Z.G. is supported by NINDS R01NS100801.

Animal experiments were approved by UCSF Institutional Animal Care and Use Committee and were conducted in accordance with the NIH Guide for the Care and Use of Laboratory animals. Adult C57BL/6 mice weighing 20-30 g was used in this study. The von Frey assessment was performed between 1:00 pm and 3:00 pm.
1. Anesthesia and mouse preparation
<ol>
	<li>Place a mouse in a plastic chamber filled with 2% isoflurane in O2 at a flow rate of 1.0 L/min until it is fully anesthetized.</li>
	<li>Cover the eyes with ophthalmic ointment using a cotton-wool applicator.</li>
	<li>Place the snout of the mouse into a flexible nose cone with 2% isoflurane flow throughout the surgical procedure.</li>
	<li>Place the mouse in a right lateral position. Keep the left leg up with knee flexion and secure it with adhesive tape.</li>
	<li>Remove the hair around the thigh and knee area with an electric shaver and hair removal cream. Disinfect the skin with 2% chlorhexidine and 70% alcohol.</li>
	<li>Ensure sufficient anesthesia depth before surgery by testing no response to pinch stimulus on the hind limb or tail with tweezers as the standard. Provide thermal support to the mouse. 
	NOTE: No local anesthetic or NSAID was used before and after performing the SNIt model because local anesthetic and NSAID significantly reduce the neuropathic pain behavior after SNIt12.</li>
</ol>
2. Modified SNIt surgery
<ol>
	<li>Cut a 1 cm incision starting at the first 1/3 of the horizontal line crossing the knee with an approximately 30&#176; angle from the vertical line with a scalpel (Figure 2A).</li>
	<li>Two white lines can be visualized under the biceps femoris muscle (BFM) after separating skin incision, with the medial (cephalad) thick line as the femur and lateral (caudal) thin line as sciatic nerve (Figure 2B).</li>
	<li>Blunt dissect BFM along the caudal white line with curved micro forceps and micro scissors to expose the sciatic nerve. Avoid blood vessel damage during blunt dissection. If accidental vessel damage occurs, use sterile cotton-wool swabs to absorb blood and apply proper pressure to stop bleeding.</li>
	<li>Differentiate the three branches from the sciatic nerve at the superior edge of the gastrocnemius muscle. The tibial nerve is the one with the biggest diameter passing under the gastrocnemius muscle, whereas the sural nerves (lateral, the smallest diameter) and the common peroneal nerve (medial) run above the gastrocnemius muscle (Figure 2C and Figure 3A).</li>
	<li>Depending on how BFM is dissected and opened, visualize the common peroneal nerve as lateral (Figure 2C) or medial (Figure 3A) to the tibial nerve.</li>
	<li>Separate the common peroneal and the sural nerves from the neighboring tissues using curve micro forceps.</li>
	<li>Ligate the common peroneal and sural nerves together with a 6-0 suture, as both nerves run above the gastrocnemius muscle, but the tibial nerve passes under the gastrocnemius muscle (Figure 2D and Figure 3B,C). Observe the limb for contraction following the tight ligature. For the traditional method, ligate the common peroneal and sural nerves separately.</li>
	<li>Cut nerve at a distal part within 2-4 mm of the ligation with a pair of micro scissors. Ensure that the tibial nerve remains untouched during the whole procedure. For the traditional method, cut the common peroneal and sural nerves at the distal part within 2-4 mm of the ligation and remove a 2 mm section, separately.</li>
	<li>Close the muscular layer with a 6-0 silk suture and the skin incision with wound clips.</li>
	<li>After surgery, return the mice to the animal room until full recovery from anesthesia. Check daily for intact incisions, normal food intake, water consumption, general body condition, regular movements, and grooming. Remove wound clips 7-14 days after surgery.</li>
</ol>
3. von Frey assessment for mechanical threshold
<ol>
	<li>Acclimatize the mice for 6 days to the testing room environment and testing materials to perform the von-Frey assessment. Place the mice in clear plastic cylinders on an elevated wire mesh grid for 1 h of habituation. Place white papers between each cylinder to prevent any visual cue from each testing animal.</li>
	<li>During this period, perform habituation every 2 days, and measure the baseline of von Frey monofilaments under the mid-plantar of the hind paw after the last habituation.</li>
	<li>Perform von Frey assessment with a blind tester. Stimulate the mid-plantar of the hind paw with von Frey filaments by using the up-down paradigm13. Apply the von Frey filament to the plantar surface perpendicularly with applied force to cause slight curling.</li>
	<li>Verify positive responses as sudden paw withdrawal, sudden flinching, or sudden paw licking. Exert the next stimulus at an interval of 5 s to avoid the influence of the previous stimulus.</li>
	<li>Confirm mechanical threshold per paw by taking an average of 3 sessions.</li>
	<li>Based on the 50% hind paw withdrawal threshold decided by the up-down method, use the percent response method with 0.16 g filament to assess the difference further.</li>
	<li>Record the percentage of the positive responses after 10 stimuli of 0.16 g filament applied to the mid-plantar of the hind paw regardless of the responses.</li>
	<li>Perform the von Frey assessments on pre-surgery day 1, and post-surgery on days 1, 3, 5, 7, and 14.</li>
</ol>
4. Statistical Analysis
<ol>
	<li>Report normally distributed continuous data as mean &#177; standard error of the mean (SEM). Analyze the continuous data with 2-tailed t-test or two-way repeated-measures ANOVA. Process all data using statistical analysis software, with statistical significance at the level of p &#60; 0.05.</li>
</ol>

