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>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+post-stroke+pain+syndrome:+yet+another+use+for+gabapentin?.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allodynia+in+patients+with+post-stroke+central+pain+(CPSP)+studied+by+statistical+quantitative+sensory+testing+within+individuals.">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. 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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 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 ...

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

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

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The approach allows unbiased identification and functional characterization of individual colorectal afferents. Extracellular recordings of propagated action potentials (APs) that originate from one or a few afferent (i.e., single-fiber) receptive fields (RFs) in the colorectum are made from teased nerve fiber fascicles. The colorectum is removed with either the pelvic (PN) or lumbar splanchnic (LSN) nerve attached and opened longitudinally. The tissue is placed in a recording chamber, pinned flat and perfused with oxygenated Krebs solution. Focal electrical stimulation is used to locate the colorectal afferent endings, which are further tested by three distinct mechanical stimuli (blunt probing, mucosal stroking and circumferential stretch) to functionally categorize the afferents into five mechanosensitive classes. Endings responding to none of these mechanical stimuli are categorized as mechanically-insensitive afferents (MIAs). Both mechanosensitive and MIAs can be assessed for sensitization (i.e., enhanced response, reduced threshold, and/or acquisition of mechanosensitivity) by localized exposure of RFs to chemicals (e.g., inflammatory soup (IS), capsaicin, adenosine triphosphate (ATP)). We describe the equipment and colorectum&#8211;nerve recording preparation, harvest of colorectum with attached PN or LSN, identification of RFs in the colorectum, single-fiber recording from nerve fascicles, and localized application of chemicals to the RF. In addition, challenges of the preparation and application of standardized mechanical stimulation are also discussed.

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation.

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The ...

The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The current treatments for OA pain such as NSAIDS or opiates are neither sufficiently effective nor devoid of detrimental side effects. Animal models of OA are being developed to improve our understanding of OA-related pain mechanisms and define novel pharmacological targets for therapy. Currently available models of OA in rodents include surgical and chemical interventions into one knee joint. The monoiodoacetate (MIA) model has become a standard for modelling joint disruption in OA in both rats and mice. The model, which is easier to perform in the rat, involves injection of MIA into a knee joint that induces rapid pain-like responses in the ipsilateral limb, the level of which can be controlled by injection of different doses. Intra-articular injection of MIA disrupts chondrocyte glycolysis by inhibiting glyceraldehyde-3-phosphatase dehydrogenase and results in chondrocyte death, neovascularization, subchondral bone necrosis and collapse, as well as inflammation. The morphological changes of the articular cartilage and bone disruption are reflective of some aspects of patient pathology. Along with joint damage, MIA injection induces referred mechanical sensitivity in the ipsilateral hind paw and weight bearing deficits that are measurable and quantifiable. These behavioral changes resemble some of the symptoms reported by the patient population, thereby validating the MIA injection in the knee as a useful and relevant pre-clinical model of OA pain.
The aim of this article is to describe the methodology of intra-articular injections of MIA and the behavioral recordings of the associated development of hypersensitivity with a mind to highlight the necessary steps to give consistent and reliable recordings.

Osteoarthritis (OA), or degenerative joint disease, is a debilitating condition associated with pain that remains only partially controlled by available analgesics. Animal models are being developed to improve our understanding of OA-related pain mechanisms. Here we describe the methodology for the monoiodoacetate model of OA pain in the mouse.

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The ...

Patch Angioplasty in the Rat Aorta or Inferior Vena Cava

Pericardial patches are commonly used in vascular surgery to close vessels. To facilitate studies of the neointimal hyperplasia that forms on the patch, we developed a rat model of patch angioplasty that can be used in either a vein or an artery, creating a patch venoplasty or a patch arterioplasty, respectively. Technical aspects of this model are discussed. The infra-renal IVC or aorta are dissected and then clamped proximally and distally. A 3 mm venotomy or arteriotomy is performed in the infrarenal inferior vena cava or aorta of 6 to 8 week-old Wistar rats. A bovine pericardial patch (3 mm x 1.5 mm x 0.6 mm) is then used to close the site using a 10-0 nylon suture. Compared to arterial patches, venous patches show increased neointimal thickness on postoperative day 7. This novel model of pericardial patch angioplasty can be used to examine neointimal hyperplasia on vascular biomaterials, as well as to compare the differences between the arterial and venous environments.

We have established a model of pericardial patch angioplasty that can be used in either small-diameter veins or arteries. This model can be used to compare venous and arterial neointimal hyperplasia formation.

Pericardial patches are commonly used in vascular surgery to close vessels. To facilitate studies of the neointimal hyperplasia that forms on the patch, ...

