The authors have received funding for research, writing, and publication of this paper from the National Natural Science Foundation of China (Nos. 82074573 and 82274675) and the Beijing Natural Science Foundation (No. 7232278).

The Committee on Animal Protection and Use of Beijing University of Chinese Medicine (BUCM-4-2022082605-3043) approved all procedures used in this study.
1. Animals and study design
<ol>
	<li>Obtain 49 male Sprague-Dawley (SD) rats of 8-week-old, weighing 200 &#177; 10 g.</li>
	<li>Using a random number table method, divide the rats into a sham operation group (n = 7), a model group (n = 7), and immediately after tuina group (n = 7), 6 h after tuina group (n = 7), 12 h after tuina group (n = 7), 18 h after tuina group (n = 7), and 24 h after tuina group (n = 7).</li>
</ol>
2. Establishment of a rat model of minor CCI (Figure 1)
NOTE: The method of modeling the minor CCI was as described in previous studies22,23,24.
<ol>
	<li>Allow adaptive feeding of SD rat for 7 days. Let the rat fast for 2-3 h before modeling.</li>
	<li>Place the rat in a box filled with 3-5% isoflurane for anesthesia by an anesthesia machine. After anesthesia, apply an ophthalmic ointment to both eyes to prevent drying.</li>
	<li>Fix the rat in a prone position on a table, shave the fur of the right hip joint area, and disinfect the area with iodine (Figure 1B).</li>
	<li>Confirm the surgical plane of anesthesia by a toe pinch. Make a skin incision of approximately 0.5-1 cm along the walking direction of the sciatic nerve, bluntly separate the muscle layer, and fully expose the lower edge of the piriformis muscle (Figure 1C).</li>
	<li>Loosely tie an absorbable chrome intestinal suture (4-0 type) around the nerve without interrupting the blood circulation of extraneural vessels. Expose the sciatic nerve of sham group rats for 3 min without ligation (Figure 1A,D,E).</li>
	<li>Use a syringe without a needle to apply 1 drop of 0.9% sodium chloride solution to the sciatic nerve to help reset it. Suture layer by layer, give an appropriate amount of anti-inflammatory drug such as Amoxicillin powder, suture the muscle layer with 1 stitch, and suture the skin with 2 stitches (Figure 1F). 
	NOTE: From step 2.3 to step 2.6, keep 2%-3% isoflurane flowing into the mouths and noses of the rats to maintain anesthesia.</li>
	<li>Keep the rat warm and wait for it to wake up. Return the rats to the breeding room.</li>
</ol>
3. Intelligent tuina manipulation simulator intervention (Figure 2)
NOTE: The treatment began on the 7th day after the model was established.
<ol>
	<li>Adjust the instrument parameters to a stimulation force of 4 N, a stimulation frequency of 60 times/min, and a temperature of 36 &#176;C on the computer operation interface. Place the rat in the rat fixator and expose the location of the hind limb acupoints (Figure 2A, self-developed machine, patent No. ZL 2023 20511277.5)25.</li>
	<li>Activate the instrument to perform the point-pressing, plucking, and kneading methods (Table 1). Click on the Point-Pressing, Plucking, and Kneading displayed on the computer screen in the sequence on the Yinmen (BL37), Chengshan (BL57), and Yanglingquan (GB34) points (Table 2) on the model side of the rat of the tuina group (Figure 2B, C)26.
	<ol>
		<li>For the tuina group, perform one intervention once a day for 1 day, with a force of 4 N, a frequency of 60 times/min, and a total of 9 min per point per method.</li>
		<li>Restrain the sham and model groups for 9 min, with the same number of times as the tuina group.</li>
	</ol>
	</li>
</ol>
4. Behavioral measurement
NOTE: After the intervention, the threshold cold sensitivity threshold (CST), mechanical withdrawal threshold (MWT), and thermal withdrawal latency (TWL) were tested in the 5 tuina subgroups, model group, and sham operation group, respectively. The testing time for the model and sham groups is the same as that for the tuina group (i.e., 24 h).
<ol>
	<li>Cold sensitivity threshold (CST)
	<ol>
		<li>Place the rat on an intelligent cold and hot plate pain detector with a surface temperature of 4 &#177; 1 &#176;C (Click on START &#62; SET), and cover it with a transparent plastic cage.</li>
