Name: Autoradiographic Measurements of 14C -Iodoantipyrine in Rat Brain Following Central Post-Stroke Pain (Video) | JoVE Journal
Uploaded: 2016-07-18T15:00:00.000+00:00
Duration: 7 min 27 s
Description: Learn autoradiographic measurements of [14C]-iodoantipyrine uptake to identify neural substrates in central post-stroke pain using a rat model. This efficient radioisotope tracer method enables cost-effective brain mapping.

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5 minutes

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 rats to adapt to the environment in their home cages for 1 week before the experiments. During adaptation, perform the von Frey and plantar tests to establish baselines.</li>
	<li>von Frey Test
	<ol>
		<li>Place the rat in an acrylic enclosure (30 cm &#215; 30 cm &#215; 80 cm) for 30 min for habitation.</li>
		<li>Obtain von Frey filaments (filament no. 11 - 20) that have the same length but varying diameters to provide a range of forces of 2 - 100 g.</li>
		<li>To assess the paw withdrawal threshold in the rat&#39;s hindpaw, use von Frey filaments to stimulate the center of the hindpaws through a net-like port on the acrylic plate at 5 min intertrial intervals. To record the maximum applied pressure, use the filaments in ascending order, from low to high, until the maximum applied pressure is recorded.10</li>
		<li>When rats exhibit a paw withdrawal response to the stimulation, record the filament number. Determine the withdrawal response in this trial according to the lowest stimulus.</li>
		<li>Repeat the same procedure immediately, for a total of three times in succession on the same rat. Convert the filament number into the corresponding force (in grams) and average the values.</li>
	</ol>
	</li>
	<li>Plantar Test
	<ol>
		<li>Place the rat in a transparent Plexiglas box (divided into four frames, 80 cm &#215; 30 cm &#215; 15 cm) for 30 min for habitation.</li>
		<li>Use an infrared beam to stimulate the center of the hindpaw through a glass plate. Adjust the infrared light intensity to obtain an average paw withdrawal response latency of approximately 10 sec. Conduct a trial by depressing a key that turns on the infrared light source and starts a digital solid-state timer. Manipulate the duration of the infrared light beam.</li>
		<li>Record the duration of the infrared light when the rats exhibit a paw withdrawal response. The longest duration should not exceed 20 sec in each trial to avoid tissue damage. Use an intertrial interval of at least 5 min to avoid successive stimulation.</li>
		<li>Repeat the test with three trials for the left and right hindpaws, and calculate the average for each hindpaw for each rat.</li>
	</ol>
	</li>
	<li>Collagenase Lesion Surgery
	<ol>
		<li>Anesthetize the rat with 4% isoflurane until loss of the toe-pinch response and somatic responses to surgical stimuli occur. Maintain anesthesia with 1.5 - 2% isoflurane for the duration of surgery.</li>
		<li>Place the rat in a stereotaxic device with a simple heating pad to maintain body temperature at 36.5 - 37.5 &#176;C. Apply eye cream on the eyes to prevent dryness while under anesthesia.</li>
		<li>Shave the fur with sterilized electric clippers, and make a smooth incision (approximately 2-2.5 cm) with a scalpel along the midline of the scalp. Clean the skin and skull with alternating providence iodine and 75% alcohol solution for disinfection. During the surgical phase, use 75% alcohol to sterilize all devices, tools, and the workbench to maintain sterile conditions. Before that, all surgical materials must be sterilized in a steam autoclave.</li>
		<li>Use the sterile gloves and instruments, and drill a small hole (3 mm diameter) in the skull with an electric drill over the VB (including ventral posteromedial thalamic nucleus [VPM] and ventral posterolateral thalamic nucleus [VPL]) of the thalamus (3.0-3.5 mm posterior, 3.0-3.5 mm lateral to bregma, and 5.5-5.8 mm depth from skull surface).</li>
		<li>Microinject 0.5 &#956;l of normal saline or&#160;0.125 U of type 4 collagenase solution in the control and experimental groups, respectively.</li>
		<li>Keep the injection needle in place for an additional 5 min to allow for drug diffusion.</li>
		<li>Use dental cement to fill the hole in the skull, and suture the incision. After suture the incision, apply the local analgesic anesthetics (lidocaine ointment) to the wound, and return them to their home cages.</li>
		<li>After surgery, singly house the rats in plastic cages until they maintain sternal recumbency, and keeps warm until recovered from anesthesia.</li>
	</ol>
	</li>
	<li>Behavioral Tests after Surgical Recovery
	<ol>
		<li>After 7 days of recovery from surgery, repeat the procedures in sections 2.2 and 2.3 for the test phase. Perform the behavioral tests over 4 weeks while monitoring the health and developmental status of the animals.</li>
