The authors gratefully acknowledge support by T. Akay, Dalhousie University, during a visit of MG to his lab. This work was supported by funding from the Friebe Foundation (T0498/28960/16) and the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) - Project-ID 431549029 - SFB 1451.

All experiments were conducted in compliance with European and National animal care laws and institutional guidelines,&#160;and were approved by the Landesamt f&#252;r Natur-, Umwelt-, und Verbraucherschutz North Rhine-Westphalia (Az: 81-02.04.2019.A309). The protocol is optimized for adult mice (approx. 8-16 weeks old C57Bl/6J mice) and the forelimb recording. It can be easily adapted by stimulating the respective nerves of the hindlimb and recording hindpaw muscles (Figure 1B). A description of the recording and stimulation electrodes is added in the Table of Materials. Note that the protocol is used for a terminal measurement only.
1. Preparation
<ol>
	<li>Weigh the animal and start anesthesia by injecting via i.p. a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg).</li>
	<li>Keep the mouse in the warming box until surgical tolerance is reached. Wait a few minutes until the mouse is calm, the breathing is stable, and reflexes are absent. Check the depth of anesthesia by measuring the lack of response to the toe pinch.</li>
	<li>Turn the mouse on its back and place it on a heating pad (optimal would be feedback-controlled heating using a rectal temperature probe). Fix the forepaws with tape. Here, ensure that the tape is positioned in such a way that the measuring electrodes are easily inserted into the forepaws.</li>
	<li>Insert a rectal probe to measure the temperature of the animal and fix it with tape. Apply eye ointment to prevent the eyes from drying out.</li>
</ol>
2. Surgery
NOTE: The stable condition of the anesthetized animal, i.e., respiration, temperature, and loss of reflexes, should be monitored regularly throughout the procedure. The direct nerve H-wave measurement procedure is shown for the radial/ulnar/median nerve of the forepaw (Figure 3A). The measurement can also be adapted to the hindpaw (sciatic/tibial nerve) with modifications.
<ol>
	<li>For a better overview of the surgical area, remove the hair with an electric razor or a pair of scissors in a seperate place beforehand. For more experienced surgeons this is only optional. 
	NOTE: Disinfection is not necessary here, as this is an end experiment. The animal will be euthanized later.</li>
	<li>Lift the skin with tweezers and make an incision of approximately 1 cm in the skin along the ventro-posterior axis of the forepaw (area above the armpit and thorax) with a pair of fine rounded scissors.</li>
	<li>Carefully remove the connective tissue and expose the muscle and nerve underneath. Remove the exposed pectoralis profundus muscle using forceps, to access the median&#160;nerve (Figure 3E,F). Remove small amounts of blood and tissue fluid with soft tissue.</li>
	<li>In the next step, carefully cut the breast or axillary muscle from the top to the bottom to expose the nerve bundle underneath. Free the nerve bundle from the connective and muscle tissue over a length of approximately 1.5 cm. 
	NOTE: Here, particular care should be taken not to damage the blood vessels that run in parallel to the median nerve. When cutting the tissue, always cut along the nerve to avoid injuring it. If this happens, remove leaking fluid and blood with a swab. Astereo microscope is not necessary for the entire experiment, however it can be useful for the preparation of the nerves.</li>
	<li>Separate the ulnar and median nerve of the forepaw carefully using a bent glass pipette (Figure 3E). The upper of the two nerves is the ulnar nerve, and the lower is the median nerve. 
	&#8203;NOTE: When separating the nerves from each other, make sure that the blood vessel underneath is not injured.</li>
</ol>
3. Electrode placement
<ol>
	<li>Arrange the stimulation hook electrodes in parallel at a distance of 0.5-1.0 mm, and use a micromanipulator to position the double hook in close proximity to the nerve.</li>
	<li>Use the glass hook as a tool to lift the ulnar nerve onto the stimulation hook electrodes. Pull back the electrode with the nerve and separate it from other nerves by approximately 1-2 mm using the micromanipulator (Figure 3D,F).</li>
	<li>Place the electrodes along the long axis of the paw to reduce the crosstalk between the muscles. 
	NOTE: The placement of the electrodes is very important but difficult to standardize due to the individual anatomy. It requires an experienced surgeon to place the electrodes correctly. The wires can also be repositioned should the signal amplitude be unsatisfactory.</li>
	<li>Superficially dry the electrode hooks attached to the nerve and apply petroleum jelly using a syringe to provide electrical insulation from the adjacent tissue. 
	&#8203;NOTE: Care should be taken to apply enough petroleum jelly to the electrode and also between the two hooks to ensure electrical insulation and prevent the nerves from drying out.</li>
</ol>
4. Placement of the recording and reference electrodes
<ol>
	<li>To measure the H-reflex, position the EMG electrodes intramuscularly in the forepaw. In addition, position the reference electrode subcutaneously in the hindlimb (e.g., using a minute pin), held by a miniature alligator clip (Figure 3B).</li>
	<li>When the stimulator is switched on, observe a successful stimulation as tiny twitches of the forepaw. The minimum stimulation current to elicit the M-wave and tiny visible twitches in the forepaw should be in the range of 10-50 &#181;A. 
	NOTE: If no twitches are visible at 50 &#181;A, adjust the stimulation electrodes and re-apply petroleum jelly. Also, in mice, it is not uncommon that the M-wave appears at lower stimulation intensities than the H-wave5.</li>
</ol>
5. Measurement
<ol>
	<li>Repeat the stimulation of the nerve 15 times with 0.2 ms long pulses each. With pauses of 2 min between sets of stimuli, the frequency is increased from 0.1, 0.5, 1.0, 2.0, to 5 Hz. 
	NOTE: These frequencies are necessary if calculating the RDD afterwards. All EMG data are recorded, digitized, and analyzed using software e.g., Spike2 software (CED, version 7.19). The largest H-wave amplitude is expected at 0.1 Hz. The higher the frequency, the smaller the amplitude of the H-wave becomes due to the RDD.</li>
	<li>After the experiment, sacrifice the animal as per the IACUC protocol of the institute. In this experiment mouse transcardial perfusion was performed using PBS and 4% PFA under deep anesthesia.</li>
</ol>

