown to be involved in pain-related perception.8 Data on the pathophysiological mechanisms of CPSP are diverse, and the activation of neural substrates in CPSP needs further scrutiny.
[14C]-Iodoantipyrine (IAP) uptake is used to indirectly observe regional cerebral blood flow (rCBF), assuming a relationship between brain activity and CBF. Although [14C]-IAP cannot assess brain activity in real time, such as with functional magnetic resonance imaging (fMRI), it has several advantages. For example, [14C]-IAP is suitable for measuring spontaneously occurring brain events during pathological states.9 Moreover, [14C]-IAP uptake is measured without anesthesia. It also costs less than other imaging methods, including fMRI and positron emission tomography (PET). The [14C]-IAP method has been suggested to be appropriate for measuring spontaneous pain (e.g., CPSP) that is induced by lesions of the ventral basal nucleus (VB) of the thalamus.9
The present protocol describes how to perform the [14C]-IAP method to assess the involvement of neural substrates of CPSP that is induced by lesions of the VB of the thalamus in an animal model. The technique offers a way of determining the pathophysiological mechanisms that underlie CPSP symptoms at the behavioral and neuronal levels.

<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.

Figure 1A depicts the experimental timeline. Rats were assigned to the sham and CPSP groups for the behavioral tests (i.e., von Frey test and plantar test). The first day of the experiment served as baseline, and tests were repeated at weeks 1 - 5. PE-50 catheterization was performed in the external jugular vein at week 4. Heparin (20 U/ml, 0.1 ml/day) was injected during weeks 4 and 5. Five minutes after the heparin injection, [14C]-IAP was injected, ...

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.

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 ...

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