Thanks to the Department of Neurosurgery of the Second Affiliated Hospital of Army Medical University for site support.&#160;Funding: This work was supported by the Natural Science Foundation of China (82104696).

The experiments in this study comply with the &#34;3Rs&#34; principle of animal ethics and have been approved by the Laboratory Animal Welfare and Ethnic Committee of the Army Medical University (AMUWEC20234543).
1. Preparation of rodent body fixation device
<ol>
	<li>Design a small animal holder based on the Chinese utility model patent9.</li>
	<li>Produce the device through 3D printing technology.</li>
	<li>Prepare a 2 m long rope and divide it into 6 ropes. Thread these 6 ropes through the holes in the fixator. Utilize the spring-loaded buckle to fasten the rope ends, facilitating loop size adjustment. 
	&#8203;NOTE: The fixator could be manufactured by any company equipped with 3D printing technology based on a design drawing. The design drawing of the device is shown in Figure 1. The fixator used in this study was produced by a 3D printer by a printing method called fused deposition modeling (FDM), also known as fused filament fabrication (FFF)10. This additive manufacturing method prints the parts of the design layer by layer by selectively depositing the material in the melted form in a predetermined path. The process uses thermoplastic polymers that come in filaments to create the final physical structure. The immobilization apparatus utilized in this study can secure three animals simultaneously, necessitating six ropes, with each animal requiring a pair of ropes: one for the neck and one for the trunk.</li>
</ol>
2. Preparation of rodents
<ol>
	<li>Purchase 3 Sprague-Dawley rats (10-40 g, 7-45 days old) and 3 C57 BL/C mice (5-40 g).</li>
	<li>Breed animals in a room with a 12 h day/night cycle, an ambient temperature of 21-23 &#176;C, and a humidity of 45%-60%. Disinfect and ventilate the feeding environment regularly, and allow the animals to eat and drink freely.</li>
</ol>
3. Fixing rodents to the fixator (prone position)
<ol>
	<li>Place the head of the rodent in the U-shaped hole in front of the fixator, with the rodent in a prone position. Pass the rodent&#39;s body through two rope loops.</li>
	<li>Fasten the ropes around the neck and abdomen of the rodent, ensuring that it cannot escape, but not so tightly as to cause respiratory distress and death.
	<ol>
		<li>The key step is to fix the neck in place. Leave a gap of 2-5 mm between the rodent&#39;s neck and the rope. Ensure that the rope is not too tight for the animals to strangle or too loose to escape.</li>
	</ol>
	</li>
	<li>Fix the other rodents in the same way in the device.</li>
	<li>Repeat the above fixation steps (3.1-3.2) for 3 days to minimize the potential stress response caused by the fixation procedure. Repeat the fixation procedure for 3 consecutive days, with each session lasting 5 min per day. 
	NOTE: To ensure that rodents were not over-stressed during the fixation procedure, we repeated the procedure for 3 consecutive days. Each session lasted 5 min per day, providing ample time for mice or rats to acclimate to the procedure and minimizing any potential discomfort.</li>
	<li>Strat EA from Day 4.</li>
	<li>To prevent the spread of disease, immerse a sterile cotton swab or ball in 75% alcohol and thoroughly wipe the holder to clean and disinfect it before and after each use. Use gloves and other protective equipment to avoid potential health risks when handling rodents.</li>
</ol>
4. Performing EA in mice or young rats (MS6)
<ol>
	<li>Choose 0.25 mm x 13 mm disposable sterile acupuncture needles.</li>
	<li>Find the acupoints by anatomical location and confirm the insertion depth at the acupoint. 
	NOTE: MS6 is located on the lateral side of the head. This region in the head passes through the connection between Shencong (Ex-hn1) and Xuanli (GB6), also known as the anterior parietal (Du21). MS6 corresponds to the expression of the precentral gyrus of the cerebral cortex on the scalp. Ex-hn1 is located 0.5 cm behind the midpoint of the anterior-posterior midline of the head. GB6 is located at the midpoint of the front of the ear and the end angle of the eye. The superior supratrochlear nerve, supraorbital nerve, occipital nerve, and auriculotemporal nerve are distributed in it. MS6 is mainly used to treat stroke and encephalopathy, such as motor disorders.</li>
	<li>Slowly twist and insert sterile needles bilateral into MS6 at a depth of 3/5 from the anterior Shenchong (Exhn1) to the Xuanli (GB6) along the subcutaneous in 3 rodents, respectively.</li>
	<li>Connect one side of the connection cable to the EA instrument.</li>
	<li>Connect another side of the connection cable containing the positive and negative EA clips to the acupuncture needle shanks on the left and right MS6.</li>
	<li>Turn on the EA instrument.
	<ol>
		<li>Rotate the button (Function/ Choose Switch) to set the frequency and function mode.</li>
		<li>Set the Frequency to 10 Hz and choose a Continuous Wave mode.</li>
		<li>Rotate the corresponding button (Adjust Intensity) to set the intensity. Each adjust intensity button controls the current strength of the rodents of the wire connected below it. Adjust the current intensity to the acceptable range of rodents, usually 1-2 mA, lasting 5 min. 
		&#8203;NOTE: It is important to monitor the breathing and behavior of the rodents during the procedure to ensure that they are not experiencing any distress or discomfort. If the rat appears to be struggling or in pain, the procedure should be stopped immediately.</li>
	</ol>
	</li>
</ol>
5. Fixing rodents to the fixator (supine position)
<ol>
	<li>Place the heads of the rodent in the U-shaped hole in front of the fixator, with the rodent in a prone position. Pass the rodent&#39;s body through two rope loops.</li>
	<li>Fasten the rope around the neck and abdomen of the rodent, ensuring that it cannot escape, but not so tight as to cause respiratory distress and death. 
	NOTE: The points to be noted during fixation are the same as in step 3.2.</li>
	<li>Fix the other rodents in the same way.</li>
	<li>As in step 3.4, repeat the above fixation steps for 3 days to reduce the stress response of rodents.</li>
	<li>Strat EA from Day 4. 
	&#8203;NOTE: Disinfection is performed as mentioned in step 3.6.</li>
</ol>
6. Performing EA (ST25)
<ol>
	<li>Choose 0.25 mm x 13 mm disposable sterile acupuncture needles.</li>
	<li>Find the acupoints by anatomical location and confirm the insertion depth at the acupoint. 
	NOTE: ST25 is located at the level of the umbilicus. Positioning this point in rodents requires first measuring the length of the rodent&#39;s paw. ST25 is located at 2/3 the length of the paw next to the navel. For example, if the measured paw of the rodent is 0.6 cm, then ST25 is located 0.4 cm to the right and left of the umbilicus.</li>
	<li>Slowly twist and penetrate sterile needles into ST25 bilateral at a depth of 4-8 mm on 3 rodents, respectively.</li>
	<li>Connect one side of the connection cable to the EA instrument.</li>
	<li>Connect another side of the connection cable containing the positive and negative EA clips to the acupuncture needle shanks on the left and right ST25.</li>
	<li>Turn on the EA instrument.
	<ol>
		<li>Rotate the button (Function/ Choose Switch) to set the frequency and function mode.</li>
		<li>Set the Frequency to 10 Hz and choose Continuous Wave Mode.</li>
		<li>Rotate the corresponding button (Adjust Intensity) to set the intensity. Each adjust intensity button controls the current strength of the rat of the wire connected below it. Adjust the current intensity to the acceptable range of rodents, usually 1-2 mA, lasting 5 min. 
		NOTE: The observations were the same as in step 4.6.</li>
	</ol>
	</li>
</ol>

