Name: autoradiographic measurements of [14c]-iodoantipyrine in rat brain following central post-stroke pain
Uploaded: 2016-07-18T15:00:00.000+00:00
Duration: 7 min 27 s
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 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+central+neuropathic+pain+states.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+central+post-stroke+pain.">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. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Levetiracetam+in+patients+with+central+neuropathic+post-stroke+pain:+a+randomized,+double-blind,+placebo-controlled+trial.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+tactile+allodynia+in+the+rat+paw.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+local+cerebral+blood+flow+with+[14C]iodoantipyrine+in+the+mouse.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+[14C]iodoantipyrine+study+of+inter-regional+correlations+of+neural+substrates+following+central+post-stroke+pain+in+rats.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+brain+activation+during+retrieval+of+visceral+pain-conditioned+passive+avoidance+in+the+rat.">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. 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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 environmental background signal was defined as 25.811 - 46.979 CPM. The signal of the [14C]-IAP 0.001 &#181;Ci filter paper was defined as 42 CPM. The pixel signal intensity was &#60; 0.001 &#181;Ci, serving as the background intensity. The pixel intensity of five filter papers was determined for 0.001, 0.01, 0.1, and 10 &#181;Ci, serving as the pixel gray scale for each of the brain images. [14C]-IAP radioactivity showed a positive correlation with pixel intensity and radioactivity count on a logarithmic scale. Therefore, the above procedure can be followed for the calibration of [14C]-IAP radioactivity to pixel intensity.
When performing the [14C]-IAP experimental protocol, some points need to be considered. For example, the external jugular vein can become blocked, and experimenters need to ensure the patency of the PE- 50 tubing with heparin every day. Additionally, the location of the lesion sites can sometimes be misplaced, resulting in nonsignificant CPSP symptoms. Before the injections, the accuracy of the injection sites and locations relative to bregma must be confirmed. The angle and volume of each injection must also be accurately determined.
Limitations of brain images also need to be considered. Distortions of the brain images can occur after exposing brain slices in the cassettes to a phosphor screen. The brain images need to be normalized to a standard brain atlas using an image analysis program to avoid potential distortions in the brain images. Furthermore, different isotopes can yield different results because of their different mechanisms and action. For example, the metabolite and mechanism of action of [18F]-fludeoxyglucose (FDG) are similar to glucose. Therefore, the [18F]-FDG images were shown to be similar to the pathway of glucose metabolism. Additionally, the half-life of [18F]-FDG is short; therefore, it must be combined with PET to generate the images. [201Tl] is suitable for assessing myocardial blood flow perfusion using single-photon emission computed tomography. Therefore, choosing a suitable isotope for assessing brain images is important.
The application of the [14C]-IAP method to assess brain activation in CPSP is less costly than other brain mapping techniques (e.g., PET and fMRI). The [14C]-IAP method is suitable for spontaneously occurring events, but it cannot be used for brain mapping in real time. The method is different from other brain mapping techniques, such as PET and fMRI. Furthermore, the present [14C]-IAP protocol can measure subtle changes in rCBF in any pathological condition.
The [14C]-IAP method can be used to test conventional pain pathways, such as the spinothalamic tract (STT), medial thalamus (MT)-ACC, and mPFC-amygdala neural circuits. The activation of each of these pathways impacts the others. The activation of these pathways in CPSP has been detailed in our previous paper.12

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

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, followed 10 sec later by an overdose of anesthetic for sacrifice. One minute later, the rats were decapitated, and OCT-embedded brain slices were made. Figure 1B depicts the rats in a surgical stereotaxic device, cannula implantation sites, and histological map of the brain slices based on a rat brain atlas. Figure 1C shows the right hole of the external jugular vein in red, the location of the PE- 50 tubing, and final experimental setup.
Figure 2A shows brain slices that were exposed in the cassettes. The phosphor screen was analyzed using a variable-mode imager. Sample and standard data for the brain slices were then analyzed. Figure 2B shows the standard autoradiographic curves. The left panel shows the relationship between image intensity (pixel/mm2) and radioactivity counts per minute (CPM), thus yielding the following predicted linear equation: Y = 44.542X + 196.24. The right panel shows that the resolution (pixel/mm2) was enhanced as the exposure time increased (in days). The optimal resolution was observed on Day 4.
Figure 3A shows the experimental setup for the plantar test, which assesses thermal pain. The CPSP group exhibited a significant decrease in the paw withdrawal threshold (i.e., less heat tolerance) compared with the sham group at baseline and weeks 1 - 5 (all p &#60;&#160; 0.05). Figure 3B shows the experimental setup for the von Frey test, which assesses mechanical pain. The CPSP group exhibited a significant decrease in mechanical force (gw) at baseline and weeks 1 - 5 (all p &#60;&#160; 0.05).
Figure 4A shows the ROIs in an anatomical atlas. The ROI analysis showed that activation of the infralimbic cortex (IL), prelimbic cortex (PrL), and cingulate cortex area 1 (Cg1) was significantly higher in the CPSP group in the right hemisphere, with the exception of the VB (Figure 4B).
Differences in inter-regional correlations of rCBF were observed between the CPSP and sham groups in the right hemisphere (Figure 4C). The matrix (Fisher&#39;s Z-statistics) of all of the regions was analyzed using Pearson&#39;s correlation. Figure 4C shows differences in inter-regional correlations of the involvement of neural substrates in the CPSP group. Pain-related neural substrates were determined by analyzing differences in inter-regional correlations of rCBF. The red lines in Figure 4D indicate significant positive correlations, and blue lines indicate significant negative correlations.
<img alt="Figure 1" src="/files/ftp_upload/53947/53947fig1.jpg" /> 
Figure 1. Experimental Timeline of Lesioning the Ventral Basal Nucleus (VB) of the Thalamus to Induce Central Post-Stroke Pain (CPSP) and Injecting [14C]-IAP. (A) Ventral basal nuclei lesions to induce CPSP for the behavioral assessments and injections of [14C]-IAP to measure the activation of neural substrates that are involved in CPSP. (B) Location of VB. (C) [14C]-IAP injections. Scale bar = 1 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53947/53947fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53947/53947fig2.jpg" /> 
Figure 2. Standard Autoradiographic Curves. (A) Sample and standard curves were obtained for different exposure times and image resolutions. (B) Standard curve of image intensity and CPM and standard curve of exposure time and resolution. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53947/53947fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53947/53947fig3.jpg" /> 
Figure 3. The Plantar Test (Thermal Pain) and von Frey Test (Mechanical Pain) were Conducted in the Sham and CPSP Groups at Baseline and Weeks 1 - 5. (A) CPSP rats exhibited a lower paw withdrawal threshold in the plantar test (i.e., less heat tolerance) compared with sham rats, indicating greater thermal pain. (B) CPSP rats exhibited a lower paw withdrawal threshold in the von Frey test compared with sham rats, indicating greater mechanical pain. SEM, standard error of the mean. Green asterisks (*) indicate a significant difference compared with the sham group. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53947/53947fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53947/53947fig4.jpg" /> 
Figure 4. ROI Analysis and Relationship among Inter-Regional Correlations Between Neural Substrates Involved in CPSP. (A) Brain areas were determined and analyzed by the ratio formulation. (B) The ROIs in the IL, PrL, Cg1, and VB were significantly different in the right hemisphere. (C) Analysis of rCBF in the selected brain areas. (D) Inter-regional correlations between brain areas. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53947/53947fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Medicine

8.7K Views.  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.

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

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.

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

Behavior

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.

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 ...

Neuroscience

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 ...

Biochemistry

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.

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.

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.

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.

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.

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.

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

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

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