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. 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>

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). Depending on the selected nerve, validation of the amplitude of the H-wave as a function of the stimulation intensity is recommended. The amplitude can vary due to the nerve diameter and motoneuron excitability, as well as electrical contact. By measuring the H/M ratio and RDD, experimental influences such as the positioning of the needle can be reduced, which significantly increases the reliability of the values obtained.
The main limitation of the protocol presented here is the terminal application without the possibility for longitudinal measurements. Further, several methodological details should be considered. Constant anesthesia with minimal muscle relaxation is critical for reliable measurement and should be validated for each specific model/application. In contrast to isoflurane anesthesia which causes a strong suppression of muscle reflexes (i.e., the H-reflex9,10,11, the combination of ketamine-xylazine provides a safe anesthesia and is widely used for EMG recordings12. In line with measurements of motor evoked potentials in the rat13, in our experience 100 mg/kg ketamine and 10 mg/kg xylazine provides the best protocol for stable and reliable recordings. For a skilled experimenter it is possible to perform fore- and hindpaw measurements in one final experiment. The procedure as described here for the forepaw, including animal preparation, and measurement of all frequencies for rate-dependent depression, can be performed in about 30-40 min. It is strongly recommended to practice the nerve dissection technique before performing in vivo experiments. In unilateral disease models (e.g., cortical stroke), we recommend repeating the stimulation 15 times on both contralateral paws to include the non-affected paw as an internal control. As only one nerve is to be stimulated in the method shown here, special care must be taken to distribute sufficient petroleum jelly around the stimulation electrodes so that no stimulation of neighboring nerves occurs.

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 longer transit time through the spinal cord (Figure 1A and Figure 2B). Measure the amplitude of the M- and H-waves as peak-to-peak.
To evaluate the physiological changes that occur in spinal cord injury or stroke, the ratio between the H- and M-wave amplitude (H/M ratio, Figure 2) is less prone to experimental variability, which would be reflected, for example, in amplitude differences. The ratio thus provides a more reliable evaluation of disease-related electrophysiological changes. For example, in mice with a stroke in the primary and secondary motor cortex, the H-wave is increased, whereas the M-wave remains unchanged (Figure 2), suggesting an increased excitability of &#945;MN. Furthermore, there is a reduced RDD (i.e., a reduced decrease in the suppression of the H-wave with increasing stimulation frequency). The decreased RDD is the result of reduced spinal cord inhibition4. Thus, RDD can validate the activation of spinal inhibitory circuits, whose interruption may result in spasticity. To calculate the RDD of the H-reflex, the method described by Lee et al. is recommended4. Briefly, the H-reflex stimulation at 0.1 Hz is averaged and set to 100%. The H-reflex obtained for the other stimulation frequencies is expressed as relative values to 0.1 Hz. From each stimulation train, the first three stimulations are discarded.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63304/63304fig01.jpg" /> 
Figure 1: Illustration of the recording setup and pathways to measure the Hoffman reflex (H-reflex) and muscle response (M-wave). (A) The H-reflex is induced by stimulation of Ia afferents, which activate corresponding alpha motoneurons in the spinal cord and subsequently evoke muscle contractions in the innervated forepaw muscles. (B) Locations of the electrically stimulated radial/ulnar/median nerves in the forepaw and sciatic/tibial nerves in the hindpaw. Created with BioRender.com. <a href="https://www.jove.com/files/ftp_upload/63304/63304fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63304/63304fig02.jpg" /> 
Figure 2: Schematic and representative electrical recording results. (A) Schematic of a recording. The stimulus and respective stimulation artifact are set to 0 ms, which is followed by the direct muscular response (M-wave) and the subsequent smaller peak representing the H-wave. In spasticity models, the H-reflex will be larger compared to the healthy control. (B) Screenshots from a representative recording with the software showing original data with a stimulus artifact (lower traces) and the appearance of the M-wave alone vs. an example where both the M- and H-waves are visible in the recording (upper trace, middle and right panel, respectively). <a href="https://www.jove.com/files/ftp_upload/63304/63304fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63304/63304fig03.jpg" /> 
Figure 3: Positioning of the electrodes for terminal electrophysiological measurement. (A,B) Overview of the terminal H-reflex measurement with the hook stimulation electrodes, the recording electrodes within the forepaw, and the reference electrode inserted into the hindlimb. (C,D) In the hindlimb, after skin and muscle removal, the sciatic nerve becomes visible and can be divided into the sciatic and tibial nerves. (E) In the forelimb, the radial, median, and ulnar nerves become visible. (F) The ulnar nerve can be stimulated with the hook electrode without stimulation of neighboring nerves. Created with BioRender.com. <a href="https://www.jove.com/files/ftp_upload/63304/63304fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