Using a 3D Printed Holder for Bulk Electroacupuncture in Small Animals

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<li>Xin, Y. Y., Wang, J. X., Xu, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electroacupuncture+ameliorates+neuroinflammation+in+animal+models%5BTitle%5D)+AND+%22Acupuncture+in+Medicine%3A+Journal+of+the+British+Medical+Acupuncture+Society%22%5BJournal%5D)">Electroacupuncture ameliorates neuroinflammation in animal models.</a> Acupuncture in Medicine: Journal of the British Medical Acupuncture Society. 40 (5), 474-483 (2022).</li>
<li>Mengzhu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electroacupuncture+at+Tianshu+%28ST25%29+and+Zusanli+%28ST36%29+alleviates+stress-induced+irritable+bowel+syndrome+in+mice+by+modulating+gut+microbiota+and+corticotropin-releasing+factor%5BTitle%5D)+AND+%22Journal+of+traditional+Chinese+medicine%22%5BJournal%5D)">Electroacupuncture at Tianshu (ST25) and Zusanli (ST36) alleviates stress-induced irritable bowel syndrome in mice by modulating gut microbiota and corticotropin-releasing factor.</a> Journal of traditional Chinese medicine. 42 (5), 732-740 (2022).</li>
<li>Xu, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+attempt+of+re-mapping+acupoint+atlas+in+the+rat%5BTitle%5D)+AND+%22Acupuncture+Research%22%5BJournal%5D)">A new attempt of re-mapping acupoint atlas in the rat.</a> Acupuncture Research. 44 (1), 62-65 (2019).</li>
<li>Yin, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+proposed+transpositional+acupoint+system+in+a+mouse+and+rat+model%5BTitle%5D)+AND+%22Research+in+Veterinary+Science%22%5BJournal%5D)">A proposed transpositional acupoint system in a mouse and rat model.</a> Research in Veterinary Science. 84 (2), 159-165 (2008).</li>
<li>Cao, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effectiveness+of+acupuncture+for+Parkinson%27s+disease%3A+An+overview+of+systematic+reviews%5BTitle%5D)+AND+%22Complementary+Therapies+in+Medicine%22%5BJournal%5D)">The effectiveness of acupuncture for Parkinson's disease: An overview of systematic reviews.</a> Complementary Therapies in Medicine. 50, 102383(2020).</li>
<li>Yang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+warming+moxibustion+Tianshu+%28ST+25%2C+bilateral%29+and+Qihai+%28CV+6%29+for+the+treatment+of+diarrhea-dominant+irritable+bowel+syndrome%3A+a+patient-blinded+pilot+trial+with+orthogonal+design%5BTitle%5D)+AND+%22Journal+of+Traditional+Chinese+Medicine%22%5BJournal%5D)">Effect of warming moxibustion Tianshu (ST 25, bilateral) and Qihai (CV 6) for the treatment of diarrhea-dominant irritable bowel syndrome: a patient-blinded pilot trial with orthogonal design.</a> Journal of Traditional Chinese Medicine. 37 (4), 538-545 (2017).</li>
<li>Delcour, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+movement+restriction+leads+to+maladaptive+plasticity+in+the+sensorimotor+cortex+and+to+movement+disorders%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Early movement restriction leads to maladaptive plasticity in the sensorimotor cortex and to movement disorders.</a> Scientific Reports. 8 (1), 16328(2018).</li>
<li>A fixing device for mice body. China Patent. , 202220586749 (2023).</li>
<li>Ismail, K. I., Yap, T. C., Ahmed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((3D-printed+fiber-reinforced+polymer+composites+by+fused+deposition+modelling+%28FDM%29%3A+Fiber+length+and+fiber+implementation+techniques%5BTitle%5D)+AND+%22Polymers%22%5BJournal%5D)">3D-printed fiber-reinforced polymer composites by fused deposition modelling (FDM): Fiber length and fiber implementation techniques.</a> Polymers. 14 (21), 4659(2022).</li>
<li>Lu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficacy+of+electroacupuncture+with+different+frequencies+in+the+treatment+of+chemotherapy-induced+peripheral+neuropathy%3A+A+study+protocol+for+a+randomized+controlled+trial%5BTitle%5D)+AND+%22Frontiers+in+Neurology%22%5BJournal%5D)">Efficacy of electroacupuncture with different frequencies in the treatment of chemotherapy-induced peripheral neuropathy: A study protocol for a randomized controlled trial.</a> Frontiers in Neurology. 13, 843886(2022).</li>