		<li>Observe the rats&#39; exploratory activities for 5 min until they have been acclimated to the transparent plastic cage.</li>
		<li>Observe and record the number of foot lifts in the operative side of the hind limb within the next 5 min. 
		NOTE: The number of foot lifts caused by changes in the rat&#39;s activity or posture is not included.</li>
	</ol>
	</li>
	<li>Mechanical withdrawal threshold (MWT)
	<ol>
		<li>Place the rat on the bottom surface as a grid (0.5 cm &#215; 0.5 cm) in a test box for 15-30 min before testing.</li>
		<li>Move the probe of the pain (EVF 5 type) to the center of the right posterior plantar region of the rat, linearly increase the pressure by hand, and record the threshold displayed on the instrument screen when the rat lifts and licks its feet. Measure continuously 5 times, with a measurement interval of 10 min each.</li>
	</ol>
	</li>
	<li>Thermal withdrawal latency (TWL)
	<ol>
		<li>Place the rat in a test box with a glass bottom surface and allow it to adapt for 15 min before starting the test. 
		NOTE: The latent period of thermal contraction foot reflex was detected using a thermal stimulation pain instrument (PL200 type).</li>
		<li>Set parameters: the maximum test time is 30 s, and the intensity is 50%. Click on the START and direction buttons.</li>
		<li>Move the infrared probe to the center of the right posterior plantar region of the rat and start the detection.</li>
		<li>Record the latency of the foot retraction reflex when the rat lifts and licks its feet. Measure the latency continuously 5 times, with an interval of 10 min between each measurement.</li>
	</ol>
	</li>
</ol>
5. ELISA
<ol>
	<li>Anesthetize the rat with a dose of 35 mL/100 g of 4% chlorine hydrate for intraperitoneal injection anesthesia after behavioral testing. Cut the skin and muscles longitudinally along the middle of the rat&#39;s abdomen to expose its internal organs.</li>
	<li>Use a ballast and needle holder for pure separation, identify the abdominal aorta, take fresh blood, and keep a sample for testing. Use a separation tube to separate the serum. Take the serum sample for immediate detection, or pack it separately and store it in a refrigerator at -80 &#176; C. 
	NOTE: Before separating, allow the blood sample to agglutinate for 30 min.</li>
	<li>Prepare the serum of rats for the testing process and store them at room temperature (RT) for at least 30 min.</li>
	<li>Set up blank wells and standard sample wells for the sample to be tested. Add a total of 50 &#181;L of the standard samples to the enzyme-linked plate. Add 40 &#181;L of diluent first and then add 10 &#181;L of sample to the sample well to be tested. 
	NOTE: When adding samples, it is necessary to add them to the bottom of the enzyme-labeled well, ensuring that they do not touch the wall of the well as much as possible, and gently shake and mix well. The blank well does not contain samples or enzyme-labeled reagents, and the other steps are the same. The final dilution of the sample is 5 times.</li>
	<li>After sealing the plate with a sealing film, place it in a 37 &#176;C incubator for 30 min.</li>
	<li>Dilute 30 times the concentrated washing solution with distilled water and set it aside.</li>
	<li>Remove the sealing film, pour the liquid out from the well, and shake it dry. Fill each well with washing liquid, let it stand for 30 s, and then pour it out. Repeat this step 5 times, and finally, shake it dry.</li>
	<li>Add horseradish peroxidase-labeled avidin (100 &#181;L per well).</li>
	<li>Add 50 &#181;L of color developer A to each well, then add 50 &#181;L of color developer B. Gently shake and mix, and place it in a 37 &#176;C environment for 10 min in the dark.</li>
	<li>Add 50 &#181;L of the stop solution per well to terminate the reaction. 
	NOTE: At this point, the blue color will turn yellow.</li>
	<li>Zero the blank wells and measure the optical density (OD value) of each well in sequence at a wavelength of 450 nm.</li>
</ol>

Assessing Tuina Therapy's Pain Relief in Chronic Constriction Rats

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No conflicts of interest, financial or otherwise, are declared by the authors.