		<li>Monitor the animals&#39; health conditions (e.g., body weight, feeding amount, and free movement) between the control and experimental groups for the post-surgery phase.</li>
	</ol>
	</li>
	<li>Perform Cannula Implantation with PE-50 Tubing
	<ol>
		<li>Anesthetize the rat with 4% isoflurane, and maintain body temperature at 36.5 - 37.5 &#176;C with a simple heating pad.</li>
		<li>With a scalpel, cut two holes (2 cm diameter each) in the midline of the dorsal part of the forelimbs and intersection of the ventral part of the left shoulder and thoracic cavity, respectively.</li>
		<li>Dissociate the skin and muscle with a pair of scissors between the two holes.</li>
		<li>Connect one end of PE- 50 tubing (20 cm length) to the external jugular vein through the ventral hole. Connect the terminal end of the same PE- 50 tubing to the dorsal hole, and affix to the skin.</li>
		<li>Use a syringe to inject saline into the jugular vein to verify that the PE- 50 tubing is not obstructed.</li>
		<li>Suture the incision, and inject the rats with 6 mg/kg gentamycin (intraperitoneally).</li>
		<li>Flush the tubing every other day after surgery with 0.3 ml of 0.9% saline, followed by 0.1 ml of saline with 20 U/ml heparin.</li>
	</ol>
	</li>
	<li>Final Behavioral Test
	<ol>
		<li>One week after step 2.6, repeat steps 2.2-2.3 to confirm that behavior is stable compared with step 2.5 after surgical recovery.</li>
	</ol>
	</li>
	<li>Radiotracer Injection
	<ol>
		<li>Place the rat in a resting cage for 5 - 10 min for adaptation.</li>
		<li>Using a splitter, connect PE- 50 tubing to two 1 ml syringes. Fill one syringe with normal saline and fill the other with [14C]-IAP solution (125 &#181;Ci/kg in a volume of 0.3 - 0.5 ml).</li>
		<li>Inject the radiotracer into the external jugular vein, and replace the syringe with another syringe filled with 3 M potassium chloride.</li>
		<li>Ten seconds after the radiotracer injection, inject 3 M potassium chloride under an overdose of isoflurane, and sacrifice the animals according to standard euthanasia methods (i.e., isoflurane from a vaporizer for 50 min) based on the guidelines of the Academia Sinica Institutional Animal Care and Utilization Committee in Taiwan.</li>
		<li>After 1 min, expose the skull, and trim off the remaining muscle. Using rongeurs, peel away the dorsal surface of the skull from the brain. Trim away the sides of the skull using rongeurs. Next, using a spatula, cut the olfactory bulbs and nerve connections along the ventral surface of the brain, and remove the brain.</li>
		<li>Use optimal cutting temperature (OCT) compound to freeze the brain in dry ice and methylbutane (approximately -55 &#176;C). Store the brain tissue in a freezer.11, 12</li>
	</ol>
	</li>
	<li>Brain Slicing
	<ol>
		<li>Orient the tissue in a microtome, with the hindbrain facing down. Use a cryostat to slice the brain into 20 &#181;m-thick sections.</li>
		<li>Place the brain slices on microscope slides in a cryostat at -20 &#176;C, with a 240 &#181;m interval between each slice.</li>
		<li>Place the microscope slides and five standard filter papers with graded radioactivity into exposure cassettes for 3 days at -20 &#176;C. According to the sequence of the brain slices, arrange the microscope slides from top to bottom. Finally, place the filter papers in the bottom of the cassettes.12</li>
		<li>Remove the phosphor screen from the exposure cassettes, and use a variable-mode imager to read the phosphor screen and generate images to show [14C]-IAP uptake for the brain slices.</li>
	</ol>
	</li>
</ol>
3. Data Analysis
<ol>
	<li>After step 2.9.4, adjust the images using Statistical Parametric Mapping (SPM) and ImageJ software. Reconstruct all of the images using serial coronal sections. Smooth and normalize the images according to a reference rat brain model.12, 13</li>
	<li>For quantitative assessments, measure the region-of-interest (ROI) of the brain images using ImageJ software to determine the pixel signal intensity, and use statistical software for the analysis.12, 13</li>
	<li>To investigate the connections between different brain nuclei, use MATLAB correlation analysis software to display radioactivity ratio in an interregional correlation matrix, and visualize the matrices as color maps. Finally, use Pajek software for network analysis.12, 13</li>
	<li>Use 2 &#215; 5 two-way mixed analysis of variance (ANOVA), with group and weeks as factors, to compare the duration of heat tolerance in the plantar test and mechanical force in the von Frey test in the sham and CPSP groups at baseline and weeks 1 - 5. Use a 2 &#215; 31 two-way ANOVA to measure the radioactivity ratio according to group and brain area. When appropriate, conduct Tukey&#39;s Honestly Significant Difference (HSD) post hoc tests. Calculate Pearson correlation coefficients to assess correlations among all of the selected brain areas in the sham and CPSP groups.</li>
</ol>