<li>Zhang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modulation+of+colonic+function+in+irritable+bowel+syndrome+rats+by+electroacupuncture+at+ST25+and+the+neurobiological+links+between+ST25+and+the+colon%5BTitle%5D)+AND+%22Frontiers+in+Neuroscience%22%5BJournal%5D)">Modulation of colonic function in irritable bowel syndrome rats by electroacupuncture at ST25 and the neurobiological links between ST25 and the colon.</a> Frontiers in Neuroscience. 16, 930489(2022).</li>
<li>Wang, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anti-inflammation+mechanism+of+electro-scalp+acupuncture+in+treatment+of+ischemic+stroke+based+on+IL-12+mediated+JAK%2FSTAT+signaling+pathway%5BTitle%5D)+AND+%22Chinese+Acupuncture+%26+Moxibustion%22%5BJournal%5D)">Anti-inflammation mechanism of electro-scalp acupuncture in treatment of ischemic stroke based on IL-12 mediated JAK/STAT signaling pathway.</a> Chinese Acupuncture & Moxibustion. 42 (10), 1137-1144 (2022).</li>
<li>Ren, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+design+of+a+plate+for+posterolateral+tibial+plateau+fractures+through+traditional+anterolateral+approach%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">A novel design of a plate for posterolateral tibial plateau fractures through traditional anterolateral approach.</a> Scientific Reports. 8 (1), 16418(2018).</li>
<li>Wu, L., Dong, Y., Zhu, C., Chen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+and+mechanism+of+acupuncture+on+Alzheimer%27s+disease%3A+A+review%5BTitle%5D)+AND+%22Frontiers+in+Aging+Neuroscience%22%5BJournal%5D)">Effect and mechanism of acupuncture on Alzheimer's disease: A review.</a> Frontiers in Aging Neuroscience. 15, 1035376(2023).</li>
<li>Wu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electroacupuncture+for+spinal+cord+injury%3A+A+scientific+study+of+traditional+medicine%5BTitle%5D)+AND+%22Neurospine%22%5BJournal%5D)">Electroacupuncture for spinal cord injury: A scientific study of traditional medicine.</a> Neurospine. 19 (3), 770-772 (2022).</li>
<li>Gozzi, A., Zerbi, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modeling+brain+dysconnectivity+in+rodents%5BTitle%5D)+AND+%22Biological+Psychiatry%22%5BJournal%5D)">Modeling brain dysconnectivity in rodents.</a> Biological Psychiatry. 93 (5), 419-429 (2023).</li>
<li>Denic, A., Macura, S. I., Mishra, P., Gamez, J. D., Rodriguez, M., Pirko, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MRI+in+rodent+models+of+brain+disorders%5BTitle%5D)+AND+%22Neurotherapeutics%22%5BJournal%5D)">MRI in rodent models of brain disorders.</a> Neurotherapeutics. 8 (1), 3-18 (2011).</li>
<li>Cong-hui, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficacy+observation+of+long-retaining+scalp+acupuncture+plus+interactive+training+for+upper-extremity+dysfunction+after+cerebral+stroke%5BTitle%5D)+AND+%22Journal+of+Acupuncture+and+Tuina+Science%22%5BJournal%5D)">Efficacy observation of long-retaining scalp acupuncture plus interactive training for upper-extremity dysfunction after cerebral stroke.</a> Journal of Acupuncture and Tuina Science. 19, 43-48 (2021).</li>
<li>Kvist, L. J., Hall-Lord, M. L., Rydhstroem, H., Larsson, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+randomised-controlled+trial+in+Sweden+of+acupuncture+and+care+interventions+for+the+relief+of+inflammatory+symptoms+of+the+breast+during+lactation%5BTitle%5D)+AND+%22Midwifery%22%5BJournal%5D)">A randomised-controlled trial in Sweden of acupuncture and care interventions for the relief of inflammatory symptoms of the breast during lactation.</a> Midwifery. 23 (2), 184-195 (2007).</li>
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The authors declared that no competing conflicts of interest exist.