<ol>
	<li>Palmieri, R. M., Ingersoll, C. D., Hoffman, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hoffmann+reflex:+methodologic+considerations+and+applications+for+use+in+sports+medicine+and+athletic+training+research.">The Hoffmann reflex: methodologic considerations and applications for use in sports medicine and athletic training research.</a> Journal of Athletic Training. 39 (3), 268-277 (2004).</li><li>Henneman, E., Somjen, G., Carpenter, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitability+and+inhibitibility+of+motoneurons+of+different+sizes.">Excitability and inhibitibility of motoneurons of different sizes.</a> Journal of Neurophysiology. 28 (3), 599-620 (1965).</li><li>Toda, T., Ishida, K., Kiyama, H., Yamashita, T., Lee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Down-regulation+of+KCC2+expression+and+phosphorylation+in+motoneurons,+and+increases+the+number+of+in+primary+afferent+projections+to+motoneurons+in+mice+with+post-stroke+spasticity.">Down-regulation of KCC2 expression and phosphorylation in motoneurons, and increases the number of in primary afferent projections to motoneurons in mice with post-stroke spasticity.</a> PLoS ONE. 9 (12), 114328 (2014).</li><li>Lee, S., Toda, T., Kiyama, H., Yamashita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Weakened+rate-dependent+depression+of+Hoffmann&#8217;s+reflex+and+increased+motoneuron+hyperactivity+after+motor+cortical+infarction+in+mice.">Weakened rate-dependent depression of Hoffmann&#8217;s reflex and increased motoneuron hyperactivity after motor cortical infarction in mice.</a> Cell Death &amp; Disease. 5 (1), 1007 (2014).</li><li>Knikou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+H-reflex+as+a+probe:+Pathways+and+pitfalls.">The H-reflex as a probe: Pathways and pitfalls.</a> Journal of Neuroscience Methods. 171 (1), 1-12 (2008).</li><li>Wieters, 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=Introduction+to+spasticity+and+related+mouse+models.">Introduction to spasticity and related mouse models.</a> Experimental Neurology. 335, 113491 (2020).</li><li>Pearson, K. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depressive+effect+of+isoflurane+anesthesia+on+motor+evoked+potentials.">Depressive effect of isoflurane anesthesia on motor evoked potentials.</a> Neurosurgery. 26 (6), 993 (1990).</li><li>Chang, H. -. Y., Havton, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+urethane+and+isoflurane+on+external+urethral+sphincter+electromyography+and+cystometry+in+rats.">Differential effects of urethane and isoflurane on external urethral sphincter electromyography and cystometry in rats.</a> American Journal of Physiology-Renal Physiology. 295 (4), 1248-1253 (2008).</li><li>Nolan, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Section+4:+Nervous+system+(Br.+J.+Pharmacol).">Section 4: Nervous system (Br. J. Pharmacol).</a> Clinical pharmacology. , 295-310 (2012).</li><li>Struck, M. B., Andrutis, K. A., Ramirez, H. E., Battles, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+a+short-term+fast+on+ketamine-xylazine+anesthesia+in+rats.">Effect of a short-term fast on ketamine-xylazine anesthesia in rats.</a> Journal of the American Association for Laboratory Animal Science: JAALAS. 50 (3), 344-348 (2011).</li><li>Zandieh, S., Hopf, R., Redl, H., Schlag, M. 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The authors declare no competing financial interests.