<ol><li>Finnerup, N. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+review+of+central+neuropathic+pain+states%5BTitle%5D)+AND+%22Curr+Opin+Anaesthesiol%22%5BJournal%5D)">A review of central neuropathic pain states.</a> Curr Opin Anaesthesiol. 21 (5), 586-589 (2008).</li>
<li>Andersen, G., Vestergaard, K., Ingeman-Nielsen, M., Jensen, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Incidence+of+central+post-stroke+pain%5BTitle%5D)+AND+%22Pain%22%5BJournal%5D)">Incidence of central post-stroke pain.</a> Pain. 61 (2), 187-193 (1995).</li>
<li>Chen, B., Stitik, T. P., Foye, P. M., Nadler, S. F., DeLisa, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Central+post-stroke+pain+syndrome%3A+yet+another+use+for+gabapentin%3F%5BTitle%5D)+AND+%22Am+J+Phys+Med+Rehabil%22%5BJournal%5D)">Central post-stroke pain syndrome: yet another use for gabapentin?</a> Am J Phys Med Rehabil. 81 (9), 718-720 (2002).</li>
<li>Greenspan, J. D., Ohara, S., Sarlani, E., Lenz, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Allodynia+in+patients+with+post-stroke+central+pain+%28CPSP%29+studied+by+statistical+quantitative+sensory+testing+within+individuals%5BTitle%5D)+AND+%22Pain%22%5BJournal%5D)">Allodynia in patients with post-stroke central pain (CPSP) studied by statistical quantitative sensory testing within individuals.</a> Pain. 109 (3), 357-366 (2004).</li>
<li>Klit, H., Finnerup, N. B., Jensen, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Central+post-stroke+pain%3A+clinical+characteristics%2C+pathophysiology%2C+and+management%5BTitle%5D)+AND+%22Lancet+Neurol%22%5BJournal%5D)">Central post-stroke pain: clinical characteristics, pathophysiology, and management.</a> Lancet Neurol. 8 (9), 857-868 (2009).</li>
<li>Kumar, G., Soni, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Central+post-stroke+pain%3A+current+evidence%5BTitle%5D)+AND+%22J+Neurol+Sci%22%5BJournal%5D)">Central post-stroke pain: current evidence.</a> J Neurol Sci. 284 (1-2), 10-17 (2009).</li>
<li>Denk, F., McMahon, S. B., Tracey, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pain+vulnerability%3A+a+neurobiological+perspective%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">Pain vulnerability: a neurobiological perspective.</a> Nat Neurosci. 17 (2), 192-200 (2014).</li>
<li>Ji, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cognitive+impairment+in+pain+through+amygdala-driven+prefrontal+cortical+deactivation%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Cognitive impairment in pain through amygdala-driven prefrontal cortical deactivation.</a> J Neurosci. 30 (15), 5451-5464 (2010).</li>
<li>Jungehulsing, G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Levetiracetam+in+patients+with+central+neuropathic+post-stroke+pain%3A+a+randomized%2C+double-blind%2C+placebo-controlled+trial%5BTitle%5D)+AND+%22Eur+J+Neurol%22%5BJournal%5D)">Levetiracetam in patients with central neuropathic post-stroke pain: a randomized, double-blind, placebo-controlled trial.</a> Eur J Neurol. 20 (2), 331-337 (2013).</li>
<li>Chaplan, S. R., Bach, F. W., Pogrel, J. W., Chung, J. M., Yaksh, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+assessment+of+tactile+allodynia+in+the+rat+paw%5BTitle%5D)+AND+%22J+Neurosci+Methods%22%5BJournal%5D)">Quantitative assessment of tactile allodynia in the rat paw.</a> J Neurosci Methods. 53, 55-63 (1994).</li>
<li>Jay, T. M., Luciqnani, G., Crane, A. M., Jehle, J., Sokoloff, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measurement+of+local+cerebral+blood+flow+with+%5B14C%5Diodoantipyrine+in+the+mouse%5BTitle%5D)+AND+%22J+Cereb+Blood+Flow+Metab%22%5BJournal%5D)">Measurement of local cerebral blood flow with [14C]iodoantipyrine in the mouse.</a> J Cereb Blood Flow Metab. 8 (1), 121-129 (1988).</li>
<li>Lu, H. C., Chang, W. J., Kuan, Y. H., Huang, A. C., Shyu, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+%5B14C%5Diodoantipyrine+study+of+inter-regional+correlations+of+neural+substrates+following+central+post-stroke+pain+in+rats%5BTitle%5D)+AND+%22Mol+Pain%22%5BJournal%5D)">A [14C]iodoantipyrine study of inter-regional correlations of neural substrates following central post-stroke pain in rats.</a> Mol Pain. 11 (1), (2015).</li>
<li>Wang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+brain+activation+during+retrieval+of+visceral+pain-conditioned+passive+avoidance+in+the+rat%5BTitle%5D)+AND+%22Pain%22%5BJournal%5D)">Functional brain activation during retrieval of visceral pain-conditioned passive avoidance in the rat.</a> Pain. 152, 2746-2756 (2011).</li>
<li>Wasserman, J. K., Koeberle, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+and+characterization+of+a+hemorrhagic+rat+model+of+central+post-stroke+pain%5BTitle%5D)+AND+%22Neuroscience%22%5BJournal%5D)">Development and characterization of a hemorrhagic rat model of central post-stroke pain.</a> Neuroscience. 161 (1), 173-183 (2009).</li>
</ol>