Electroacupuncture (EA) is a form of acupuncture that involves the use of electrical stimulation on acupuncture needles11. This technique involves the use of micro-pulse currents of specific intensity and frequency to stimulate acupuncture points and achieve enhanced therapeutic effects3. However, the mechanism of EA for healing is limited and requires extensive basic research to prove1,2,<sup class="xref...

<a href="https://app.jove.com/t/6632">This corrects</a> the article <a href="https://dx.doi.org/10.3791/65648">10.3791/65648</a>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/65648">Bulk Electroacupuncture Operation for Mice or Young Rats</a>. Figure 3 was updated.

Erratum: Bulk Electroacupuncture Operation for Mice or Young Rats

Electroacupuncture (EA) therapy is a unique method in which acupuncture needles are inserted into the scalp and connected to an electro-machine to stimulate specific points1. Unlike manual stimulation, EA allows better control of stimulation by stabilizing specific frequencies and waveforms to achieve optimal therapeutic effects2. According to a survey, 81.2% of medical institutions in China use EA or manual acupuncture to treat cerebral palsy, neuralgia, facial palsy, and other conditions. Despite its popularity, the specific mechanism of efficacy of EA is still unknown, which has hindered its promotion and application in the rehabilitation treatment of neurological diseases1,2,3. Further research is needed to fully understand the mechanism of EA&#39;s effectiveness and to promote its use in the rehabilitation treatment of neurological diseases.
As the influence of acupuncture expands worldwide, many studies have already investigated the mechanisms of EA performed on rodent models, such as rat or mouse models1,2,3. Several issues are often encountered in EA&#39;s rodent experiments. The first is how to immobilize rodents without anesthesia, as acupuncture performed on awake subjects is more reflective of clinical practice. In addition, some experiments require animals to be awake to observe treatment effects2,3. Another challenge is accurately locating the acupoints in mice or rats that correspond to those in humans. The precise localization of acupoints in experimental rodents is currently being studied by many scholars4,5. In this protocol, MS6 and ST25 were selected, which have been clearly defined in rodents by the transformation of human anatomical localization. MS6 is commonly used for the treatment of some brain diseases, such as Parkinson&#39;s disease6. ST25 is usually used to treat gastrointestinal problems, such as diarrhea7. These two acupoints were chosen primarily to demonstrate how rodents can be effectively immobilized in both supine and prone positions. Moreover, these acupoints have been extensively studied and offer significant insights for research purposes6,7.
The previous method of immobilizing a single rat for experiments is not only time-consuming but also difficult to handle by a single person8. Additionally, due to the non-cooperation of animals, the success rate is relatively low in practice. Therefore, there is a critical need to create an easily established animal model with stable characteristics to improve experiment efficiency. In this article, a 3D printed holder for small animals was introduced that could easily immobilize multiple rodents, leading to motor restriction. The aim of this paper is to administer EA treatments to a batch of young rats or mice, focusing on the strategies for bulk restriction of mice, identification, and stimulation of MS6 and ST25 acupoints.

<table><tbody><tr><td>3D printing batch mice fixator</td><td>MESH INVENT</td><td>Custom made</td><td>The fixator produced by 3D printer. The printing method is called fused deposition modeling (FDM), also known as fused filament fabrication (FFF).&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Electroacupuncture instrument</td><td>Hwato, Suzhou Medical Appliance Factory</td><td>SDZ-III</td><td>www.Hwato-med.com</td></tr><tr><td>Disposable sterile acupuncture needle</td><td>Suzhou Medical Appliance Factory</td><td>N/A</td><td>0.25 mm x 13 mm</td></tr></tbody></table>

bulk electroacupuncture operation for mice or young rats

Electroacupuncture (EA) is widely used to treat various health conditions. However, the underlying mechanism of EA treatment remains unclear, hindering its promotion. The mechanistic study requires mouse or rat models to address this issue. However, these animals are not obedient to the experimental process, which is time-consuming. To solve these problems, we designed a 3D-printed small animal body bulk fixator to improve the efficiency of EA's animal experiments. This video shows in detail how to use the fixator to perform bulk EA on mice or young rats. For the selection of acupoints, the anterior oblique line of vertex temporal (MS6 head) and Tianshu point (ST25 belly) were chosen to verify the effect of the fixation device with prone positioning and supine positioning. Using the 3D-printed small animal holder allows multiple rodents to be immobilized and treated simultaneously, reducing the time and resources required for the experiment. This technique could be applied to other animal models by 3D printing different sizes and could potentially be used for various fixing conditions. The device is beneficial for the promotion of experimental scientific research in EA.

The floor plan of the animal body fixator we designed is shown in Figure 1. In addition, a 3D graphic model of this fixator was submitted to provide a comprehensive view of the design (Supplementary File 1). This is a device that allows 3 rodents to be immobilized and perform EA simultaneously. Rats and mice were restricted to the fixator in the awake state, and both prone and supine positions could be firmly fixed without causing injury to the rodents (...