<ol>
	<li>Chung, J. M., Kim, H. K., Chung, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segmental+spinal+nerve+ligation+model+of+neuropathic+pain.">Segmental spinal nerve ligation model of neuropathic pain.</a> Methods in Molecular Medicine. 99, 35-45 (2004).</li><li>Vissers, K., Adriaensen, H., De Coster, R., De Deyne, C., Meert, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chronic-constriction+injury+of+the+sciatic+nerve+reduces+bilaterally+the+responsiveness+to+formalin+in+rats:+a+behavioral+and+hormonal+evaluation.">A chronic-constriction injury of the sciatic nerve reduces bilaterally the responsiveness to formalin in rats: a behavioral and hormonal evaluation.</a> Anesthesia and Analgesia. 97 (2), 520-525 (2003).</li><li>Malmberg, A. B., Basbaum, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+sciatic+nerve+injury+in+the+mouse+as+a+model+of+neuropathic+pain:+behavioral+and+neuroanatomical+correlates.">Partial sciatic nerve injury in the mouse as a model of neuropathic pain: behavioral and neuroanatomical correlates.</a> Pain. 76 (1-2), 215-222 (1998).</li><li>Moore, K. A., 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=Partial+peripheral+nerve+injury+promotes+a+selective+loss+of+GABAergic+inhibition+in+the+superficial+dorsal+horn+of+the+spinal+cord.">Partial peripheral nerve injury promotes a selective loss of GABAergic inhibition in the superficial dorsal horn of the spinal cord.</a> The Journal of Neuroscience: the official journal of the Society for Neuroscience. 22 (15), 6724-6731 (2002).</li><li>Decosterd, I., Woolf, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spared+nerve+injury:+an+animal+model+of+persistent+peripheral+neuropathic+pain.">Spared nerve injury: an animal model of persistent peripheral neuropathic pain.</a> Pain. 87 (2), 149-158 (2000).</li><li>Cichon, J., Sun, L., Yang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spared+Nerve+Injury+Model+of+Neuropathic+Pain+in+Mice.">Spared Nerve Injury Model of Neuropathic Pain in Mice.</a> Bio-Protocol. 8 (6), 2777 (2018).</li><li>Shields, S. D., Eckert, W. A., Basbaum, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spared+nerve+injury+model+of+neuropathic+pain+in+the+mouse:+a+behavioral+and+anatomic+analysis.">Spared nerve injury model of neuropathic pain in the mouse: a behavioral and anatomic analysis.</a> The Journal of Pain. 4 (8), 465-470 (2003).</li><li>Guan, 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=Injured+sensory+neuron-derived+CSF1+induces+microglial+proliferation+and+DAP12-dependent+pain.">Injured sensory neuron-derived CSF1 induces microglial proliferation and DAP12-dependent pain.</a> Nature Neuroscience. 19 (1), 94-101 (2016).</li><li>Richner, M., Bjerrum, O. J., Nykjaer, A., Vaegter, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spared+nerve+injury+(SNI)+model+of+induced+mechanical+allodynia+in+mice.">The spared nerve injury (SNI) model of induced mechanical allodynia in mice.</a> Journal of Visualized Experiments: JoVE. (54), e3092 (2011).