A Model of Free Tissue Transfer: The Rat Epigastric Free Flap

Free tissue transfer has been increasingly used in clinical practice since the 1970s, allowing reconstruction of complex and otherwise untreatable defects resulting from tumor extirpation, trauma, infections, malformations or burns. Free flaps are particularly useful for reconstructing highly complex anatomical regions, like those of the head and neck, the hand, the foot and the perineum. Moreover, basic and translational research in the area of free tissue transfer is of great clinical potential. Notwithstanding, surgical trainees and researchers are frequently deterred from using microsurgical models of tissue transfer, due to lack of information regarding the technical aspects involved in the operative procedures. The aim of this paper is to present the steps required to transfer a fasciocutaneous epigastric free flap to the neck in the rat.
This flap is based on the superficial epigastric artery and vein, which originates from and drain into the femoral artery and vein, respectively. On average the caliber of the superficial epigastric vein is 0.6 to 0.8 mm, contrasting with the 0.3 to 0.5 mm of the superficial epigastric artery. Histologically, the flap is a composite block of tissues, containing skin (epidermis and dermis), a layer of fat tissue (panniculus adiposus), a layer of striated muscle (panniculus carnosus), and a layer of loose areolar tissue.
Succinctly, the epigastric flap is raised on its pedicle vessels that are then anastomosed to the external jugular vein and to the carotid artery on the ventral surface of the rat's neck. According to our experience, this model guarantees the complete survival of approximately 70 to 80% of epigastric flaps transferred to the neck region. The flap can be evaluated whenever needed by visual inspection. Hence, the authors believe this is a good experimental model for microsurgical research and training.

This paper describes the steps required to raise a fasciocutaneous epigastric free flap and transfer it to the neck in the rat.

Free tissue transfer has been increasingly used in clinical practice since the 1970s, allowing reconstruction of complex and otherwise untreatable defects ...

Reproducible Motor Deficit Following Aortic Occlusion in a Rat Model Of Spinal Cord Ischemia

Spinal cord ischemia is a fatal complication following thoracoabdominal aortic aneurysm surgery. Researchers can investigate the strategies for preventing and treating this complication using experimental models of spinal cord ischemia. The model described here demonstrates varying degrees of paraplegia that relate to the length of occlusion following thoracic aortic occlusion in a rat spinal cord ischemia model.
A 2-Fr. balloon-tipped catheter was advanced through the femoral artery into the descending thoracic aorta until the catheter tip was placed at the left subclavian artery in anesthetized male Sprague-Dawley rats. Spinal cord ischemia was induced by inflating the catheter balloon. After a set period of occlusion (9, 10, or 11 min), the balloon was deflated. Neurologic assessment was performed using the motor deficit index at 24 h after surgery, and the spinal cord was harvested for histopathological examination.
Rats that underwent 9 min of aortic occlusion showed mild and reversible motor impairment in the hind limb. Rats subjected to 10 min of aortic occlusion presented with moderate but reversible motor impairment. Rats subjected to 11 min of aortic occlusion displayed complete and persistent paralysis. The motor neurons in the spinal cord sections were more preserved in rats subjected to shorter duration of aortic occlusion.
Researchers can achieve a reproducible hind limb motor deficit following thoracic aortic occlusion using this spinal cord ischemia model.

This study demonstrates the technique to make a minimally invasive and easily reproducible model of spinal cord ischemia in rats. Various degrees of hind limb motor deficit can be produced by controlling the aortic occlusion time.

Spinal cord ischemia is a fatal complication following thoracoabdominal aortic aneurysm surgery. Researchers can investigate the strategies for preventing ...

Spontaneous and Evoked Measures of Pain in Murine Models of Monoarticular Knee Pain

Pain is the main cause of disability from arthritis. There is currently an unmet need for adequate treatments for arthritis pain. Pre-clinical models are necessary and useful for studying the mechanisms of pain and for evaluating efficacy of arthritis therapies. Measuring pain in animal models of arthritis is challenging. We have developed methods for measuring evoked and spontaneous pain in three models of murine arthritis. We quantitate the evoked pain responses of mice subjected to firm palpation of a painful knee. We also evaluate spontaneous pain by the proportion of weight and the amount of time placed on each of their 4 limbs after induction of arthritis pain in one knee. Joint pain in these mouse models produces a significant increase in evoked pain responses and an alteration in weight bearing. Since mice are quadrupeds, they offload the painful limb to the contralateral limb, to the forelimbs, or some combination. These methods are simple, require minimal equipment, and are reproducible and sensitive for detecting pain. They are useful for studying both disease-modifying arthritis treatments and analgesics in mice.

We have developed an evoked measure of arthritis pain and coupled it with a standardized method for measuring spontaneous pain in different murine models of chemically induced arthritis. These measures are sensitive and reproducible for different types of joint pain.

Pain is the main cause of disability from arthritis. There is currently an unmet need for adequate treatments for arthritis pain. Pre-clinical models are ...