Watch this Scientific Journal Video about Bulk Electroacupuncture Operation for Mice or Young Rats at JoVE.com

Author Spotlight: Using a 3D-Printed Holder for Simultaneous Multiple Electroacupuncture Operation

Here, we present a protocol for efficient and rapid electroacupuncture (EA) in mice or young rats using a three-dimensional (3D) printed holder. This technique allows simultaneous operation on multiple animals, saving time and increasing experimental efficiency.

Bulk Electroacupuncture Operation for Mice or Young Rats

Electroacupuncture (EA) is widely used to treat various health conditions. However, the underlying mechanism of EA treatment remains unclear, hindering ...

bulk-electroacupuncture-operation-for-mice-or-young-rats

Research

JoVE Journal

Biology

650 Views.  Army Medical University. Here, we present a protocol for efficient and rapid electroacupuncture (EA) in mice or young rats using a three-dimensional (3D) printed holder. This technique allows simultaneous operation on multiple animals, saving time and increasing experimental efficiency.

Video: Author Spotlight: Using a 3D-Printed Holder for Simultaneous Multiple Electroacupuncture Operation

A Murine Model of Muscle Training by Neuromuscular Electrical Stimulation

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional capacity. Transcutaneous surface stimulation of skeletal muscle involves a current flow between a cathode and an anode, thereby inducing excitement of the motor unit and the surrounding muscle fibers. 
NMES is an attractive modality to evaluate skeletal muscle adaptive responses for several reasons. First, it provides a reproducible experimental model in which physiological adaptations, such as myofiber hypertophy and muscle strengthening6, angiogenesis7-9, growth factor secretion9-11, and muscle precursor cell activation12 are well documented. Such physiological responses may be carefully titrated using different parameters of stimulation (for Cochrane review, see 13). In addition, NMES recruits motor units non-selectively, and in a spatially fixed and temporally synchronous manner14, offering the advantage of exerting a treatment effect on all fibers, regardless of fiber type. Although there are specified contraindications to NMES in clinical populations, including peripheral venous disorders or malignancy, for example, NMES is safe and feasible, even for those who are ill and/or bedridden and for populations in which rigorous exercise may be challenging. 
Here, we demonstrate the protocol for adapting commercially available electrodes and performing a NMES protocol using a murine model. This animal model has the advantage of utilizing a clinically available device and providing instant feedback regarding positioning of the electrode to elicit the desired muscle contractile effect. For the purpose of this manuscript, we will describe the protocol for muscle stimulation of the anterior compartment muscles of a mouse hindlimb.

A murine model of neuromuscular electrical stimulation (NMES), a safe and inexpensive clinical modality, to the anterior compartment muscles is described. This model has the advantage of modifying a readily available clinical device for the purpose of eliciting targeted and specific muscle contractions in mice.

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional ...

Medicine

Manipulation of Epileptiform Electrocorticograms (ECoGs) and Sleep in Rats and Mice by Acupuncture

Ancient Chinese literature has documented that acupuncture possesses efficient therapeutic effects on epilepsy and insomnia. There is, however, little research to reveal the possible mechanisms behind these effects. To investigate the effect of acupuncture on epilepsy and sleep, several issues need to be addressed. The first is to identify the acupoints, which correspond between humans, rats, and mice. Furthermore, the depth of insertion of the acupuncture needle, the degree of needle twist in manual needle acupuncture, and the stimulation parameters for electroacupuncture (EA) need to be determined. To evaluate the effects of acupuncture on epilepsy and sleep, a feasible model of epilepsy in rodents is required. We administer pilocarpine into the left central nucleus of the amygdala (CeA) to simulate focal temporal lobe epilepsy (TLE) in rats. Intraperitoneal (IP) injection of pilocarpine induces generalized epilepsy and status epilepticus (SE) in rats. Five IP injections of pentylenetetrazol (PTZ) with a one-day interval between each injection successfully induces spontaneous generalized epilepsy in mice. Recordings of electrocorticograms (ECoGs), electromyograms (EMGs), brain temperature, and locomotor activity are used for sleep analysis in rats, while ECoGs, EMGs, and locomotor activity are employed for sleep analysis in mice. ECoG electrodes are implanted into the frontal, parietal, and contralateral occipital cortices, and a thermistor is implanted above the cerebral cortex by stereotactic surgery. EMG electrodes are implanted into the neck muscles, and an infrared detector determines locomotor activity. The criteria for categorizing vigilance stages, including wakefulness, rapid eye movement (REM) sleep, and non-REM (NREM) sleep are based on information from ECoGs, EMGs, brain temperature, and locomotor activity. Detailed classification criteria are stated in the text.

Manipulation of Epileptiform Electrocorticograms ECoGs and Sleep in Rats and Mice by Acupuncture

This paper demonstrates the performance of acupuncture, epilepsy models, and the analysis of sleep in rodents. The acupuncture procedure and the identification of acupoints are described. Pilocarpine or pentylenetetrazol (PTZ) is used to induce epilepsy. Electrocorticogram (ECoG), electromyogram (EMG), brain temperature, and locomotor activity recordings are employed for sleep analysis.

