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* These authors contributed equally
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 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.
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 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 (αMNs) in the spinal cord, which in turn elicit action potentials that travel down the αMN 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 αMNs 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 αMN 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 αMN 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 αMN 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.
All experiments were conducted in compliance with European and National animal care laws and institutional guidelines, and were approved by the Landesamt für 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
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.
3. Electrode placement
4. Placement of the recording and reference electrodes
5. Measurement
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...
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 authors declare no competing financial interests.
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.
Name | Company | Catalog Number | Comments |
Absorbent underpad | VWR | 115-0684 | |
AD converter | Cambridge Electronic Design, UK | CED 1401micro | |
Amplifier | Workshop Zoological Institute, UoC | - | |
Digital stimulator | Workshop Zoological Institute, UoC | MS 501 | |
EMG electrodes | Workshop Zoological Institute, UoC | Two twisted, insulated copper wires (50 µm outer diameter) were soldered to a male plug and connected to a differential amplifier. | |
Eye ointment | Bayer | Bepanthen | |
Glass pipette | Workshop Zoological Institute, UoC | - | Prepare a glass pipette bent into a simple glass hook in the flame of a Bunsen burner. |
Heating box | MediHeat | MediHeat V1200 | |
Heating pad | WPI | 61840 Heating pad | |
Hook electrodes | Workshop Zoological Institute, UoC | - | 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. |
Ketamine | Pfizer | Ketavet | |
Rectal probe | WPI | RET-3 | |
Stimulator isolation unit | Workshop Zoological Institute, UoC | MI 401 | |
Sterilizer | CellPoint Scientific | Germinator 500 | 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°C). |
Temperature controller | WPI | ATC200 | |
Vaseline | Bayer | - | |
Xylazine | Bayer | Rompun |
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