The study used the minor CCI model to simulate pNP caused by clinical sciatic nerve injury. The minor CCI model involves the continuous, chronic compression and restraint of the nerve trunk through ligation, accompanied by the gradual swelling of the ligature, resulting in edema within the sciatic nerve and forming stable chronic pain in 3-5 days27,28. In the preliminary study, the research group found that the minor CCI model group was more stable than the class...

Peripheral neuropathic pain (pNP) refers to pain caused by a lesion or disease of the peripheral somatosensory nervous system, manifested as a series of symptoms and signs, with hyperalgesia as one of the main symptoms1,2. Hyperalgesia is a heightened experience of pain caused by a noxious stimulus, including pinprick, cold, and heat3. Large epidemiological studies have been conducted, which show pNP is the most common, with a prevalence rate of 6.9%-10% in neuropathic pain4. pNP can be caused by multiple diseases, including injury of the nerve, postherpetic neuralgia, painful diabetic polyneuropathy, multiple sclerosis, stroke, cancer, etc.5. Nowadays, the main method for treating pNP is medication, but the effect is not ideal; and the side effects are significant, which is the main reason for the high economic burden on individuals and society6.
Tuina is a green, economical, safe, and effective external treatment method of traditional Chinese medicine7. Many clinical studies have proven the analgesic effect of tuina on pNP, and basic studies have verified the immediate and cumulative analgesic effects of tuina8,9. The main cumulative analgesic mechanism of tuina is to reduce the levels of inflammatory factors and inhibit the activation of glial cells10,11. The previous research confirmed the cumulative analgesic effect of tuina and found differentially expressed genes (DEGs) in the dorsal root ganglia (DRG) and spinal dorsal horn (SDH) of rats with sciatic nerve injury after 20 times of tuina treatment, mainly related to protein binding, pressure response, and neuronal projection12. In recent studies, it has been confirmed that tuina has an immediate analgesic effect and that 1-time tuina intervention could alleviate the hyperalgesia of minor CCI rats and especially ease thermal hyperalgesia more effectively13. However, the optimal time point for the analgesic effect of tuina may vary depending on the injury sensation (cold, heat, mechanical), affecting the exploration of the initiation mechanism of tuina analgesia.
Inflammatory mediators can sensitize and activate pain receptors, leading to decreased discharge thresholds and ectopic discharges, thereby contributing to peripheral sensitization14,15. After peripheral nerve injury, TNF-&#945; is an initiator of the inflammatory response, which can promote the synthesis of inflammatory factors such as IL-10 and IL-1&#946;, causing direct tissue inflammatory injury, stimulating local nerve endings, and causing pain16,17,18. Tuina can achieve analgesic effects by reducing the expression of inflammatory factors such as TNF-&#945;, IL-10, IL-6, and IL-1&#946;19,20,21. The study selected minor CCI model rats to simulate clinical pNP and selected different time points for behavioral testing of cold, thermal, and mechanical stimulation pain after a 1-time tuina intervention by cold sensitivity threshold (CST), mechanical withdrawal threshold (MWT), thermal withdrawal latency (TWL), and choose IL-10 and TNF-&#945; in the serum by enzyme-linked immunosorbent assay (ELISA), in order to select the time point at which the analgesic effect of tuina is significant, providing a basis for the study of the initiation mechanism of tuina analgesia in the later stage.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anesthesia machine</td>
			<td>Ruiwode Life Technology Co., Ltd., Shenzhen, China</td>
			<td>R500</td>
			<td>Animal respiratory anesthesia related equipment</td>
		</tr>
		<tr>
			<td>Chromic intestinal suture</td>
			<td>Shandong Boda Medical Products Co., Ltd., China</td>
			<td>BD210903</td>
			<td>An absorbable surgical suture mainly made from collagen protein processed from the intestines of healthy young goats</td>
		</tr>
		<tr>
			<td>Electronic Von Frey instrument</td>
			<td>Bioseb, USA</td>
			<td>BIO-EVF5</td>
			<td>An instrument for detecting mechanical withdrawal threshold</td>
		</tr>
		<tr>
			<td>Intelligent cold and hot plate pain detector</td>
			<td>Anhui Zhenghua Biological Instrument Equipment Co., Ltd,China.</td>
			<td>ZH-6C</td>
			<td>An instrument for detecting cold sensitivity threshold</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Ruiwode Life Technology Co., Ltd., Shenzhen, China</td>
			<td>R510-22-10</td>
			<td>An anesthetic</td>
		</tr>
		<tr>
			<td>Multi-function full-wavelength microplate reader</td>
			<td>Molecular Devices (Shanghai) Co., Ltd.</td>
			<td>SpectraMax M2</td>
			<td>An instrument for detecting optical density (OD)</td>
		</tr>
		<tr>
			<td>Thermal analgesia device</td>
			<td>Chengdu Techman Software Co., Ltd., China</td>
			<td>PL-200</td>
			<td>An instrument for detecting thermal withdrawal latency</td>
		</tr>
	</tbody>
</table>