Ancient Chinese literature has documented that acupuncture possesses efficient therapeutic effects on epilepsy and insomnia. There is, however, little ...

Behavior

High-density Electroencephalographic Acquisition in a Rodent Model Using Low-cost and Open-source Resources

Advanced electroencephalographic analysis techniques requiring high spatial resolution, including electrical source imaging and measures of network connectivity, are applicable to an expanding variety of questions in neuroscience. Performing these kinds of analyses in a rodent model requires higher electrode density than traditional screw electrodes can accomplish. While higher-density electroencephalographic montages for rodents exist, they are of limited availability to most researchers, are not robust enough for repeated experiments over an extended period of time, or are limited to use in anesthetized rodents.1-3 A proposed low-cost method for constructing a durable, high-count, transcranial electrode array, consisting of bilaterally implantable headpieces is investigated as a means to perform advanced electroencephalogram analyses in mice or rats.

Procedures for headpiece fabrication and surgical implantation necessary to produce high signal to noise, low-impedance electroencephalographic and electromyographic signals are presented. While the methodology is useful in both rats and mice, this manuscript focuses on the more challenging implementation for the smaller mouse skull. Freely moving mice are only tethered to cables via a common adapter during recording. One version of this electrode system that includes 26 electroencephalographic channels and 4 electromyographic channels is described below.

Instructions for the low-cost construction and surgical implantation of a chronic transcranial high-density electroencephalographic montage into mice are provided. Signal recording, extraction, and processing techniques are also described.

Advanced electroencephalographic analysis techniques requiring high spatial resolution, including electrical source imaging and measures of network ...

Neuroscience

Electroconvulsive Seizures in Rats and Fractionation of Their Hippocampi to Examine Seizure-induced Changes in Postsynaptic Density Proteins

Electroconvulsive seizure (ECS) is an experimental animal model of electroconvulsive therapy, the most effective treatment for severe depression. ECS induces generalized tonic-clonic seizures with low mortality and neuronal death and is a widely-used model to screen anti-epileptic drugs. Here, we describe an ECS induction method in which a brief 55-mA current is delivered for 0.5 s to male rats 200 - 250 g in weight via ear-clip electrodes. Such bilateral stimulation produced stage 4 - 5 clonic seizures that lasted about 10 s. After the cessation of acute or chronic ECS, most rats recovered to be behaviorally indistinguishable from sham &#34;no seizure&#34; rats. Because ECS globally elevates brain activity, it has also been used to examine activity-dependent alterations of synaptic proteins and their effects on synaptic strength using multiple methods. In particular, subcellular fractionation of the postsynaptic density (PSD) in combination with Western blotting allows for the quantitative determination of the abundance of synaptic proteins at this specialized synaptic structure. In contrast to a previous fractionation method that requires large amount of rodent brains, we describe here a small-scale fractionation method to isolate the PSD from the hippocampi of a single rat, without sucrose gradient centrifugation. Using this method, we show that the isolated PSD fraction contains postsynaptic membrane proteins, including PSD95, GluN2B, and GluA2. Presynaptic marker synaptophysin and soluble cytoplasmic protein &#945;-tubulin were excluded from the PSD fraction, demonstrating successful PSD isolation. Furthermore, chronic ECS decreased GluN2B expression in the PSD, indicating that our small-scale PSD fractionation method can be applied to detect the changes in hippocampal PSD proteins from a single rat after genetic, pharmacological, or mechanical treatments.

Electroconvulsive seizure (ECS) is an experimental animal model of electroconvulsive therapy for severe depression. ECS globally stimulates activity in the hippocampus, leading to synaptogenesis and synaptic plasticity. Here, we describe methods for ECS induction in rats and for subcellular fractionation of their hippocampi to examine seizure-induced changes in synaptic proteins.

Electroconvulsive seizure (ECS) is an experimental animal model of electroconvulsive therapy, the most effective treatment for severe depression. ECS ...

Transcranial Electrical Brain Stimulation in Alert Rodents

Transcranial electrical brain stimulation can modulate cortical excitability and plasticity in humans and rodents. The most common form of stimulation in humans is transcranial direct current stimulation (tDCS). Less frequently, transcranial alternating current stimulation (tACS) or transcranial random noise stimulation (tRNS), a specific form of tACS using an electrical current applied randomly within a pre-defined frequency range, is used. The increase of noninvasive electrical brain stimulation research in humans, both for experimental and clinical purposes, has yielded an increased need for basic, mechanistic, safety studies in animals. This article describes a model for transcranial electrical brain stimulation (tES) through the intact skull targeting the motor system in alert rodents. The protocol provides step-by-step instructions for the surgical set-up of a permanent epicranial electrode socket combined with an implanted counter electrode on the chest. By placing a stimulation electrode into the epicranial socket, different electrical stimulation types, comparable to tDCS, tACS, and tRNS in humans, can be delivered. Moreover, the practical steps for tES in alert rodents are introduced. The applied current density, stimulation duration, and stimulation type may be chosen depending on the experimental needs. The caveats, advantages, and disadvantages of this set-up are discussed, as well as safety and tolerability aspects.