In contrast to previously described transcutaneous H-reflex measurements in the mouse6, we provide a more direct and nerve-specific measurement. This new approach can be applied to the nerves of the fore- and hindlimb (e.g., the median, ulnar, and radial nerves, and the tibial, and sciatic nerves, respectively), rendering this method adaptable as a diagnostic tool to many disease models (e.g., stroke, multiple sclerosis, amyotrophic lateral sclerosis, traumatic brain injury, and spinal cord injury...

The Hoffmann reflex (H-reflex), named after the physiologist Paul Hoffmann, can be evoked by electrical stimulation of peripheral nerves, which carry axons of sensory and motor neurons arising from and leading to&#160;the same muscles. It is the electrically induced analog of the monosynaptic stretch reflex, and shares the same pathway1. Unlike the muscle stretch, the H-reflex results from electrical stimulation. When peripheral nerves are electrically stimulated at low current intensity, the Ia afferent fibers are typically depolarized first due to their large axon diameter2. Their action potentials excite alpha motorneurons (&#945;MNs) in the spinal cord, which in turn elicit action potentials that travel down the &#945;MN axons toward the muscle (Figure 1). This cascade generates a muscular response with small amplitude, reflected in the so-called H-wave. By gradually increasing the stimulus intensity, the amplitude of the H-wave increases due to the recruitment of additional motor units. From a certain stimulus intensity, action potentials in the thinner axons of the &#945;MNs are elicited directly, which is recorded as the M-wave. This M-wave appears with a shorter latency than the H-wave (Figure 2). If the stimulation intensity is further increased, the amplitude of the M-wave becomes larger due to the recruitment of more &#945;MN axons, whereas the H-wave gradually becomes smaller. The H-wave can be suppressed at high stimulus intensities due to antidromic backpropagation of action potentials in the &#945;MN axons. These triggered action potentials collide with those from the Ia stimulation and can thus cancel each other out. At supramaximal stimulus intensities, orthodromic (toward the muscle) and antidromic (toward the spinal cord) action potentials occur in all MN axons; the former gives rise to the maximal M-wave amplitude (Mmax), whereas the latter results in complete abolition of the H-reflex3.
For the evaluation of post-stroke spasticity (PSS) or spinal cord injury (SCI), the H-reflex has been used to assess the neural basis of movement and spasticity in humans1. An improved quantification of the change in the H-reflex between measurements and between subjects is achieved by using the ratio of the H- and M-wave (H/M ratio). Alternatively, the rate-dependent depression (RDD) is measured, using a set of ascending frequencies (e.g., 0.1, 0.5, 1.0, 2.0, and 5.0 Hz). The RDD reflects the integrity of inhibitory circuits that may be disturbed by stroke or SCI. When all neural circuits are intact, there is a uniform, frequency-independent suppression of the H-reflex. However, if there is reduced neural inhibition as a result of stroke or SCI, the suppression of the H-reflex decreases with increasing stimulation frequency4.