characteristics of pain changes in rats with nerve injury within 24 hours after one-time tuina intervention

Tuina, as an external treatment method of traditional Chinese medicine, has been proven to have an analgesic effect on peripheral neuropathic pain (pNP) in clinical and basic research. However, the optimal time point for the analgesic effect of tuina may vary according to different injury sensations, affecting the exploration of the initiation mechanism of tuina analgesia. 
The research used minor chronic constriction injury (minor CCI) model rats to simulate pNP and used the intelligent tuina manipulation simulator to simulate the three methods (point-pressing, plucking, and kneading) and three acupoints (Yinmen BL37, Chengshan BL57, and Yanglingquan GB34) for performing tuina therapy. The study evaluated the changes in pain within 24 h and the optimal time point for the efficacy of tuina analgesia in rats with minor CCI models by testing cold sensitivity threshold (CST), mechanical withdrawal threshold (MWT), and thermal withdrawal latency (TWL). Furthermore, the study evaluated IL-10 and TNF-&#945; expression changes through Elisa detection. The results show that tuina has both immediate and sustained analgesic effects. For the three different injury sensitivity thresholds of CST, MWT, TWL, and two cytokines of IL-10 and TNF-&#945;, the analgesic efficacy of tuina within 24 h after intervention is significantly different at different time points.

CST: Compared with the model group, the number of foot lifts in the group 6 h after tuina was significantly reduced, and the difference was statistically significant (P &#60; 0.05). Compared with the sham group, the number of foot lifts in the model group was significantly increased, and the difference was statistically significant (P &#60; 0.05) (Table 3, Figure 3). 
MWT: Compar...

Read this Scientific Article Protocol about Author Spotlight: Exploring the Initiation Mechanism of 'Three-Method and Three-Acupoint' Tuina Analgesia in Rats with Nerve Injury at JoVE.com

Author Spotlight: Exploring the Initiation Mechanism of 'Three-Method and Three-Acupoint' Tuina Analgesia in Rats with Nerve Injury

We present a protocol using the tuina manipulation simulator to perform the "Three-Manipulation and Three-Acupoint" tuina therapy for minor chronic constriction injury rats and evaluate the effective analgesic time points of tuina within 24 h by testing pain changes through behavioral analysis and changes in inflammatory factor expression using enzyme-linked immunosorbent assay.

Characteristics of Pain Changes in Rats with Nerve Injury Within 24 hours After One-Time Tuina Intervention

Tuina, as an external treatment method of traditional Chinese medicine, has been proven to have an analgesic effect on peripheral neuropathic pain (pNP) ...

characteristics-pain-changes-rats-with-nerve-injury-within-24-hours

Research

JoVE Journal

Neuroscience

394 Views.  Beijing University of Chinese Medicine. We present a protocol using the tuina manipulation simulator to perform the "Three-Manipulation and Three-Acupoint" tuina therapy for minor chronic constriction injury rats and evaluate the effective analgesic time points of tuina within 24 h by testing pain changes through behavioral analysis and changes in inflammatory factor expression using enzyme-linked immunosorbent assay.

