Nerve–muscle physiology constitutes a very important portion of undergraduate teaching curriculum which comprises of about 8-12 didactic lectures in most medical colleges. The practical sessions on this system are mostly on amphibian (frog) nerve-muscle preparation. The purpose of the practical teaching is to develop new psycho-motor skills as well as reinforcement of theoretical concepts. The concepts and practical skills on nerve￾muscle physiology are indispensable part of undergraduate teaching that develops the basic understanding of electrophysiology. But the availability of frog is a limiting issue in most of the medical colleges. Here we propose an alternative approach by using mammalian (rat) model for nerve-muscle physiology practical experiments.

Several studies using rat’s nerve-muscle preparation are available. By using nerve-muscle preparation of rat, Norenberg et al. in 2004 demonstrated that isotonic resistance exercise training increases muscle twitch tension in soleus and gastrocnemius muscles. Various physiological contractile properties of skeletal muscle (gastrocnemius) in rats, such as post-tetanic potentiation (MacIntosh et al., 1987), force-frequency relationship (Dormer et al., 2009), force-length relationship (MacNaughton et al., 2006), force-velocity relationship (Devrome et al., 2007) and fatigue (MacNaughton et al., 2006) were studied in in-situ nerve-muscle preparation.

Fatigue may be defined as a progressive loss of the ability to generate maximum force during (or following) repeated or sustained muscle contractions or the loss of force generation during a task (Davis et al., 2010). Fatigue is divided into central and peripheral fatigue. Central fatigue consists of impaired muscle performance due to central (CNS) cause where CNS drive is reduced or which prevents complete muscle group recruitment. Peripheral fatigue may be because of muscle fatigue or fatigue at neuromuscular junction because of depletion of neurotransmitter. Muscle fatigue predominately involves muscle bioenergetics or excitation-contraction coupling (Davis et al., 2010).

To study the effect of neuro-muscular blocking agent on neuromuscular transmission, Pancuronium bromide can be used. It is a long acting, non-depolarizing acetylcholine receptor blocking agent. It is used clinically to facilitate endotracheal intubation and to provide skeletal muscle relaxation during surgery (Larijani et al., 2011).

So the objective of this practical was to study of neuromuscular transmission under (i) phenomenon of fatigue, (ii) site of fatigue, (iii) neuromuscular blocking in an in-situ rat nerve-muscle preparation.

Material required

Dissection board, dissection instruments, glass seeker, dental spatula, povidone iodine solution, cotton, tissue paper, thread, mammalian ringer, normal saline, liquid paraffin, 1 ml syringe, Sodium Thiopental, Pancuronium bromide, stand, clamp, pulley, force transducer (MLTF500/ST), stimulating electrodes and digital recording system - Power Lab
26T with LabChart™ software version 8.1 (AD Instruments, Australia).

Experimental animals

Adult male wistar rats (body weight 200-250 gm) were obtained from institutional (All India Institute of Medical Sciences, New Delhi, India) central animal facility. They were housed in a temperature-controlled room at 24±2% C with a light:dark cycle of 14:10 hours and provided ad libitum food and water. Rats were food deprived overnight to avoid mucous and salivary secretion during experiment.

Ethics statement

The present study was approved by the institutional ethics committee, AIIMS, New Delhi ((65/IAEC-1/ 2018) and was performed in accordance with the Laboratory Animal Welfare Act, the Guide for the Care and Use of Laboratory Animal (National Institutes of Health, Bethesda, MD, USA).

Specific learning objective 1:

To demonstrate the phenomenon and site of fatigue in an in situ nerve–muscle preparation of rat.

