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

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

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

<ol>
	<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. 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. S. <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:+clinical+characteristics,+pathophysiology,+and+management.">Central post-stroke pain: clinical characteristics, pathophysiology, and management.</a> Lancet Neurol. 8 (9), 857-868 (2009).</li><li>Kumar, G., Soni, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+post-stroke+pain:+current+evidence.">Central post-stroke pain: current evidence.</a> J Neurol Sci. 284 (1-2), 10-17 (2009).</li><li>Denk, F., McMahon, S. B., Tracey, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pain+vulnerability:+a+neurobiological+perspective.">Pain vulnerability: a neurobiological perspective.</a> Nat Neurosci. 17 (2), 192-200 (2014).</li><li>Ji, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cognitive+impairment+in+pain+through+amygdala-driven+prefrontal+cortical+deactivation.">Cognitive impairment in pain through amygdala-driven prefrontal cortical deactivation.</a> J Neurosci. 30 (15), 5451-5464 (2010).</li><li>Jungehulsing, G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/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. 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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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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.

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	<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 ...

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

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Medicine

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 ...

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 ...

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 ...

Improving FSN Therapy in the Rat Model&amp;#58; Minimizing Discomfort and Maximizing Efficiency

Efficacy of Fu's Subcutaneous Needling on Sciatic Nerve Pain: Behavioral and Electrophysiological Changes in a Chronic Constriction Injury Rat Model

Fu&#39;s subcutaneous needling (FSN), an invented acupuncture technique from traditional Chinese medicine, is used worldwide for pain relief. However, the mechanisms of action are still not fully understood. During FSN treatment, the FSN needle is inserted and retained in the subcutaneous tissues for a long duration with a swaying movement. However, challenges arise from maintaining a posture while manipulating FSN in animal models (e.g., rats) for researchers. Uncomfortable treatment can lead to fear and resistance to FSN needles, increasing the risk of injury and may even affect research data. Anesthesia may also affect the study results too. Hence, there is a need for strategies in FSN therapy on animals that minimize injury during the intervention. This study employs a chronic constriction injury model in Sprague-Dawley rats to induce neuropathic pain. This model replicates the pain induced by nerve injury observed in humans through surgical constriction of a peripheral nerve, mimicking the compression or entrapment seen in conditions such as nerve compression syndromes and peripheral neuropathies. We introduce an appropriate manipulation for easily inserting an FSN needle into the subcutaneous layer of the animal&#39;s body, including needle insertion and direction, needle retention, and swaying movement. Minimizing the rat&#39;s discomfort prevents the rat from being tense, which causes the muscle to contract and hinder the entry of the needle and improves the study efficiency.

Author Spotlight: Unveiling the Therapeutic Effects of FSN Treatment &amp;#8211; Bridging Research and Clinical Applications in Neuropathic Pain

We present a protocol for using Fu's subcutaneous needling in a chronic constriction injury model to induce sciatic nerve pain in rats.

Fu&#39;s subcutaneous needling (FSN), an invented acupuncture technique from traditional Chinese medicine, is used worldwide for pain relief. However, the ...

Standardized Tuina Protocol for Rat DRG Compression Model

Analgesic Effect of Tuina on Rat Models with Compression of the Dorsal Root Ganglion Pain

Neuropathic pain is a prevalent condition that affects 6.9%-10% of the population and results from nerve damage due to various etiologies, such as lumbar disc herniation, spinal canal stenosis, and intervertebral foramen stenosis. Although Tuina, a traditional Chinese manual therapy, has shown analgesic effects in clinical practice for the treatment of neuropathic pain, its underlying neurobiological mechanisms remain unclear. Animal models are essential for elucidating the basic principles of Tuina. In this study, we propose a standardized Tuina protocol for rats with compression of the dorsal root ganglion (DRG), which involves inducing DRG compression by inserting a stainless steel rod into the intervertebral foramen, performing Tuina manipulation with specific parameters of location, intensity, and frequency in a controlled environment, and assessing the behavioral and histopathological outcomes of Tuina treatment. This article also discusses the potential clinical implications and limitations of the study and suggests directions for future research on Tuina.

Author Spotlight: Analgesic Effect of Tuina on Rat Models with Compression of the Dorsal Root Ganglion Pain  

This article presents a manipulation for treating chronic compression of the dorsal root ganglion in rats using Tuina therapy, along with a method for evaluating its effectiveness based on pain behavior and histopathological results.

Neuropathic pain is a prevalent condition that affects 6.9%-10% of the population and results from nerve damage due to various etiologies, such as lumbar ...

Assessing Blood Hypercoagulation of EV-Rich Plasma

Determination of the Procoagulant Activity of Extracellular Vesicle (EV) Using EV-Activated Clotting Time (EV-ACT)

The role of extracellular vesicles (EV) in various diseases is gaining increased attention, particularly due to their potent procoagulant activity. However, there is an urgent need for a bedside test to assess the procoagulant activity of EV in clinical settings. This study proposes the use of thrombin activation time of EV-rich plasma as a measure of EV's procoagulant activity. Standardized procedures were employed to obtain sodium-citrated whole blood, followed by differential centrifugation to obtain EV-rich plasma. The EV-rich plasma and calcium chloride were added to the test cup, and the changes in viscoelasticity were monitored in real-time using an analyzer. The natural coagulation time of EV-rich plasma, referred to as EV-ACT, was determined. The results revealed a significant increase in EV-ACT when EV was removed from plasma obtained from healthy volunteers, while it significantly decreased when EV was enriched. Furthermore, EV-ACT was considerably shortened in human samples from preeclampsia, hip fracture, and lung cancer, indicating elevated levels of plasma EV and promotion of blood hypercoagulation. With its simple and rapid procedure, EV-ACT shows promise as a bedside test for evaluating coagulation function in patients with high plasma EV levels.

Author Spotlight: Deciphering Coagulation Disorders in Traumatic Brain Injury Patients 

This protocol investigates the use of extracellular vesicle (EV)-rich plasma as an indicator of the coagulative ability of EV. EV-rich plasma is obtained through a process of differential centrifugation and subsequent recalcification.

The role of extracellular vesicles (EV) in various diseases is gaining increased attention, particularly due to their potent procoagulant activity. ...