Anatomy & physiology of neuromuscular junction & monitoring

Presented by

Dr.Rashmi.GChairperson Dr.M.Jajoor

MD,DA

PROFESSOR & HOD

INTRODUCTIONRed Indians used arrow poison to hunt their

food, using Curare as the poison. Unknown to them they laid the foundation of blocking the NMJ.

Through subsequent ages new methods have been studied and technique refined to produce a blockade where we control the patient.

Curare was first used clinically in 1942 by Griffith and Johnstone.

DEFINITIONThe NMJ is a synapse at which an electrical

impulse travelling down a motor nerve, releases chemical transmitter which cause the muscle to contract.

PHYSIOLOGY OF NEUROMUSCULAR TRANSMISSION

NMJ is specialised on the nerve side and on the muscle side to transmit and recieve chemical messages.

Each motor neuron runs without interruption from the ventral horn of spinal cord to NMJ as a large myelinated axon.

As it approaches muscle it branches to contact many muscle cells together into functional group known as Motor unit.

PARTS OF NMJThe anatomy of NMJ consist of following

parts:1.Pre-synaptic membrane2.Synaptic cleft3.Post-Synaptic membrane4.Contractile apparatus• The nerve is separated from the surface of

the muscle by a gap of about 20nm called junctional cleft.

• Presynaptic membrane contains prejunctional acetylcholine receptors and active zone.

Synaptic cleft: Lies Between the muscle endplate and nerve terminal which are held in tight alignment by basal lamina.

Post synaptic membrane – acetylcholine receptors: At the post synaptic membrane the area overlying the nerve terminal is called muscle end plate. The membrane here is thrown into primary and secondary clefts.

At the shoulder of these clefts numerous acetylcholine receptors are present.

The acetylcholine receptors are nicotinic and are of following types

a.Junctional or mature

b.Extra junctional or immature

Acetyl choline receptors/Post junctional receptors:

Present in the post junctional membrane of the motor end plate & are of nicotinic type. These receptors exist in pairs.

It consists of protein made up of 1000 amino acids, made up of 5 protein subunits designated as alpha, beta, delta and epsilon joined to form a channel that penetrates through and projects on each side of the membrane.

In the fetus gama replaces epsilon subunit.

Each receptor has central funnel shaped core which is an ion channel, 4 nm in diameter at entrance narrowing to less than 0.7nm within the membrane.

The receptor is 11 nm in length and extends 2nm into the cytoplasm of the muscle cell.

The receptor has 2 gates, an Upper voltage- dependent and a Lower time-dependent.

When acetylcholine receptors bind to the pentameric complex, they induce a conformational change in the proteins of the alpha subunits which opens the channel and occurs only if it binds to both the alpha binding sites.

For ions to pass through the channel both the gates should be open.

Cations flow through the open channel, sodium and calcium in and potassium out, thus generating end plate potential.

Na ions are attracted to the inside of the cell which induces depolarisation.

PREJUNCTIONAL RECEPTORSThese are nicotinic receptors that control ion

channel specific for calcium which is essential for synthesis and mobilization of acetylcholine.

They contain protein subunits that are blocked by non depolarising muscle relaxants resulting in fade and exhaustion.

They are also blocked by aminoglycosides and polymyxin antibiotics

EXTRAJUNCTIONAL RECEPTORThese tend to be concentrated around the end plate,

where they mix with post junctional receptors but may be found anywhere on the muscle membrane. In them, the adult epsilon subunit is replaced by the fetal gamma subunit.

They are not found in normal active muscle, but appear very rapidly after injury or whenever muscle activity has ended.

They can appear within 18hrs of injury and an altered response to neuromuscular blocking drugs can be detected in 24hrs of the insult.

When a large number of extrajunctional receptors are present, resistance to non-depolarising muscle relaxants develops, yet there is an increased sensitivity to depolarising muscle relaxants.

In most extreme cases, increased sensitivity to succinylcholine results in lethal hyperkalemic receptors with an exaggerated efflux of intracellular potassium.

The longer opening time of the ion channel on the extrajunctional receptor also results in larger efflux.

CONTRACTILE APPARATUSIt is formed by thin actin , thick myosin

filaments tropomyosin & troponin.