Procedure

1. An adult wistar rat was anaesthetized by injecting Sodium Thiopental intraperitoneally (dose 50 mg/ kg). Depth of anesthesia was confirmed by tail pinch.
2. Rat was fixed on dissection table by tying all the four limbs with cotton thread as shown in Fig. 1.
3. One lower limb was shaved, cleaned with povidone iodine solution and a skin incision was given in midline between knee and ankle joint.
4. Soleus muscle is flat and red in appearance and lies with the surface of tibia. The soleus muscle was identified and detached from gastrocnemius and plantaris muscle by dental scalpel. As the tendons of three above mentioned muscles are attached, tendon of soleus muscle was isolated from them without damaging any blood vessels. The distal end (tendon) was isolated from ankle joint while the proximal end still remained attached.
5. One end of cotton thread was tied to the isolated tendon of soleus muscle and other end was attached to force transducer as shown in Fig. 1.
6. The skin incision was extended proximally in the thigh region to expose the gluteus muscle. Sciatic nerve was then located deep to the gluteus muscle and isolated using glass seeker.
7. Stimulating hook electrodes were applied to the sciatic nerve and used to stimulate the nerve (Fig. 1).
8. Liquid paraffin was used to prevent drying of muscle and nerve.
9. Force transducer (MLTF500/ST) and stimulating electrodes were connected to the digital recording system. Power Lab 26T with LabChart™ software version 8.1 (AD Instruments, Australia) were used for real time digital data acquisition and analysis.
10. Since the force transducer measures contraction strength in Volts, it was essential to calibrate the same before the start of the experiment to get values in grams. Transducer channel was identified and zeroing was done before commencement of calibration. Two-point calibration was done by applying weights from 10-50 g in a step up and step down manner. Unit conversion was done to get the subsequent values in Newton.
11. A simple muscle twitch was recorded by applying supramaximal strength of stimulus. The amplitude of contraction and total twitch duration were measured.
12. For demonstrating the phenomenon of fatigue, the nerve-muscle preparation was stimulated with supramaximal stimuli at a frequency [(1/total twitch duration) per second] till the amplitude of contraction reached to 50% of the first recording.
13. The changes in latent, contraction and relaxation periods after the onset of fatigue were analyzed. Further the phenomenon of beneficial effect during initial phase and contraction remainder were observed.
14. The stimulating electrodes were applied directly to the muscle belly immediately after fatigue sets in and a muscle twitch was recorded. The amplitude of contraction was observed.
15. After the data collection, the rat was sacrificed with the over dose of anaesthesia or cervical dislocation while still under anaesthesia.
    

Specific learning objective 2:

To demonstrate the neuromuscular blocking effect of Pancuronium bromide in an in situ nerve-muscle preparation of rat

Procedure

1. Two nerve-muscle preparations were prepared in both hind limbs of a rat as per the procedure mentioned earlier.
2. Simple muscle twitch was recorded from both the preparations by stimulating the sciatic nerve. The amplitude of contraction and total twitch duration were measured.
3. 0.5 ml of 0.9% saline was injected into one muscle (control), and 0.5 ml of Pancuronium bromide (10 mg/ml) was injected into the center and periphery of the other muscle belly by multiple punctures.
4. Sciatic nerves of both the preparations were stimulated every minute with supramaximal stimulus strength and the muscle twitch responses were recorded. The muscles in both the preparations were also stimulated directly and twitch responses were recorded.
5. After the data collection, the rat was sacrificed with the over dose of anesthesia or cervical dislocation while still under anesthesia.

The present study proposes an innovative practical teaching experiment to demonstrate the phenomenon of fatigue and site of fatigue. In this in-situ nerve-muscle preparation there are three possible sites of fatigue: the nerve, neuromuscular junction and the muscle. Nerve is practically unfatiguable. Direct muscle stimulation after development of fatigue showed same muscle twitch amplitude as initial, which confirms that muscle is not the primary site of fatigue. So by exclusion it can be demonstrated that in a nerve-muscle preparation, neuromuscular junction is the site of fatigue. With further extension, recording of compound action potential from sciatic nerve in a fatigued nerve-muscle preparation can demonstrate that nerve is not the site of fatigue.

We also demonstrated the effect of neuromuscular blocker, Pancuronium bromide, on muscle twitch amplitude, which decreased 3 to 4 minutes after injection. As Pancuronium bromide is a known neuromuscular blocker, twitch amplitude remained same as initial when muscle was stimulated directly. By demonstrating this practical, students can easily understand the phenomenon of fatigue, the site of fatigue, phenomenon of beneficial effect, contraction remainder and the effect of neuromuscular blocker on muscle twitch responses of a nerve-muscle preparation.

In conclusion, this novel approach of using mammalian nerve-muscle preparation for demonstrating the phenomenon and site of fatigue and effect of neuromuscular blocking is an effective undergraduate practical teaching module in physiology. The recordings from the present experiment can be modified as ‘objective structured practical examination’ (OSPE) questions for undergraduate practical assessment examinations.

Further, these experiments can be included as an individual practical module in post graduate assessment examination as it requires dissection skills, arrangement of stimulating and recording instruments and demonstration of several physiological phenomena in nerve-muscle physiology.