A New Best Practice for Validating Tail Vein Injections in Rat with Near-infrared-Labeled Agents

Intravenous (IV) administration of agents into the tail vein of rats can be both difficult and inconsistent. Optimizing tail vein injections is a key part of many experimental procedures where reagents need to be introduced directly into the bloodstream. Unwittingly, the injection can be subcutaneous, possibly altering the scientific outcomes. Utilizing a nanoemulsion-based biological probe with an incorporated near-infrared fluorescent (NIRF) dye, this method offers the capability of imaging a successful tail vein injection in vivo. With the use of a NIRF imager, images are taken before and after the injection of the agent. An acceptable IV injection is then qualitatively or quantitatively determined based on the intensity of the NIRF signal at the site of injection.

Here we present a method to validate tail vein injections in rats by utilizing near-infrared fluorescence imaging data from dyes incorporated into agents or biological probes. The tail is imaged before and after the injection, the fluorescent signal is quantified, and an assessment of the injection quality is made.

Intravenous (IV) administration of agents into the tail vein of rats can be both difficult and inconsistent. Optimizing tail vein injections is a key part ...

Blood Collection Through Subclavian Vein Puncture in Mice

The mouse is the foremost mammalian model for studying human disease and human health. However, blood sample collection from mice is challenging in research work. Tail blood collection is a popular method when a small amount of blood sample is needed. Orbital artery could be considered if a large amount of blood is needed but this blood collection method has ethical issues. Formerly, we demonstrated the feasibility and safety of blood sample collection through subclavian vein puncture in rats, and here we investigate whether this method could be used in mice. We report that this method is safe and practical for blood collection in mice. Blood collection through the subclavian vein puncture in mice can be a convenient method in daily research works.

Here, we present a protocol to take blood samples from the subclavian vein of mice.

The mouse is the foremost mammalian model for studying human disease and human health. However, blood sample collection from mice is challenging in ...

Functional and Physiological Methods of Evaluating Median Nerve Regeneration in the Rat

The main goal of this investigation is to show how to create and repair different types of median nerve (MN) lesions in the rat. Moreover, different methods of simulating postoperative physiotherapy are presented. Multiple standardized strategies are used to assess motor and sensory recovery using an MN model of peripheral nerve lesion and repair, thus permitting easy comparison of the results. Several options are included for providing a postoperative physiotherapy-like environment to rats that have undergone MN injuries. Finally, the paper provides a method to evaluate the recovery of the MN using several noninvasive tests (i.e., grasping test, pin prick test, ladder rung walking test, rope climbing test, and walking track analysis), and physiological measurements (infrared thermography, electroneuromyography, flexion strength evaluation, and flexor carpi radialis muscle weight determination). Hence, this model seems particularly appropriate to replicate a clinical scenario, facilitating extrapolation of results to the human species.
Although the sciatic nerve is the most studied nerve in peripheral nerve research, analysis of the rat MN presents various advantages. For example, there is a reduced incidence of joint contractures and automutilation of the affected limb in MN lesion studies. Furthermore, the MN is not covered by muscle masses, making its dissection easier than that of the sciatic nerve. In addition, MN recovery is observed sooner, because the MN is shorter than the sciatic nerve. Also, the MN has a parallel path to the ulnar nerve in the arm. Hence, the ulnar nerve can be easily used as the nerve graft for repairing MN injuries. Finally, the MN in rats is located in the forelimb, akin to the human upper limb; in humans, the upper limb is the site of most peripheral nerve lesions.

Presented is a protocol to produce different types of median nerve (MN) lesions and repair in the rat. Additionally, the protocol shows how to evaluate the functional recovery of the nerve using several noninvasive behavioral tests and physiological measurements.

The main goal of this investigation is to show how to create and repair different types of median nerve (MN) lesions in the rat. Moreover, different ...

Protocol for Developing a Femur Osteotomy Model in Wistar Albino Rats

Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts. Osteoanabolic drugs have the potential to augment the repair of fractures but have constraints like high costs or undesirable side effects. The bone healing potential of a drug can initially be determined by in vitro studies, but in vivo studies are needed for the final proof of concept. Our objective was to develop a femur osteotomy rodent model that could help researchers understand the development of callus formation following fracture of the shaft of the femur and that could help establish whether a potential drug has bone healing properties. Adult male Wistar albino rats were used after Institutional Animal Ethics Committee clearance. The rodents were anesthetized, and under aseptic conditions, complete transverse fractures at the middle one-third of the shafts of the femurs were created using open osteotomy. The fractures were reduced and internally fixed using intramedullary K-wires, and secondary fracture healing was allowed to take place. After surgery, intraperitoneal analgesics and antibiotics were given for 5 days. Sequential weekly x-rays assessed callus formation. The rats were sacrificed based on radiologically pre-determined time points, and the development of the fracture callus was analyzed radiologically and using immunohistochemistry.

Here, we present a protocol to iatrogenically fracture the shaft of the femur of Wistar albino rats and follow up on the development of the callus. This femur osteotomy model can help researchers evaluate the process of fracture healing and to study how a drug could influence fracture healing.

Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts. ...