This protocol describes a surgical set-up for a permanent epicranial electrode socket and an implanted chest electrode in rodents. By placing a second electrode into the socket, different types of transcranial electrical brain stimulation can be delivered to the motor system in alert animals through the intact skull.

Transcranial electrical brain stimulation can modulate cortical excitability and plasticity in humans and rodents. The most common form of stimulation in ...

An Invasive Method for the Activation of the Mouse Dentate Gyrus by High-frequency Stimulation

Electrical high-frequency stimulation (HFS), using implanted electrodes targeting various brain regions, has been proven as an effective treatment for various neurological and psychiatric disorders. HFS in the deep region of the brain, also named deep-brain stimulation (DBS), is becoming increasingly important in clinical trials. Recent progress in the field of high-frequency DBS (HF-DBS) surgery has begun to spread the possibility of utilizing this invasive technique to other situations, such as treatment for major depression disorder (MDD), obsessive-compulsive disorder (OCD), and so on. 
Despite these expanding indications, the underlying mechanisms of the beneficial effects of HF-DBS remain enigmatic. To address this question, one approach is to use implanted electrodes that sparsely activate distributed subpopulations of neurons by HFS. It has been reported that HFS in the anterior nucleus of the thalamus could be used for the treatment of refractory epilepsy in the clinic. The underlying mechanisms might be related to the increased neurogenesis and altered neuronal activity. Therefore, we are interested in exploring the physiological alterations by the detection of neuronal activity as well as neurogenesis in the mouse dentate gyrus (DG) before and after HFS treatment. 
In this manuscript, we describe methodologies for HFS to target the activation of the DG in mice, directly or indirectly and in an acute or chronic manner. In addition, we describe a detailed protocol for the preparation of brain slices for c-fos and Notch1 immunofluorescent staining to monitor the neuronal activity and signaling activation and for bromodeoxyuridine (BrdU) labeling to determine the neurogenesis after the HF-DBS induction. The activation of the neuronal activity and neurogenesis after the HF-DBS treatment provides direct neurobiological evidence and potential therapeutic benefits. Particularly, this methodology can be modified and applied to target other interested brain regions such as the basal ganglia and subthalamic regions for specific brain disorders in the clinic.

This protocol shows how to set up a reliable HFS method in mice. Neurons throughout the hippocampal dentate gyrus are electrically stimulated by HFS directly and indirectly in vivo. Neuronal activity and molecular signaling are examined by c-fos and Notch1 immunofluorescent staining, respectively; neurogenesis is quantified by bromodeoxyuridine labeling assay.

Electrical high-frequency stimulation (HFS), using implanted electrodes targeting various brain regions, has been proven as an effective treatment for ...

Using a Bipolar Electrode to Create a Temporal Lobe Epilepsy Mouse Model by Electrical Kindling of the Amygdala

The amygdala is one of the most common origins of seizures, and the amygdala mouse model is essential for the illustration of epilepsy. However, few studies have described the experimental protocol in detail. This paper illustrates the whole process of amygdala electrical kindling epilepsy model making, with the introduction of a method of bipolar electrode fabrication. This electrode can both stimulate and record, reducing brain injury caused by implanting separate electrodes for stimulation and recording. For long-term electroencephalogram (EEG) recording purposes, slip rings were used to eliminate the record interruption caused by cable tangles and falling off.
After periodic stimulation (60 Hz, 1 s every 15 min) of the basolateral amygdala (AP: 1.67 mm, L: 2.7 mm, V: 4.9 mm) for 19.83 &plusmn; 5.742 times, full kindling was observed in six mice (defined as induction of three continuous grade V episodes classified by Racine's scale). An intracranial EEG was recorded throughout the entire kindling process, and an epileptic discharge in the amygdala lasting 20-70 s was observed after kindling. Therefore, this is a robust protocol for modeling epilepsy originating from the amygdala, and the method is suitable for revealing the role of the amygdala in temporal lobe epilepsy. This research contributes to future studies on the mechanisms of mesial temporal lobe epilepsy and novel antiepileptogenic drugs.

The amygdala plays a key role in temporal lobe epilepsy, which originates in and propagates from this structure. This article provides a detailed description of the fabrication of deep brain electrodes with both recording and stimulating functions. It introduces a model of medial temporal lobe epilepsy originating from the amygdala.

The amygdala is one of the most common origins of seizures, and the amygdala mouse model is essential for the illustration of epilepsy. However, few ...

 Fabricating Epimysial EMG Electrodes for Preclinical Rodent Models 

Development of a Low-cost Epimysial Electromyography Electrode: A Simplified Workflow for Fabrication and Testing