The correct electrophysiological recording using surface electrodes can be challenging and may be affected by motor tasks, inhibitory mechanisms, and &#945;MN excitability5. In the transcutaneous recording in rodents, a stimulus electrode is placed near the tibial nerve, and a recording electrode is placed near the related muscles in the forepaw. According to our experience, however, the correct placement of the transcutaneous electrodes (Figure 1A) is even more complex and variable in rodents than surface electrode placement in humans. This can lead to differences in length, frequency, and stimulation intensity necessary to elicit the H-reflex. These methodological challenges could explain why there are only a very limited number of H-reflex measurement studies (e.g., in experimental stroke models3,4, and other spasticity models6. A precise (long-term) stimulation and recording of the H-reflex on individual nerves could, in principle, be achieved using implantable electrodes surrounding the target nerve7,8. Due to the challenging surgery with potential side effects for the animal and potential instability of the probe, this approach has not become a standard in the field. The method presented here also requires some surgical expertise. However, it allows a novel, precise stimulation and recording of isolated nerves in vivo using low stimulation intensities, which avoids simultaneous stimulation of neighboring nerves.

<table><tbody><tr><td>Absorbent underpad</td><td>VWR</td><td>115-0684</td><td></td></tr><tr><td>AD converter</td><td>Cambridge Electronic Design, UK</td><td>CED 1401micro</td><td></td></tr><tr><td>Amplifier</td><td>Workshop Zoological Institute, UoC</td><td>-</td><td></td></tr><tr><td>Digital stimulator</td><td>Workshop Zoological Institute, UoC</td><td>MS 501</td><td></td></tr><tr><td>EMG electrodes</td><td>Workshop Zoological Institute, UoC</td><td></td><td>Two twisted, insulated copper wires (50 &amp;micro;m outer diameter) were soldered to a male plug and connected to a differential amplifier.</td></tr><tr><td>Eye ointment</td><td>Bayer</td><td>Bepanthen</td><td></td></tr><tr><td>Glass pipette</td><td>Workshop Zoological Institute, UoC</td><td>-</td><td>Prepare a glass pipette bent into a simple glass hook in the flame of a Bunsen burner.</td></tr><tr><td>Heating box</td><td>MediHeat</td><td>MediHeat V1200</td><td></td></tr><tr><td>Heating pad</td><td>WPI</td><td>61840 Heating pad</td><td></td></tr><tr><td>Hook electrodes</td><td>Workshop Zoological Institute, UoC</td><td>-</td><td>To produce the electrodes, bend stainless steel miniature pins into hooks at one end and insert into blunt cannulas to create direct mechanical contact. Solder the end of the cannula to copper wires (length approx. 50 cm), which are connected to either stimulation or recording device.</td></tr><tr><td>Ketamine</td><td>Pfizer</td><td>Ketavet</td><td></td></tr><tr><td>Rectal probe</td><td>WPI</td><td>RET-3</td><td></td></tr><tr><td>Stimulator isolation unit</td><td>Workshop Zoological Institute, UoC</td><td>MI 401</td><td></td></tr><tr><td>Sterilizer</td><td>CellPoint Scientific</td><td>Germinator 500</td><td>Routine pre- and post-operative disinfection of the surgical equipment should be done by heat sterilization. Decontaminate instruments for 15 s in the heated glass bead bath (260&amp;deg;C).</td></tr><tr><td>Temperature controller</td><td>WPI</td><td>ATC200</td><td></td></tr><tr><td>Vaseline</td><td>Bayer</td><td>-</td><td></td></tr><tr><td>Xylazine</td><td>Bayer</td><td>Rompun</td><td></td></tr></tbody></table>

