Indian Journal of Physiology and Pharmacology
Medical Education / Method Paper
EStudy of Neuromuscular Transmission Under (i) Phenomenon of Fatigue, (ii) Site of Fatigue, (iii) Neuromuscular Blocking in an in-situ Rat Nerve Muscle Preparation: A Novel Approach to Nerve Muscle Physiology Experiment Teaching
Arani Das, Arpita Chakraborty, Shival Srivastav, Simran Kaur, Suman Jain and Geetanjali Bade*
Department of Physiology,
AIIMS, New Delhi
Dr. Geetanjali Bade, Department of Physiology, AIIMS, New Delhi, Email: firstname.lastname@example.org
Purpose of the study: Nerve-muscle physiology is a very basic and vital module in undergraduate physiology curriculum. The practical sessions on this system are demonstrated on amphibian (frog) nerve-muscle preparation, which becomes a limiting factor in most of the medical colleges of India. In this study, we propose an alternative approach by using mammalian (rat) model for nerve-muscle physiology undergraduate practical experiments.
Materials and methods: Rat in-situ sciatic nerve-soleus muscle preparation was used to study neuromuscular transmission. Stimulation of nerve and recording of muscle contraction (force) were done by using digital recording system. To demonstrate fatigue, repetitive electrical stimulation was applied to nerve-muscle preparation and muscle twitches were recorded. A reduction in amplitude of contraction to 50% of their basal recording was considered as onset of fatigue. To demonstrate site of fatigue, the muscle was stimulated directly after the fatigue was observed with nerve stimulation and muscle twitch recorded. To observe the effect of neuromuscular blocking drug Pancuronium bromide, on neuromuscular transmission, in a separate set up the drug was injected in muscle belly at multiple sites and nerve was stimulated to elicit muscle twitch. The response (amplitude of muscle twitch) was compared with control (injection of 0.9% saline).
Main findings: On repeated stimulation of nerve muscle preparation, initially there was an increase in amplitude of contraction but progressively amplitude went on decreasing. After development of fatigue on direct muscle stimulation, amplitude recorded was same as the initial twitch amplitude. This demonstrates that the site of fatigue is not the muscle. Further, as nerve is also non- fatigable, site of fatigue was neuromuscular junction.
Injection of Pancuronium bromide showed that the twitch amplitude decreased substantially than the control (saline injection) on stimulation of nerve, but on direct muscle stimulation amplitude of contraction remained same as initial, confirming the effect of drug on neuromuscular junction.
Conclusion: In-situ rat nerve-muscle preparation can be used as an alternative approach to amphibian experiments for effective demonstration of neuromuscular transmission.
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 nervemuscle 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.
Materials and Methods
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
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.
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.
Specific learning objective 2:
To demonstrate the neuromuscular blocking effect of Pancuronium bromide in an in situ nerve-muscle preparation of rat
The representative records show the muscle twitch responses before and after fatigue followed by direct muscle stimulation and after injection of normal saline or Pancuronium bromide in Fig. 3 to 7.
On repeated stimulation of nerve muscle preparation, for the first few contractions, increase in amplitude was recorded which is termed as beneficial effect (Fig. 2). As stimulation was continued, there was progressive increase in latent period and decrease in amplitude (Fig. 3). As the fatigue started developing relaxation became incomplete and there was shifting of baseline, which is called as contraction remainder as shown in Fig. 2. After development of fatigue (twitch amplitude decreased to 50% of initial) when muscle was stimulated directly amplitude recorded was the same as the initial twitch amplitude (Fig. 4).
Injection of Pancuronium bromide decreased twitch amplitude substantially as compared to control (0.9% saline injection) secondary to nerve stimulation, but on direct muscle stimulation, twitch amplitude recorded was the same as initial (Fig. 5 to 7).
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.