Nanyang Technological University

Research

JoVE Journal

Biology

4.9K 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

Preparation and Culture of Rat Lens Epithelial Explants for Studying Terminal Differentiation

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  Following division, these cells migrate posteriorly, where FGF diffusing from the retina induces them to differentiate into a posterior array of elongated lens fiber cells, which compose the bulk of the lens.  Differentiation of lens epithelial cells into lens fibers can be induced in vitro by culturing explants of the central region of the anterior epithelium in the presence of FGF-2.  Explants are prepared from lenses of neonatal rats by removing the lens from the eye and grasping the lens capsule on the posterior side with dissecting tweezers.  The posterior capsule is then gently torn open and pressed down into the plastic bottom of a tissue culture dish. The peripheral regions of the explant are removed with a scalpel and the central area is then cultured in the presence of 100ng/ml FGF-2 for as long as 2-3 weeks, depending on the parameters to be studied.  Since epithelial cells in cultured explants differentiate in approximate synchrony over a period of days to weeks, the time course of signaling and gene expression can be determined using molecular, biochemical, and pharmacological techniques.  Immunofluorescence microscopy is a powerful adjunct to these methods as it demonstrates the subcellular localization of proteins of interest and can reveal the physiological consequences of experimental manipulations of signaling pathways.

Explants of the central region of rat lens epithelia differentiate synchronously when cultured in the presence of FGF-2. Immunofluorescence microscopy of such cultures can provides novel information about gene expression and signaling events associated with terminal differentiation.

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  ...

A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated procedures to obtain such measurements in the same animal are generally not feasible.  Here, we demonstrate protocols for obtaining from mice repeated measurements of AHR and bronchoalveolar lavage fluid samples.  Mice were challenged intranasally seven times over 14 days with a potent allergen or sham treated.  Prior to the initial challenge, and within 24 hours following each intranasal challenge, the same animals were anesthetized, orally intubated and mechanically ventilated.  AHR, assessed by comparing dose response curves of respiratory system resistance (RRS) induced by increasing intravenous doses of acetylcholine (Ach) chloride between sham and allergen-challenged animals, were determined.  Afterwards, and via the same intubation, the left lung was lavaged so that differential enumeration of airway cells could be performed.  These studies reveal that repeated measurements of AHR and BAL fluid collection are possible from the same animals and that maximal airway hyperresponsiveness and airway eosinophilia are achieved within 7-10 days of initiating allergen challenge.  This novel technique significantly reduces the number of mice required for longitudinal experimentation and is applicable to diverse rodent species, disease models and airway physiology instruments.  

Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated ...