terminal h-reflex measurements in mice

The Hoffmann reflex (H-reflex), as an electrical analog to the stretch reflex, allows electrophysiological validation of the integrity of neural circuits after injuries such as spinal cord damage or stroke. An increase of the H-reflex response, together with symptoms like non-voluntary muscle contractions, pathologically augmented stretch reflex, and hypertonia in the corresponding muscle, is an indicator of post-stroke spasticity (PSS).
In contrast to rather nerve-unspecific transcutaneous measurements, here, we present a protocol to quantify the H-reflex directly at the ulnar and median nerves of the forepaw, which is applicable, with minor modifications, to the tibial and sciatic nerve of the hindpaw. Based on the direct stimulation and the adaptation to different nerves, the method represents a reliable and versatile tool to validate electrophysiological changes in spasticity-related disease models.

From the n = 15 stimulation trials per stimulation frequency and paw, select at least n = 10 successful recordings for the analysis. Trials with measurement errors (e.g., missing M-wave) are excluded from the analysis. Analyze each trial separately and generate an average for group/time comparisons later on. The latency between stimulation and appearance of the M-wave and H-wave is recorded for each trial. In our experience, the M-wave occurs approximately 2 ms after stimulation, and the H-wave after 6-8 ms, due to the l...

Watch this Scientific Journal Video about Terminal H-reflex Measurements in Mice at JoVE.com

Terminal H-reflex Measurements in Mice

The clinical evaluation of spasticity based on the Hoffmann reflex (H-reflex) and using electrical stimulation of peripheral nerves is an established method. Here, we provide a protocol for a terminal and direct nerve stimulation for H-reflex quantification in the mouse forepaw.

The Hoffmann reflex (H-reflex), as an electrical analog to the stretch reflex, allows electrophysiological validation of the integrity of neural circuits ...

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

5.0K Views.  University of Cologne. The clinical evaluation of spasticity based on the Hoffmann reflex (H-reflex) and using electrical stimulation of peripheral nerves is an established method. Here, we provide a protocol for a terminal and direct nerve stimulation for H-reflex quantification in the mouse forepaw.

Video: Terminal H-reflex Measurements in Mice

Electrophysiological Motor Unit Number Estimation (MUNE) Measuring Compound Muscle Action Potential (CMAP) in Mouse Hindlimb Muscles

Compound muscle action potential (CMAP) and motor unit number estimation (MUNE) are electrophysiological techniques that can be used to monitor the functional status of a motor unit pool in vivo. These measures can provide insight into the normal development and degeneration of the neuromuscular system. These measures have clear translational potential because they are routinely applied in diagnostic and clinical human studies. We present electrophysiological techniques similar to those employed in humans to allow recordings of mouse sciatic nerve function. The CMAP response represents the electrophysiological output from a muscle or group of muscles following supramaximal stimulation of a peripheral nerve. MUNE is an electrophysiological technique that is based on modifications of the CMAP response. MUNE is a calculated value that represents the estimated number of motor neurons or axons (motor control input) supplying the muscle or group of muscles being tested. We present methods for recording CMAP responses from the proximal leg muscles using surface recording electrodes following the stimulation of the sciatic nerve in mice. An incremental MUNE technique is described using submaximal stimuli to determine the average single motor unit potential (SMUP) size. MUNE is calculated by dividing the CMAP amplitude (peak-to-peak) by the SMUP amplitude (peak-to-peak). These electrophysiological techniques allow repeated measures in both neonatal and adult mice in such a manner that facilitates rapid analysis and data collection while reducing the number of animals required for experimental testing. Furthermore, these measures are similar to those recorded in human studies allowing more direct comparisons.