The authors have nothing to disclose.

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><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

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, ...

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.

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

The overall goal of this experimental procedure is to assess the involvement of neural substrates of thalamic lesion induced central post-stroke pain using the radio isotope tracer method. This method can help answer key questions in which brain area involved in central post-stroke pain. The main advantage of this tactic is that it is a less expensive and more efficient to perform compared with the other brain mapping techniques.In this procedure, perform the von Frey and Plantar tests to establish baselines by placing the rat in an acrylic enclosure for 30 minutes. Obtain von Frey filaments that have the same length but varying diameters to provide a range of forces of two to 100 grams. Next, use the filaments to stimulate the center of the animal's hindpaw through a net-like port on the acrylic plate.When the rats exhibit a paw-withdrawal response to the stimulation, record the filament number. Then use ascending filaments and determine the lowest value for the withdrawal response. Repeat the test for a total of three times in succession on the same rat until the maximum applied pressure is recorded.Using a reference list, convert the filament number to the corresponding force, and then average the values. Use an infrared beam to stimulate the center of the rat's hindpaw through a glass plate. Press the red bottom of the Plantar device twice to measure the withdrawal response latency.Set the inter-trial interval to at least five minutes to avoid successive stimulation. Record the duration of the infrared light when the rat exhibits a paw-withdrawal response. The longest duration should not exceed 20 seconds in each trial to avoid tissue damage.Repeat the rest with three trials for the left and right hindpaws and calculate the average of the withdrawal responses for each hindpaw. In this step, transfer the anesthetized rat to a stereotaxic device with a heating pad to maintain its body temperature. Apply ointment to its eyes to prevent dryness while under anesthesia.Subsequently, shave the animal's head with a sterilized electric clipper. Make an incision with a scalpel along the mid-line of the scalp. Then, clean the skin and skull with alternating alcohol and povidone iodine.Afterward, sterile gloves and instruments are used. Drill a small hole with a diameter of three millimeters in the skull over the ventral basal nucleus of the thalamus. Lower the needle and micro-inject 0.5 microliters of normal saline or 0.125 units of type four collagenase solution.When it is completed, keep the injection needle in place so that the skull landmark with a dark line has been marked for identification. The injection needle allows for drug diffusion for an additional five minutes. Then, use dental cement to fill the hole in the skill and suture the incision.In this procedure, place the rat in a resting cage for five to 10 minutes for adaptation. Using a splitter, connect to two one milliliter syringes. Fill one syringe with normal saline, and the other with IAP solution.Connect a PE-50 tubing to the splitter. Then, inject the radiotracer in the external jugular vein of the animal. After that, fill the syringe with three molar potassium chloride.10 seconds after the radiotracer injection, inject potassium chloride under an overdose of isoflurane before sacrificing the animal. After one minute, remove the brain and use OCT compound to freeze it in dry ice and methylbutane. Subsequently, store the brain tissue in the freezer.To prepare brain sections, orient the brain in a microtome with the hindbrain facing down. Using a cryostat, slice the brain into 20 micrometer