</li><li>Challa, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+animal+models+of+neuropathic+pain:+Pros+and+Cons.">Surgical animal models of neuropathic pain: Pros and Cons.</a> The International Journal of Neuroscience. 125 (3), 170-174 (2015).</li><li>Kosaka, Y., 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=Development+and+persistence+of+neuropathic+pain+through+microglial+activation+and+KCC2+decreasing+after+mouse+tibial+nerve+injury.">Development and persistence of neuropathic pain through microglial activation and KCC2 decreasing after mouse tibial nerve injury.</a> Brain Research. 1733, 146718 (2020).</li><li>Parisien, M., Lima, L. V., Dagostino, C., El-Hachem, N., Drury, G. L., Grant, A. V., Huising, J., Verma, V., Meloto, C. B., Silva, J. R., Dutra, G. C. S., Markova, T., Dang, H., Tessier, P. A., Slade, G. D., Nackley, A. G., Ghasemlou, N., Mogil, J. S., Allegri, M., Diatchenko, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+inflammatory+response+via+neutrophil+activation+protects+against+the+development+of+chronic+pain.">Acute inflammatory response via neutrophil activation protects against the development of chronic pain.</a> Sci Transl Med. 14 (644), eabj9954 (2022).</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> Journal of Neuroscience Methods. 53 (1), 55-63 (1994).</li><li>Bourquin, A. F., 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=Assessment+and+analysis+of+mechanical+allodynia-like+behavior+induced+by+spared+nerve+injury+(SNI)+in+the+mouse.">Assessment and analysis of mechanical allodynia-like behavior induced by spared nerve injury (SNI) in the mouse.</a> Pain. 122 (1-2), 1-14 (2006).</li><li>Sulaiman, W., Gordon, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurobiology+of+peripheral+nerve+injury,+regeneration,+and+functional+recovery:+from+bench+top+research+to+bedside+application.">Neurobiology of peripheral nerve injury, regeneration, and functional recovery: from bench top research to bedside application.</a> The Ochsner Journal. 13 (1), 100-108 (2013).</li><li>Kochi, T., 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=Characterization+of+the+arterial+anatomy+of+the+murine+hindlimb:+functional+role+in+the+design+and+understanding+of+ischemia+models.">Characterization of the arterial anatomy of the murine hindlimb: functional role in the design and understanding of ischemia models.</a> PLoS One. 8 (12), 84047 (2013).</li><li>Omori, Y., 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=A+mouse+model+of+sural+nerve+injury-induced+neuropathy:+gabapentin+inhibits+pain-related+behaviors+and+the+hyperactivity+of+wide-dynamic+range+neurons+in+the+dorsal+horn.">A mouse model of sural nerve injury-induced neuropathy: gabapentin inhibits pain-related behaviors and the hyperactivity of wide-dynamic range neurons in the dorsal horn.</a> Journal of Pharmacological Sciences. 109 (4), 532-539 (2009).</li><li>Colloca, L., 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=Neuropathic+pain.">Neuropathic pain.</a> Nature Reviews. Disease Primers. 3, 17002 (2017).</li></ol>