Electrophysiological Motor Unit Number Estimation MUNE Measuring Compound Muscle Action Potential CMAP in Mouse Hindlimb Muscles

We present refined protocols that allow in vivo monitoring of motor unit function in the mouse. Techniques to measure compound muscle action potential (CMAP) and motor unit number estimation (MUNE) in the mouse hind limb muscles innervated by the sciatic nerve are described.

Compound muscle action potential (CMAP) and motor unit number estimation (MUNE) are electrophysiological techniques that can be used to monitor the ...

Behavior

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

Neuroscience

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain perception in rodents. Withdrawal thresholds can be measured first at baseline and then following drug, venom, injury, allergen,&#160;or otherwise evoked inflammation by applying an accurate force on very specific areas of the skin. An electronic von Frey apparatus allows precise assessment of mouse hind paw withdrawal thresholds that are not limited by the available filament sizes in contrast to classical von Frey measurements. The ease and rapidity of measurements allow for incorporation of assessment of tactile sensitivity outcomes in diverse models of rapid-onset inflammatory and neuropathic pain as multiple measurements can be taken within a short time period. Experimental measurements for individual rodent subjects can be internally controlled against individual baseline responses and exclusion criteria easily established to standardize baseline responses within and across experimental groups. Thus, measurements using an electronic von Frey apparatus represent a useful modification of the well-established classical von Frey filament-based assays for rodent mechanical allodynia that may also be applied to other nonhuman mammalian models.

Electronic von Frey measurements are an effective and improved alternative to classical von Frey filaments, as they allow rapid and precise measurement of changes in rodent mechanical withdrawal thresholds to continuously applied pressure stimuli before and after an inflammatory event.

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain ...

Urokinase-type Plasminogen Activator-induced Mouse Back Pain Model

A model of persisting lower back pain can be induced in mice with the simple methodology described herein. Step-by-step methods for simple, rapid induction of a persisting back pain model in mice are provided here using an injection of urokinase-type plasminogen activator (urokinase), a serine protease present in humans and other animals. The methodology for induction of persisting lower back pain in mice involves a simple injection of urokinase along the ligamentous insertion region of the lumbar spine. The urokinase inflammatory agent activates plasminogen to plasmin. Typically, the model can be induced within 10 min and hypersensitivity persists for at least 8 weeks.
Hypersensitivity, gait disturbance, and other standard anxiety- and depression-like measures can be tested in the persisting model.&#160;Back pain is the most prevalent type of pain. To improve awareness of back pain, the International Association for the Study of Pain (IASP) named 2021 the &#34;Global Year about Back Pain&#34; and 2022 the &#34;Global Year for Translating Pain Knowledge to Practice.&#34; One limitation of the therapeutic advancement of pain therapeutics is the lack of suitable models for testing persistent and chronic pain. The&#160;features of this model&#160;are suitable for testing potential therapeutics aimed at the reduction of back pain and its ancillary characteristics, contributing to IASP&#39;s naming 2022 as the Global Year for Translating Pain Knowledge to Practice.

Methods for simple, rapid induction of a back pain model in mice are provided here using an intraligament injection of urinary plasminogen activator.

A model of persisting lower back pain can be induced in mice with the simple methodology described herein. Step-by-step methods for simple, rapid ...

HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2. Consequently, investigation of structural details of the interaction between 5-HT2A and mGlu2 affecting schizophrenia-related behaviors represents a powerful translational tool. As previously shown, the head-twitch response (HTR) in mice is elicited by hallucinogenic drugs and this behavioral response is absent in 5-HT2A knockout (KO) mice3,4. Additionally, by conditionally expressing the 5-HT2A receptor only in cortex, it was demonstrated that 5-HT2A receptor-dependent signaling pathways on cortical pyramidal neurons are sufficient to elicit head-twitch behavior in response to hallucinogenic drugs3. Finally, it has been shown that the head-twitch behavioral response induced by the hallucinogens DOI and lysergic acid diethylamide (LSD) is significantly decreased in mGlu2-KO mice5. These findings suggest that mGlu2 is at least in part necessary for the 5-HT2A receptor-dependent psychosis-like behavioral effects induced by LSD-like drugs. However, this does not provide evidence as to whether the 5-HT2A-mGlu2 receptor complex is necessary for this behavioral phenotype. To address this question, herpes simplex virus (HSV) constructs to express either mGlu2 or mGlu2&#916;TM4N (mGlu2/mGlu3 chimeric construct that does not form the 5-HT2A-mGlu2 receptor complex) in the frontal cortex of mGlu2-KO mice were used to examine whether this GPCR heteromeric complex is needed for the behavioral effects induced by LSD-like drugs6.

This article describes how to inject viral vectors into the mouse frontal cortex to test behavioral assays that require GPCR heteromeric formation.

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2.  ...

Establishing a Mouse Model of a Pure Small Fiber Neuropathy with the Ultrapotent Agonist of Transient Receptor Potential Vanilloid Type 1

Patients with diabetes mellitus (DM) or those experiencing the neurotoxic effects of chemotherapeutic agents may develop sensation disorders due to degeneration and injury of small-diameter sensory neurons, referred to as small fiber neuropathy. Present animal models of small fiber neuropathy affect both large- and small-diameter sensory fibers and thus create a neuropathology too complex to properly assess the effects of injured small-diameter sensory fibers. Therefore, it is necessary to develop an experimental model of pure small fiber neuropathy to adequately examine these issues. This protocol describes an experimental model of small fiber neuropathy specifically affecting small-diameter sensory nerves with resiniferatoxin (RTX), an ultrapotent agonist of transient receptor potential vanilloid type 1 (TRPV1), through a single dose of intraperitoneal injection, referred to as RTX neuropathy. This RTX neuropathy showed pathological manifestations and behavioral abnormalities that mimic the clinical characteristics of patients with small fiber neuropathy, including intraepidermal nerve fiber (IENF) degeneration, specifically injury in small-diameter neurons, and induction of thermal hypoalgesia and mechanical allodynia. This protocol tested three doses of RTX (200, 50, and 10 &#181;g/kg, respectively) and concluded that a critical dose of RTX (50 &#181;g/kg) is required for the development of typical small fiber neuropathy manifestations, and prepared a modified immunostaining procedure to investigate IENF degeneration and neuronal soma injury. The modified procedure is fast, systematic, and economic. Behavioral evaluation of neuropathic pain is critical to reveal the function of small-diameter sensory nerves. The evaluation of mechanical thresholds in experimental rodents is particularly challenging and this protocol describes a customized metal mesh that is suitable for this type of assessment in rodents. In summary, RTX neuropathy is a new and easily established experimental model to evaluate the molecular significance and intervention underlying neuropathic pain for the development of therapeutic agents.

This study establishes an experimental model of pure small fiber neuropathy with resiniferatoxin (RTX). A unique dose of RTX (50 &#181;g/kg) is optimal for developing a small fiber neuropathy model that mimics patient characteristics and could help investigate the nociceptive molecular significance underlying neuropathic pain.

Patients with diabetes mellitus (DM) or those experiencing the neurotoxic effects of chemotherapeutic agents may develop sensation disorders due to ...

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings provide a sensitive approach to measure nerve conduction in humans and rodent models. To broaden the technical possibilities for electromyography in mice, the measurement of compound muscle action potentials (CMAPs) from the brachial plexus nerve in the forelimb using needle electrodes is described here. CMAP recordings after stimulating the sciatic nerve in hindlimbs have been previously described. The newly introduced method here allows for the evaluation of the nerve conductivity at an additional site, and thus provides a more profound overview of the neuromuscular functionality. The technique provides information on both the relative number of functional axons and the myelination level. Thereby, this method can be applied to assess both axonal diseases as well as demyelinating conditions. This minimally invasive method does not require extraction of the nerve and therefore it is suitable for repeated measurements for longitudinal follow-up in the same animal. Similar recordings are performed in clinical setups to emphasize the translational relevance of the method.

