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 descripti...

<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. G., Acharya, H., Fouad, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+electrode+configuration+for+recording+electromyographic+activity+in+behaving+mice.">A new electrode configuration for recording electromyographic activity in behaving mice.</a> Journal of Neuroscience Methods. 148 (1), 36-42 (2005).</li><li>Akay, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+measurement+of+muscle+denervation+and+locomotor+behavior+in+individual+wild-type+and+ALS+model+mice.">Long-term measurement of muscle denervation and locomotor behavior in individual wild-type and ALS model mice.</a> Journal of Neurophysiology. 111 (3), 694-703 (2014).</li><li>Haghighi, S. S., Green, D. K., Oro, J. J., Drake, R. K., Kracke, G. 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. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+ketamine/xylazine+anesthesia+on+sensory+and+motor+evoked+potentials+in+the+rat.">The effect of ketamine/xylazine anesthesia on sensory and motor evoked potentials in the rat.</a> Spinal Cord. 41 (1), 16-22 (2003).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#181;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&#176;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 ...

The H-reflex allows electrophysiological validation of the integrity of neuro circuits. We present a terminal protocol to quantify the H-reflex directly at the nerves of the forepaw in mice. Different to conventional rather nerve-unspecific transcutaneous recordings, the results are less variable between mice and measurement days.Similar to H-reflex recordings in patients, as precise quantification allows electrophysiological characterization in disease models, e.g. spinal cord injury and stroke. The protocol requires solid experimental skills in working with surgery equipment in mice.If you are unsure how to separate nerves from connective tissue and blood vessels, practice first. Demonstrating the procedure will be by Frederique Wieters, a Ph.D student from my laboratory. To begin, place the adult anesthetized 8-to 16-weeks-old C57 black/6J mouse on the covered heating pad and wait until the mouse is calm, breathing is stable, and reflexes are absent.Turn the mouse on its back. Then, fix the forepaws with tape. Position the tape such that the measuring electrodes are easily inserted into the forepaws.To measure the temperature of a mouse insert a rectal probe and fix it with tape. Apply eye ointment to prevent the eyes from drying out. Remove the fur carefully with a pair of fine, rounded scissors while lifting the skin with tweezers.Subsequently, make a one-centimeter incision in the skin along the ventroposterior axis of the forepaw with a pair of fine, rounded scissors. Carefully remove the connective tissue and expose the underneath muscle and nerve. Then remove the exposed pectoralis profundus muscle using forceps to assess the median nerve.Remove small amounts of blood and tissue fluid with soft tissue paper. Next, carefully cut the breast or axillary muscle from the top to bottom to expose the nerve bundle. Free the nerve bundle from the connective and muscle tissue over a length of approximately 1.5 centimeters.Separate the ulnar and median nerve of the forepaw carefully using a bent glass pipette. The upper nerve is the ulnar nerve and the lower is the median nerve. Arrange the stimulator hook electrodes in parallel at a distance of 0.5 to one millimeter and use a micromanipulator to position the double hook close to the nerve.Use the glass hook to lift the ulnar nerve onto the stimulation hook electrodes. Then, pull back the electrode with the nerve and separate it from the other nerves. Place the electrodes along the long axis of the paw to reduce the crosstalk between the muscles.Superficially dry the electrode hooks attached to the nerve and apply petroleum jelly using a syringe to provide electrical insulation from the adjacent tissue. To measure H-reflex, position the electromyography electrodes intramuscularly in the forepaw. Then, position the reference electrode subcutaneously in the hind limb, held by a miniature alligator clip.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 are between 10 to 50 microamperes. Perform the nerve stimulation 15 times with 0.2-milliseconds-long pulses each.With pauses of two minutes between the sets of stimuli, the frequency is increased from 0.1, 0.5, 1.0, 2.0, to 5 hertz. An illustration of the recording set up in pathways to measure the Hoffman reflex and muscle response is shown here. The ratio between H-and M-wave amplitude was evaluated.The stimulus and respective stimulation artifact were set at zero milliseconds, followed by the direct M-wave and the subsequent smaller peak representing the H-wave. The M-wave occurs approximately two milliseconds after stimulation, and the H-wave after six to eight milliseconds due to the longer transit time through the spinal cord. Next to the separation of the nerves, the application of petroleum jelly to the electrode and also between the two hooks are critical.This is a terminal experiment. In our lab, this technique provided a new gold standard reference to the transcutaneous H-reflex recordings.

Research

JoVE Journal

Biology

4.5K Views.  University of Cologne. Il riflesso H consente la validazione elettrofisiologica dell'integrità dei neurocircuiti. Presentiamo un protocollo terminale per quantificare il riflesso H direttamente ai nervi della zampa anteriore nei topi. A differenza delle registrazioni transcutanee convenzionali piuttosto nervose, i risultati sono meno variabili tra topi e giorni di misurazione.Simile alle registrazioni H-reflex nei pazienti, poiché la quantificazione precisa consente la caratterizzazione elettrofisiologica ...