The authors declare no competing interests.

Compared to the traditional mouse SNIt method that ligates the common peroneal nerve and the sural nerve separately6,7,8,9, the modified SNIt model has three advantages: (1) it has less risk of contracting or stretching sciatic or tibial nerves; (2) there is no need to remove the distal nerve stumps after nerve-cutting because by ligating the common peroneal nerve and the sural nerve together, ...

Nerve injury-induced neuropathic pain following surgery or trauma has a significant economic burden that impairs quality of life. A host of nerve injury models, including spinal nerve ligation (SNL)1, chronic constriction injury (CCI)2 to the sciatic nerve, partial sciatic nerve ligation (pSNL)3, sciatic nerve transaction (SNT)4 and spared nerve injury (SNI)5,6,7,8, were successfully developed to mimic the cardinal symptoms of peripheral nerve injury in rats and mice for studying the molecular and cellular mechanism of neuropathic pain6,7,8,9,10. However, each surgical model has its benefits and limitations, therefore particular attention should be given to exploring and developing the surgery models10.
The rodent SNI model produces long-term hypersensitivity to mechanical stimulation. However, it is somewhat confusing because there are two different SNI models. The initial SNI model was developed in Woolf&#39;s lab, in which the common peroneal and the tibial nerves were injured, leaving the sural nerve intact5,6. The second SNI model was developed in Basbaum&#39;s lab, in which the common peroneal and the sural nerves were injured, leaving the tibial nerve intact7,8. The initial Woolf&#39;s model is defined as SNIs here because the sural nerve is left intact, and Basbaum&#39;s model is defined as SNIt here because the tibial nerve is left intact. Because the sural nerve is purely sensory whereas the tibial nerve contains both motor and sensory fibers, the SNIt model has much less motor deficit than the SNIs model. However, unlike the SNIs model, mice in the SNIt model do not develop thermal hypersensitivity, but mechanical hypersensitivity develops in both models. Although the SNIt model is a relatively easy procedure, it requires the ligation of the sural and common peroneal nerves separately with the potential risk of stretching the sciatic or tibial nerves6,7,8,9.
The common peroneal, tibial, and sural nerves are three branches of the sciatic nerve and can be clearly identified at the superior edge of the gastrocnemius muscle (Figure 1): the tibial nerve goes under the gastrocnemius muscle, and the common peroneal (cephalad side) and sural nerve (caudal side) are above the gastrocnemius muscle11. Based on its anatomical features, a modified mouse SNIt surgery procedure was developed to ligate the common peroneal and sural nerves together with only one nerve-ligation and one nerve-cutting, which results in shortened procedure-time.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6-0 suture</td>
			<td>Henry Schein</td>
			<td>9007482</td>
			<td>Nerve ligation and close the muscular layer</td>
		</tr>
		<tr>
			<td>Iris Scissors</td>
			<td>Integra Miltex</td>
			<td>12460598</td>
			<td>Cut muscle and fascia</td>
		</tr>
		<tr>
			<td>Mayo dissecting scissors</td>
			<td>Fisherbrand</td>
			<td>895120</td>
			<td>Cut skin incision</td>
		</tr>
		<tr>
			<td>Micro forcep</td>
			<td>Fisherbrand</td>
			<td>16100110</td>
			<td>Blunt dissection biceps femoris muscle</td>
		</tr>
		<tr>
			<td>Micro Scissors</td>
			<td>Excelta</td>
			<td>17467496</td>
			<td>Cut nerve</td>
		</tr>
		<tr>
			<td>Microdissection Forceps</td>
			<td>Fisherbrand</td>
			<td>16100123</td>
			<td>Separate the common peroneal and the sural nerves from the neighboring tissues</td>
		</tr>
		<tr>
			<td>Needle Holder</td>
			<td>Fisherbrand</td>
			<td>8966</td>
			<td>Hold 6-0 needle</td>
		</tr>
		<tr>
			<td>Prism software</td>
			<td>Graph Pad</td>
			<td>version 8.0</td>
			<td>Statistical analysis software</td>
		</tr>
		<tr>
			<td>Wound clips</td>
			<td>Roboz Surgical</td>
			<td>NC1878744</td>
			<td>Close skin incision</td>
		</tr>
	</tbody>
</table>