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

The measurement of nerve conduction is a useful tool to assess mouse models of neurodegeneration but it is frequently only applied to stimulate the sciatic nerve in hindlimbs. Here, we describe a technique to measure compound muscle action potential (CMAP) in vivo in the mouse forelimb muscles innervated by the brachial plexus.

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings ...

Development of Recombinant Proteins to Treat Chronic Pain

Chronic pain is difficult to treat and new approaches to resolve persistent pain are urgently needed. Anti-inflammatory cytokines are promising candidates for treating debilitating pain conditions due to their capacity to regulate aberrant neuro-immune interactions. However, physiologically they work in a network of various cytokines, and therefore their therapeutic effect may not be optimal when used as stand-alone drugs. To overcome this limitation, we developed a fusion protein of the anti-inflammatory cytokines IL4 and IL10. Here, we describe the methods for production and quality control of IL4-10 recombinant fusion protein and we test the effectiveness of the IL4-10 fusion protein to resolve pain in a mouse model of persistent inflammatory pain.

In this paper we provide details for the methods of production and quality control of an IL4-10 recombinant fusion protein. We also show how to test the effectiveness of this protein to resolve pain in a mouse model of inflammatory pain.

Chronic pain is difficult to treat and new approaches to resolve persistent pain are urgently needed. Anti-inflammatory cytokines are promising candidates ...

Medicine

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...

Partial Sciatic Nerve Ligation: A Mouse Model of Chronic Neuropathic Pain to Study the Antinociceptive Effect of Novel Therapies

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. Chronic neuropathic pain results from lesions or diseases in the somatosensory system. To investigate potential therapies with reduced side effects, animal pain models are the gold standard in preclinical studies. Therefore, well-characterized and well-described models are crucial for the development and validation of innovative therapies. 
Partial ligation of the sciatic nerve (pSNL) is a procedure that induces chronic neuropathic pain in mice, characterized by mechanical and thermal hypersensitivity, ongoing pain, and changes in limb temperature, making this model a great fit to study neuropathic pain preclinically. pSNL is an advantageous model to study neuropathic pain as it reproduces many symptoms observed in humans with neuropathic pain. Furthermore, the surgical procedure is relatively fast and straightforward to perform. Unilateral pSNL of one limb allows for comparison between the ipsilateral and contralateral paws, as well as evaluation of central sensitization. 
To induce chronic neuropathic hypersensitivity, a 9-0 non-absorbable nylon thread is used to ligate the dorsal third of the sciatic nerve. This article describes the surgical procedure and characterizes the development of chronic neuropathic pain through multiple commonly used behavioral tests. As a plethora of innovative therapies are now being investigated to treat chronic pain, this article provides crucial concepts for standardization and an accurate description of surgeries required to induce neuropathic pain.

Partial sciatic nerve ligation induces long-lasting chronic neuropathic pain, characterized by exaggerated responses to thermal and mechanical stimuli. This mouse model of neuropathic pain is commonly used to study innovative therapies for pain management. This article describes in detail the surgical procedure to improve standardization and reproducibility.

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. ...

Terminal H-reflex Measurements in Mice

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Chapters in this video

Electrophysiological Motor Unit Number Estimation (MUNE) Measuring Compound Muscle Action Potential (CMAP) in Mouse Hindlimb Muscles

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus

Urokinase-type Plasminogen Activator-induced Mouse Back Pain Model

HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

Establishing a Mouse Model of a Pure Small Fiber Neuropathy with the Ultrapotent Agonist of Transient Receptor Potential Vanilloid Type 1

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

Development of Recombinant Proteins to Treat Chronic Pain

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Partial Sciatic Nerve Ligation: A Mouse Model of Chronic Neuropathic Pain to Study the Antinociceptive Effect of Novel Therapies