Electromyography (EMG) is a valuable diagnostic tool for detecting neuromuscular abnormalities. Implantable epimysial electrodes are commonly used to measure EMG signals in preclinical models. Although classical resources exist describing the principles of epimysial electrode fabrication, there is a sparsity of illustrative information translating electrode theory to practice. To remedy this, we provide an updated, easy-to-follow guide on fabricating and testing a low-cost epimysial electrode. 
Electrodes were made by folding and inserting two platinum-iridium foils into a precut silicone base to form the contact surfaces. Next, coated stainless steel wires were welded to each contact surface to form the electrode leads. Lastly, a silicone mixture was used to seal the electrode. Ex vivo testing was conducted to compare our custom-fabricated electrode to an industry standard electrode in a saline bath, where high levels of signal agreement (sine [intraclass correlation - ICC= 0.993], square [ICC = 0.995], triangle [ICC = 0.958]), and temporal-synchrony (sine [r = 0.987], square [r = 0.990], triangle [r= 0.931]) were found across all waveforms. Low levels of electrode impedance were also quantified via electrochemical impedance spectroscopy. 
An in vivo performance assessment was also conducted where the vastus lateralis muscle of a rat was surgically instrumented with the custom-fabricated electrode and signaling was acquired during uphill and downhill walking. As expected, peak EMG activity was significantly lower during downhill walking (0.008 &plusmn; 0.005 mV) than uphill (0.031 &plusmn; 0.180 mV, p = 0.005), supporting the validity of the device. The reliability and biocompatibility of the device were also supported by consistent signaling during level walking at 14 days and 56 days post implantation (0.01 &plusmn; 0.007 mV, 0.012 &plusmn; 0.007 mV respectively; p &gt; 0.05) and the absence of histological inflammation. Collectively, we provide an updated workflow for the fabrication and testing of low-cost epimysial electrodes.

Author Spotlight: Epimysial Electrode Fabrication and Testing in ACL Injury Studies

Our purpose was to provide an updated, easy-to-follow guide on the fabrication and testing of epimysial electromyography electrodes. To that end, we provide instructions for material sourcing and a detailed walkthrough of the fabrication and testing process.

Electromyography (EMG) is a valuable diagnostic tool for detecting neuromuscular abnormalities. Implantable epimysial electrodes are commonly used to ...

Rat Jackets to Enhance Suspension Fixation During Electroacupuncture on Rats

Improved Suspension Fixation Using Rat Jacket in Rat Acupuncture Experiments

In basic experimental acupuncture research, rats are commonly used as laboratory animals. However, it is difficult for them to maintain a fixed posture. During electroacupuncture procedures, proper immobilization of rats is essential. Various methods of rat fixation are currently used, including anesthesia fixation, high-platform fixation, binding fixation, and fixation with a self-made rat coat. However, these methods have their limitations, which may affect the efficiency and operability of the experiment to some extent. This protocol introduces a method of suspending and fixing rats using rat clothes. Firstly, rats are clothed with rat jackets that match their body shape, taking advantage of their preference for darkness and burrowing. The needling operation can then be carried out after the rats have worn rat clothes. When suspended, the rats are relatively still, as their limbs cannot move. This fixation method offers not only economical and user-friendly benefits but also ensures a stable and reliable fixation of the rats in a comfortably relaxed position. It also effectively minimizes time consumption, experimental space, and manpower resources. Additionally, this method allows for the exposure of most acupoints used for acupuncture in rats. This article primarily concentrates on the device's composition, encompassing a specially designed rat jacket, an elevated fixation rack, and their connecting structures. Additionally, an illustrative example will be presented to demonstrate the application of the rat clothing-based suspension fixation method in rat electroacupuncture procedures.

Author Spotlight: Efficient Rat Clothing for Improved Rat Fixation During Electroacupuncture 

Here, we present an improved suspension fixation method using a rat jacket to effectively immobilize rats during acupuncture experiments. Furthermore, the structure and application of this fixation apparatus, as well as instructions and evaluation for its utilization in electroacupuncture therapy, is elucidated.

In basic experimental acupuncture research, rats are commonly used as laboratory animals. However, it is difficult for them to maintain a fixed posture. ...

Effects of Different Connection Modes of Electroacupuncture on Electrocardiogram and Nerve Discharge in Rats

Electroacupuncture (EA) is one of the most commonly used methods in acupuncture and has a good effect on pain, depression, sensory movement disorders, and other diseases. The effectiveness of EA is influenced by many factors, such as the accuracy of acupoint selection, the duration and course of EA treatment, and EA parameters. However, it has rarely been discussed whether the positive and negative electrodes of the EA instrument with acupoints at different locations and distances have an effect on the curative effect. In this experiment, we observed the effects of connecting the EA instrument to acupoints at different positions and distances on ECG and vague nerve discharge in rats and preliminarily explained whether the electric field formed by different modes of EA connection has an effect on the function of the body. The connection modes of EA in this experiment included the same acupoints on both sides of the body, the same meridian or different meridian acupoints on the same side of the body, and two needles in the same acupoint area. The results showed that when the positive and negative poles were connected to the acupoints on both sides of the body, the recording of ECG and vagal nerve activity was disturbed (the same fore and hind limbs); when the acupoints were connected on the same side of the body, the smaller the distance between the two needles, the smaller the effect on ECG and vagal nerve activity recording, and the effect increased with increasing current; when the acupoints were in the same acupoint area, the recording of ECG and vagal nerve activity was not disturbed if the two needles did not form a short circuit.

Until now, there is no uniform method for electroacupuncture connection in clinical and fundamental research. We present a protocol that attempted to compare the effects of different connection methods on electrocardiogram (ECG) and neural discharge in rats to explore the most appropriate way to connect the electroacupuncture instrument.

Electroacupuncture (EA) is one of the most commonly used methods in acupuncture and has a good effect on pain, depression, sensory movement disorders, and ...