modified spared nerve injury surgery model of neuropathic pain in mice

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular mechanism of neuropathic pain in mice and rats. Currently, there are two types of SNI model, one to cut and ligate the common peroneal and the tibial nerves with intact sural nerve, which is defined as SNIs in this study, and another to cut and ligate the common peroneal and the sural nerves with intact tibial nerve, which is defined as SNIt in this study. Because the sural nerve is purely sensory whereas the tibial nerve contains both motor and sensory fibers, the SNIt model has much less motor deficit than the SNIs model. In the traditional SNIt mouse model, the common peroneal and the sural nerves are cut and ligated separately. Here a modified SNIt surgery method is described to damage both common peroneal and sural nerves with only one ligation and one cut with a shorter procedure time, which is easier to perform and reduces the potential risk of stretching the sciatic or tibial nerves, and produces similar mechanical hypersensitivity as the traditional SNIt model.

The comparison of procedure time between modified and traditional methods. 
The procedure time from the beginning of cutting the skin to the end of closing skin was recorded in 5 mice with the modified approach and 5 mice with the traditional approach, respectively. A minimal number of animals was used to obtain results with statistical significance. Compared with the control of the traditional approach7,14 to perform SNIt, the modified appr...

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Modified Spared Nerve Injury Surgery Model of Neuropathic Pain in Mice

The modified surgery is a simplified method for mouse or rat spared nerve injury model that requires only one ligation and one cut to injure both common peroneal and sural nerves.

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular ...

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4.3K Views.  Capital Medical University. The modified surgery is a simplified method for mouse or rat spared nerve injury model that requires only one ligation and one cut to injure both common peroneal and sural nerves.

Video: Modified Spared Nerve Injury Surgery Model of Neuropathic Pain in Mice

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.
Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20.
We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.

Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described.

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

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 ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

A Novel In Vitro Model of Blast Traumatic Brain Injury

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the detonation of explosive devices, however, the mechanisms that underlie the brain damage resulting from blast overpressure exposure are not entirely understood and are believed to be unique to this type of brain injury. Preclinical models are crucial tools that contribute to better understand blast-induced brain injury. A novel in vitro blast TBI model was developed using an open-ended shock tube to simulate real-life open-field blast waves modelled by the Friedlander waveform. C57BL/6N mouse organotypic hippocampal slice cultures were exposed to single shock waves and the development of injury was characterized up to 72 h using propidium iodide, a well-established fluorescent marker of cell damage that only penetrates cells with compromised cellular membranes. Propidium iodide fluorescence was significantly higher in the slices exposed to a blast wave when compared with sham slices throughout the duration of the protocol. The brain tissue injury is very reproducible and proportional to the peak overpressure of the shock wave applied.

A Novel In Vitro Model of Blast Traumatic Brain Injury

This paper describes a novel model of primary blast traumatic brain injury. A compressed-air driven shock tube is used to expose in vitro mouse hippocampal slice cultures to a single shock wave. This is a simple and rapid protocol generating a reproducible brain tissue injury with a high throughput.

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the ...