Video: Author Spotlight: Exploring the Initiation Mechanism of 'Three-Method and Three-Acupoint' Tuina Analgesia in Rats with Nerve Injury

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

Quantifying Acute Changes in Renal Sympathetic Nerve Activity in Response to Central Nervous System Manipulations in Anesthetized Rats

Renal sympathetic nerve activity (RSNA) and mean arterial pressure are important parameters in cardiovascular and autonomic research; however, there are limited resources directing scientists in the techniques for measuring and analyzing these variables. This protocol describes the methods for measuring RSNA and mean arterial pressure in anesthetized rats. The protocol also includes the approaches for accessing the brain during RSNA recordings for central nervous system (CNS) manipulations. The RSNA recording technique is compatible with pharmacologic, optogenetic, or electrical stimulation of the CNS. The approach is useful when an investigator will measure short-term (min to h) autonomic responses in non-survival experiments to correlate anatomically with CNS nuclei. The approach is not intended to be used to obtain chronic (survival) recordings of RSNA in rats. Discharges in RSNA, averaged rectified RSNA, and mean arterial pressure can be quantified and analyzed further using parametric statistical tests. Methods for obtaining venous access, recording mean arterial pressure telemetrically, and brain fixation for future histological analysis are also described in the article.

Methods for measuring sympathetic and cardiovascular responses to central nervous system (CNS) manipulations are important for advancing neuroscience. This protocol was developed to assist scientists with measuring and quantifying acute changes in renal sympathetic nerve activity (RSNA) in anesthetized rats (non-survival).

Renal sympathetic nerve activity (RSNA) and mean arterial pressure are important parameters in cardiovascular and autonomic research; however, there are ...

Activity-based Training on a Treadmill with Spinal Cord Injured Wistar Rats

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) on a treadmill is widely used as a rehabilitation tool in the SCI population with many benefits and improvements to daily life. We utilize this method of activity-based task-specific training (ABT) in rodents after SCI to both elucidate the mechanisms behind such improvements and to enhance and improve upon existing clinical rehabilitation protocols. Our current goal is to determine the mechanisms underlying ABT-induced improvements in urinary, bowel, and sexual function in SCI rats after a moderate to severe level of contusion. After securing each individual animal in a custom-made adjustable vest, they are secured to a versatile body weight support mechanism, lowered to a modified three-lane treadmill and assisted in step-training for 58 minutes, once a day for 10 weeks. This setup allows for the training of both quadrupedal and forelimb-only animals, alongside two different non-trained groups. Quadrupedal-trained animals with body weight support are aided by a technician present to assist in stepping with proper hind limb placement as necessary, while forelimb-only trained animals are raised at the caudal end to ensure no hind limb contact with the treadmill and no weight-bearing. One non-trained SCI group of animals is placed in a harness and rests next to the treadmill, while the other control SCI group remains in its home cage in the training room nearby. This paradigm allows for the training of multiple SCI animals at once, thus making it more time-efficient in addition to ensuring that our pre-clinical animal model mimics the clinical representation as close as possible, particularly with respect to the body weight support with manual assistance.

This protocol demonstrates our model of activity-based locomotor treadmill training for rats with spinal cord injury (SCI). Included is both quadrupedal and forelimb-only groups, in addition to two distinct types of non-trained control groups. Investigators are able to assess training effects on SCI rats using this protocol.

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) ...

Investigating Alterations in Caecum Microbiota After Traumatic Brain Injury in Mice

Increasing evidence shows that the microbiota-gut-brain axis plays an important role in the pathogenesis of brain diseases. Several studies also demonstrate that traumatic brain injuries cause changes to the gut microbiota. However, mechanisms underlying the bidirectional regulation of the brain-gut axis remain unknown. Currently, few models exist for studying the changes in gut microbiota after traumatic brain injury. Therefore, the presented study combines protocols for inducing traumatic brain injury using a lateral fluid percussion device and analysis of caecum samples following injury for investigating alterations in the gut microbiome. Alterations of the gut microbiota composition after traumatic brain injury are determined using 16S-rDNA sequencing. This protocol provides an effective method for studying the relationships between enteric microorganisms and traumatic brain injury.