Transverse Aortic Constriction in Mice

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart failure.1 TAC initially leads to compensated hypertrophy of the heart, which often is associated with a temporary enhancement of cardiac contractility. Over time, however, the response to the chronic hemodynamic overload becomes maladaptive, resulting in cardiac dilatation and heart failure.2 The murine TAC model was first validated by Rockman et al.1, and has since been extensively used as a valuable tool to mimic human cardiovascular diseases and elucidate fundamental signaling processes involved in the cardiac hypertrophic response and heart failure development. When compared to other experimental models of heart failure, such as complete occlusion of the left anterior descending (LAD) coronary artery, TAC provides a more reproducible model of cardiac hypertrophy and a more gradual time course in the development of heart failure. Here, we describe a step-by-step procedure to perform surgical TAC in mice. To determine the level of pressure overload produced by the aortic ligation, a high frequency Doppler probe is used to measure the ratio between blood flow velocities in the right and left carotid arteries.3, 4 With surgical survival rates of 80-90%, transverse aortic banding is an effective technique of inducing left ventricular hypertrophy and heart failure in mice.

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model to study mechanisms underlying cardiac hypertrophy and heart failure development. Here, we describe procedures to constrict the aorta to create a reproducible degree of cardiac hypertrophy in mice.

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart ...

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific shapes. Drosophila tracheal terminal cells have become a powerful genetic model to identify and elucidate the roles of genes required for generating cellular morphologies. Terminal cells are a component of a branched tubular network, the tracheal system that functions to supply oxygen to internal tissues. Terminal cells are an excellent model for investigating questions of cell shape as they possess two distinct cellular architectures. First, terminal cells have an elaborate branched morphology, similar to complex neurons; second, terminal cell branches are formed as thin tubes and contain a membrane-bound intracellular lumen. Quantitative analysis of terminal cell branch number, branch organization and individual branch shape, can be used to provide information about the role of specific genetic mechanisms in the making of a branched cell. Analysis of tube formation in these cells can reveal conserved mechanisms of tubulogenesis common to other tubular networks, such as the vertebrate vasculature. Here we describe techniques that can be used to rapidly fix, image, and analyze both branching patterns and tube formation in terminal cells within Drosophila larvae. These techniques can be used to analyze terminal cells in wild-type and mutant animals, or genetic mosaics. Because of the high efficiency of this protocol, it is also well suited for genetic, RNAi-based, or drug screens in the Drosophila tracheal system.

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Here, we present a method for light microscopy analysis of tracheal terminal cells in Drosophila larvae. This method allows for quick examination of branch and lumen morphology in whole animals and would be useful for analysis of individual mutants or screens for mutations affecting terminal cell development.

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Non-Terminal Blood Sampling Techniques in Guinea Pigs

Guinea pigs possess several biological similarities to humans and are validated experimental animal models1-3. However, the use of guinea pigs currently represents a relatively narrow area of research and descriptive data on specific methodology is correspondingly scarce. The anatomical features of guinea pigs are slightly different from other rodent models, hence modulation of sampling techniques to accommodate for species-specific differences, e.g., compared to mice and rats, are necessary to obtain sufficient and high quality samples. As both long and short term in vivo studies often require repeated blood sampling the choice of technique should be well considered in order to reduce stress and discomfort in the animals but also to ensure survival as well as compliance with requirements of sample size and accessibility. Venous blood samples can be obtained at a number of sites in guinea pigs e.g., the saphenous and jugular veins, each technique containing both advantages and disadvantages4,5. Here, we present four different blood sampling techniques for either conscious or anaesthetized guinea pigs. The procedures are all non-terminal procedures provided that sample volumes and number of samples do not exceed guidelines for blood collection in laboratory animals6. All the described methods have been thoroughly tested and applied for repeated in vivo blood sampling in studies within our research facility.

Though a known model, the guinea pig currently represents a niche in experimental animal sciences and limited data is available on the execution of most procedures. Here we present four different approaches to non-terminal in vivo blood sampling techniques in either conscious or anaesthetized guinea pigs.