thick sections. Next, place the brain slices on microscope slides at negative 20 degrees Celsius.Subsequently, place the microscope slides and five standard filter papers with graded radioactivity in the exposure cassettes. According to this sequence of brain slices, arrange the microscope slides from top to bottom. Then, place the filter places in the bottom of the cassettes.Afterward, remove the phosphor screen from the exposure cassettes. Use a variable mode imager to read the phosphor screen and generate images to show the IAP uptake for the brain slices. Shown here are the regions of interest in an anatomical atlas.The ROI analysis showed that activation of infralimbic cortex, prelimbic cortex, and cingulate cortex area one was significantly higher in the CPS-P group in the right hemisphere with the exception of the ventral-basal nucleus. Differences in inter-regional correlations of rCBF were observed between the CPSP and sham groups in the right hemisphere. And pain-related neural substrates were determined by analyzing the differences in the inter-regional correlations of rCBF.The red lines indicate significant positive correlations, and the blue lines indicate significant negative correlations. this technique can be done in two hours if it is performed properly. After its development, this technique paved the way for researcher in the field of brain mapping to expose central post-stroke brain in hemorrhagic stroke.Following this procedure, other masses like FMRI can be performed in order to answer additional questions like real time neural activity and neural network. After watching this video, you should have a good understanding of how to exam which brain areas involve in the central post-stroke pain.

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Research

JoVE Journal

Medicine

Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

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.

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

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.

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

Protein synthesis is 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.

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

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.

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

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

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.

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

This protocol has the aim of monitoring in vivo myelin changes (demyelination and remyelination) by positron emission tomography (PET) imaging in an animal model of multiple sclerosis.

Positron Emission Tomography Imaging for In Vivo Measuring of Myelin Content in the Lysolecithin Rat Model of Multiple Sclerosis

The current protocol describes a rat model of middle cerebral artery occlusion/reperfusion that preserves the anatomical structure of cerebral vessels without causing damage.

A Rat Model of Middle Cerebral Artery Occlusion/Reperfusion Without Damaging the Anatomical Structure of Cerebral Vessels

Langendorff-mode isolated heart perfusion, in conjunction with 31P NMR spectroscopy, combines the fields of biochemistry and physiology into one experiment.  The protocol allows for the dynamic measurement of high energy phosphate content and turnover in the heart while concurrently monitoring physiologic function.  When performed correctly, this is a valuable technique in the assessment of cardiac energetics.

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Learn autoradiographic measurements of [14C]-iodoantipyrine uptake to identify neural substrates in central post-stroke pain using a rat model. This efficient radioisotope tracer method enables cost-effective brain mapping.

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

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