Presented here is a protocol to induce diffuse traumatic brain injury using a lateral fluid percussion device followed by the collection of the caecum content for gut microbiome analysis.

Increasing evidence shows that the microbiota-gut-brain axis plays an important role in the pathogenesis of brain diseases. Several studies also ...

Advanced Diffusion Imaging in The Hippocampus of Rats with Mild Traumatic Brain Injury

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous variability and heterogeneity (age, gender, type of trauma, other possible pathologies, etc.), animal models play a key role in unraveling factors that are limitations in clinical research. They provide a standardized and controlled setting to investigate the biological mechanisms of injury and repair following TBI. However, not all animal models mimic the diffuse and subtle nature of mTBI effectively. For example, the commonly used controlled cortical impact (CCI) and lateral fluid percussion injury (LFPI) models make use of a craniotomy to expose the brain and induce widespread focal trauma, which are not commonly seen in mTBI. Therefore, these experimental models are not valid to mimic mTBI. Thus, an appropriate model should be used to investigate mTBI. The Marmarou weight drop model for rats induces similar microstructural alterations and cognitive impairments as seen in patients who sustain mild trauma; therefore, this model was selected for this protocol. Conventional computed tomography and magnetic resonance imaging (MRI) scans commonly show no damage following a mild injury, because mTBI induces often only subtle and diffuse injuries. With diffusion weighted MRI, it is possible to investigate microstructural properties of brain tissue, which can provide more insight into the microscopic alterations following mild trauma. Therefore, the goal of this study is to obtain quantitative information of a selected region-of-interest (i.e., hippocampus) to follow up disease progression after obtaining a mild and diffuse brain injury.

The overall goal of this procedure is to obtain quantitative microstructural information of the hippocampus in a rat with mild traumatic brain injury. This is done using an advanced diffusion-weighted magnetic resonance imaging protocol and region-of-interest based analysis of parametric diffusion maps.

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous ...

Laser-Induced Brain Injury in the Motor Cortex of Rats

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) occlusion of the middle cerebral artery (MCA) using a catheter. This generally accepted technique, however, has some limitations, thereby limiting its extensive use. Stroke induction by this method is often characterized by high variability in the localization and size of the ischemic area, periodical occurrences of hemorrhage, and high death rates. Also, the successful completion of any of the transient or permanent procedures requires expertise and often lasts for about 30 minutes. In this protocol, a laser irradiation technique is presented that can serve as an alternative method for inducing and studying brain injury in rodent models.
When compared to rats in the control and MCAO groups, the brain injury by laser induction showed reduced variability in body temperature, infarct volume, brain edema, intracranial hemorrhage, and mortality. Furthermore, the use of a laser-induced injury caused damage to the brain tissues only in the motor cortex unlike in the MCAO experiments where destruction of both the motor cortex and striatal tissues is observed.
Findings from this investigation suggest that laser irradiation could serve as an alternative and effective technique for inducing brain injury in the motor cortex. The method also shortens the time for completing the procedure and does not require expert handlers.

The protocol presented here shows a technique to create a rodent model of brain injury. The method described here uses laser irradiation and targets motor cortex.

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) ...

Purification of Fibroblasts and Schwann Cells from Sensory and Motor Nerves in Vitro

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and motor phenotypes involved in different patterns of neurotrophic factor gene expression and other biological processes, affecting nerve regeneration. The present study has established a protocol to obtain highly purified rat sensory and motor SCs and fibroblasts more rapidly. The ventral root (motor nerve) and the dorsal root (sensory nerve) of neonatal rats (7-days-old) were dissociated and the cells were cultured for 4-5 days, followed by isolation of sensory and motor fibroblasts and SCs by combining differential digestion and differential adherence methods sequentially. The results of immunocytochemistry and flow cytometry analyses showed that the purity of the sensory and motor SCs and fibroblasts were &#62;90%. This protocol can be used to obtain a large number of sensory and motor fibroblasts/SCs more rapidly, contributing to the exploration of sensory and motor nerve regeneration.