Guinea pigs possess several biological similarities to humans and are validated experimental animal models1-3. However, the use of guinea pigs currently ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The contractile characteristics can be evaluated independently of external influences such as vascular tonus or neurohumoral status. It depicts a scientific approach between single cell measurements with isolated cardiac myocytes and in vivo studies like echocardiography. Thus, papillary muscle preparations serve as an excellent model to study cardiac physiology/pathophysiology and can be used for investigations like the modulation by pharmacological agents or the exploration of transgenic animal models. Here, we describe a method of isolating the murine left anterior papillary muscle to investigate cardiac contractility in an organ bath setup. In contrast to a muscle strip preparation isolated from the ventricular wall, the papillary muscle can be prepared in toto without damaging the muscle tissue severely. The organ bath setup consists of several temperature-controlled, gassed and electrode-equipped organ bath chambers. The isolated papillary muscle is fixed in the organ bath chamber and electrically stimulated. The evoked twitch force is recorded using a pressure transducer and parameters such as twitch force amplitude and twitch kinetics are analyzed. Different experimental protocols can be performed to investigate the calcium- and frequency-dependent contractility as well as dose-response curves of contractile agents such as catecholamines or other pharmaceuticals. Additionally, pathologic conditions like acute ischemia can be simulated.

Murine left ventricular papillary muscle can be used to investigate cardiac contractility in vitro. This article describes in detail the isolation and experimental protocols to study cardiac contractile characteristics.

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

Cystometric and External Urethral Sphincter Measurements in Awake Rats with Implanted Catheter and Electrodes Allowing for Repeated Measurements

Lower urinary tract function is mainly assessed by means of cystometric bladder function analysis in rodents. Conventional cystometries are usually performed as terminal analysis under urethane anesthesia. It is well known that anesthetic drugs can influence bladder function. Hence, the aim of this technique is to perform cystometric measurements of the urinary bladder and external urethral sphincter in lightly restrained awake rats. For this purpose, a bladder catheter is implanted into the bladder dome. Subsequently, two electrodes are implanted bilateral to the external urethral sphincter and a ground electrode is sutured to a non-responsive skeletal muscle. The bladder catheter and the three electrodes are finally tunneled subcutaneously to the neck region and affixed to a harness. With this technique, the lower urinary tract can be measured at multiple time points in the same animal to assess lower urinary tract function. The main application of this technique is the follow-up of simultaneous urinary bladder and external urethral sphincter function in awake healthy rats and after induction of a disease or injury. Moreover, subsequent lower urinary tract monitoring can be performed during evaluation of the disease/injury and to monitor treatment efficacy.

This protocol first describes the surgical procedure of the permanent implantation of a urinary bladder catheter combined with external urethral sphincter electrodes, and second, the measurement of the function of the urinary bladder and external urethral sphincter in implanted awake animals.

Lower urinary tract function is mainly assessed by means of cystometric bladder function analysis in rodents. Conventional cystometries are usually ...

Measurements of Physiological Stress Responses in C. Elegans

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and physiology. Thus, organisms have evolved targeted and specific stress responses dedicated to repair damage and maintain homeostasis. These mechanisms include the unfolded protein response of the endoplasmic reticulum (UPRER), the unfolded protein response of the mitochondria (UPRMT), the heat shock response (HSR), and the oxidative stress response (OxSR). The protocols presented here describe methods to detect and characterize the activation of these pathways and their physiological consequences in the nematode, C. elegans. First, the use of pathway-specific fluorescent transcriptional reporters is described for rapid cellular characterization, drug screening, or large-scale genetic screening (e.g., RNAi or mutant libraries). In addition, complementary, robust physiological assays are described, which can be used to directly assess sensitivity of animals to specific stressors, serving as functional validation of the transcriptional reporters. Together, these methods allow for rapid characterization of the cellular and physiological effects of internal and external proteotoxic perturbations.

Measurements of Physiological Stress Responses in C. Elegans

Here, we characterize cellular proteotoxic stress responses in the nematode C. elegans by measuring the activation of fluorescent transcriptional reporters and assaying sensitivity to physiological stress.

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and ...