Facial Nerve Surgery in the Rat Model to Study Axonal Inhibition and Regeneration

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial nerve can be manipulated along its entire length, from its intracranial segment to its extratemporal course. There are three primary types of nerve injury used for the experimental study of regenerative properties: nerve crush, transection, and nerve gap. The range of possible interventions is vast, including surgical manipulation of the nerve, delivery of neuroactive reagents or cells, and either central or end-organ manipulations. Advantages of this model for studying nerve regeneration include simplicity, reproducibility, interspecies consistency, reliable survival rates of the rat, and an increased anatomic size relative to murine models. Its limitations involve a more limited genetic manipulation versus the mouse model and the superlative regenerative capability of the rat, such that the facial nerve scientist must carefully assess time points for recovery and whether to translate results to higher animals and human studies. The rat model for facial nerve injury allows for functional, electrophysiological, and histomorphometric parameters for the interpretation and comparison of nerve regeneration. It thereby boasts tremendous potential toward furthering the understanding and treatment of the devastating consequences of facial nerve injury in human patients.

This protocol describes a reproducible approach to facial nerve surgery in the rat model, including descriptions of various inducible patterns of injury.

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial ...

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 ...

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 ...

Dural Stimulation and Periorbital von Frey Testing in Mice As a Preclinical Model of Headache

The cranial meninges, comprised of the dura mater, arachnoid, and pia mater, are thought to primarily serve structural functions for the nervous system. For example, they protect the brain from the skull and anchor/organize the vascular and neuronal supply of the cortex. However, the meninges are also implicated in nervous system disorders such as migraine, where the pain experienced during a migraine is attributed to local sterile inflammation and subsequent activation of local nociceptive afferents. Of the layers in the meninges, the dura mater is of particular interest in the pathophysiology of migraines. It is highly vascularized, harbors local nociceptive neurons, and is home to a diverse array of resident cells such as immune cells. Subtle changes in the local meningeal microenvironment may lead to activation and sensitization of dural perivascular nociceptors, thus leading to migraine pain. Studies have sought to address how dural afferents become activated/sensitized by using either in vivo electrophysiology, imaging techniques, or behavioral models, but these commonly require very invasive surgeries. This protocol presents a method for comparatively non-invasive application of compounds on the dura mater in mice and a suitable method for measuring headache-like tactile sensitivity using periorbital von Frey testing following dural stimulation. This method maintains the integrity of the dura and skull and reduces confounding effects from invasive techniques by injecting substances through a 0.65 mm modified cannula at the junction of unfused sagittal and lambdoid sutures. This preclinical model will allow researchers to investigate a wide range of dural stimuli and their role in the pathological progression of migraine, such as nociceptor activation, immune cell activation, vascular changes, and pain behaviors, all while maintaining injury-free conditions to the skull and meninges.

The most notable symptom of migraine is severe head pain, and it is hypothesized that this is mediated by sensory neurons innervating the meninges. Here, we present a method to locally apply substances to the dura in a minimally invasive manner while using facial hypersensitivity as an output.

The cranial meninges, comprised of the dura mater, arachnoid, and pia mater, are thought to primarily serve structural functions for the nervous system.  ...

Electromagnetic Controlled Closed-Head Model of Mild Traumatic Brain Injury in Mice

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and understanding the mechanisms of how a TBI alters brain function. The availability of multiple animal models of TBI is necessary to model the different aspects and severities of TBI seen in people. This manuscript describes the use of a midline closed head injury (CHI) to develop a mouse model of mild TBI. The model is considered mild because it does not produce structural brain lesions based on neuroimaging or gross neuronal loss. However, a single impact creates enough pathology that cognitive impairment is measurable at least 1 month after injury. A step-by-step protocol to induce a CHI in mice using a stereotaxically guided electromagnetic impactor is defined in the paper. The benefits of the mild midline CHI model include the reproducibility of the injury-induced changes with low mortality. The model has been temporally characterized up to 1 year after the injury for neuroimaging, neurochemical, neuropathological, and behavioral changes. The model is complementary to open skull models of controlled cortical impact using the same impactor device. Thus, labs can model both mild diffuse TBI and focal moderate-to-severe TBI with the same impactor.

The protocol describes mild traumatic brain injury in a mouse model. In particular, a step-by-step protocol to induce a mild midline closed head injury and the characterization of the animal model is fully explained.

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and ...