Here, we present a method to purify fibroblasts and Schwann cells from sensory and motor nerves in vitro.

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and ...

Preparation of Peripheral Nerve Stimulation Electrodes for Chronic Implantation in Rats

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. Several recent studies have demonstrated the effectiveness of chronic VNS in enhancing central nervous system plasticity to improve motor rehabilitation, extinction learning, and sensory discrimination. Construction of chronically implantable devices for use in such studies is challenging due to rats&#8217; small size, and typical protocols require extensive training of personnel and time-consuming microfabrication methods. Alternatively, commercially available implantable cuff electrodes can be purchased at a significantly higher cost. In this protocol, we present a simple, low-cost method for construction of small, chronically implantable peripheral nerve cuff electrodes for use in rats. We validate the short and long-term reliability of our cuff electrodes by demonstrating that VNS in ketamine/xylazine anesthetized rats produces decreases in breathing rate consistent with activation of the Hering-Breuer reflex, both at the time of implantation and up to 10 weeks after device implantation. We further demonstrate the suitability of the cuff electrodes for use in chronic stimulation studies by pairing VNS with skilled lever press performance to induce motor cortical map plasticity.

Existing approaches for constructing chronically implantable peripheral nerve cuff electrodes for use in small rodents often require specialized equipment and/or highly trained personnel. In this protocol we demonstrate a simple, low-cost approach for fabricating chronically implantable cuff electrodes, and demonstrate their effectiveness for vagus nerve stimulation (VNS) in rats.

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. ...

Multifactorial Assessment of Motor Behavior in Rats after Unilateral Sciatic Nerve Crush Injury

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In addition, in the research field of movement disorders, sciatic crush injury has been employed to trigger a dystonia-like phenotype in genetically predisposed DYT-TOR1A rodent models of dystonia. To achieve consistent, reproducible and comparable results after a sciatic nerve crush injury, a standardized method for inducing the nerve crush is essential, in addition to a standardized phenotypical characterization. Attention must be paid not only to the specific assortment of behavioral tests, but also to the technical requirements, the correct execution and consecutive data analysis. This protocol describes in detail how to perform a sciatic nerve crush injury and provides a behavioral test battery for the assessment of motor deficits in rats that includes the open field test, the CatWalk XT gait analysis, the beam walking task, and the ladder rung walking task.

We provide a protocol for the assessment of motor behavior via a behavioral test battery in rats after sciatic nerve crush injury.

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In ...

Burn Injury-Induced Pain and Depression-Like Behavior in Mice

Scalding water is the most common cause of burn injury in both elderly and young populations. It is one of the major clinical challenges because of the high mortality and sequelae in low- and middle-income countries. Burns frequently induce intense spontaneous pain and persistent allodynia as well as life-threatening problem. More importantly, excessive pain is often accompanied by depression, which may significantly decrease the quality of life. This article shows how to develop an animal model for the study of burn-induced pain and depression-like behavior. After anesthesia, burn injury was induced by dipping one hind paw of the mouse into hot water (65 &#176;C &plusmn; 0.5 &#176;C) for 3 s. The von Frey test and automated gait analysis were performed every 2 days after burn injury. In addition, depression-like behavior was examined using the forced swimming test, and the rota-rod test was performed to differentiate the abnormal motor function after burn injury. The main purpose of this study is to describe the development of an animal model for the study of burn injury-induced pain and depression-like behavior in mice.

A transient scald injury (65 &#176;C &plusmn; 0.5 &#176;C, 3 s) of one hind paw decreases the threshold (g) to von Frey filament stimulation of the ipsilateral side and alters gait pattern. Besides, burn injury induces depression-like behavior in the forced swimming test.

Scalding water is the most common cause of burn injury in both elderly and young populations. It is one of the major clinical challenges because of the ...