The shortening of this apparatus causes the contraction of the muscle

Anatomy_and_Physiology_Neuromuscular_Junction_TVLesson.flv

Ach (Synthesis, storage, release)Synthesized in the Presynaptic terminal from

substrate Choline and Acetyl CoA. CAT CHOLINE + ACETYL CoA ACETYL

CHOLINE COMT 50% Carrier Facilitated Transport

Release

CHOLINE + ACETYL CoA ACETYL CHOLINE

Synaptic Cleft

Different pools of acetylcholine in the nerve terminal have variable availability for release

a)The immediately releasable stores, VP2: Responsible for the maintainance of transmitter release under conditions of low nerve activity. 1% of vesicles

b)The reserve pool, VP1: Released in response to nerve impulses. 80% of vesicles

c)The stationary store: The remainder of the vesicles.

Each vesicle contains approx 12,000 molecules of acetylcholine, which are loaded into the vesicles by an active transport process in the vesicle membrane involving a magnesium dependent H+ pump ATPase.

Contents of a single vesicle constitute a quantum of acetylcholine.

Release of acetylcholine may bea)Spontaneous orb)In response to a nerve impulse.

When a nerve impulse invades the nerve terminal, calcium channels in the nerve terminal membrane are opened up.

Calcium enters the nerve terminal and there is calcium dependant synchronous release of the contents from 50-100 vesicles.

The number of quanta released by each nerve impulse is very sensitive to extracellular ionized calcium concentrations. Increased calcium concentration results in increased quanta released.

To enable this, vesicle must be docked at special release sites (active zones) in that part of the terminal where the axonal membrane faces the postjunctional acetylcholine receptors.

These are vesicle from the immediately releasable stores

Once the contents have been discharged, they are rapidly refilled from the reserve stores.

The reserve vesicles are anchored to actin

fibrils in the cytoskeleton, by vesicular proteins called synapsins

Some calcium that enters the axoplasm, on the arrival of the nerve impulse binds to calmodulin, which activates protein kinase-2 which phosphorylates synapsins, which, in turn dissociates the vesicle from the actin fibrils allowing it to move forward to the release site.

Docking of the vesicle and subsequent discharge of acetylcholine by exocytosis, involves several other proteins.

Membrane protein called SNAREs ( Soluble N-ethylmatrimide sensitive attachment proteins) are involved in fusion, docking, and release of acetylcholine at the active zone.

SNARE includes – synaptic vesicle protein synaptobrevin, synataxin and SNAP-25.

The released acetylcholine diffuses to the muscle type nicotinic acetylcholine receptors which are concentrated at the tops of junctional folds of membrane of the motor end plate.

Binding of acetylcholine to these receptors increases Na and K conductance of membrane and resultant influx of Na produces a depolarising potential, end plate potential.

The current created by the local potential depolarise the adjacent muscle membrane to firing level.

Acetylcholine is then removed by acetylcholinesterase from synaptic cleft, which is present in high concentration at NMJ.

Action potential generated on either side of end plate and are conducted away from end plate in both directions along muscle fiber.

The muscle action potential in turn initiates muscle contraction

STRUCTURE OF NA CHANNELThis Na channel is cylindrical Has membrane proteinIts two ends act as gatesBoth should be open to allow passage of ions.Voltage dependent gate is closed in resting

state and opens only on application of a depolarising voltage, remains open as long as the voltage persists

The time dependent gate is normally open at rest closing a few milliseconds after the voltage gate opens and remains closed as long as the voltage gate is open

It reopens after the voltage gate closes.

The channel is patent, allowing sodium ions only when the gates are open.

POSSIBLE CONFIGURATION OF Na CHANNELS• Resting state: Voltage gate closed Time gate open Channel closed

• Depolarization: Voltage gate open Time gate open Channel open

• With in a few milliseconds: Voltage gate open Time gate closed Channel closed

• End of depolarization: Voltage gate closed Time gate open Channel closed

ROLE OF CALCIUMThe concentration of calcium and the length of

time during which it flows into the nerve ending, determines the number of quanta release.

Calcium current is normally stopped by the out flow of potassium.

Calcium channels are specialized proteins, which are opened by voltage change accompanying action potentials

Part of calcium is captured by proteins in the endoplasmic reticulum & are sequestrated.

Remaining part is removed out of the nerve by the Na/Ca antiport system

The sodium is eventually removed from the cell by ATPase

ACETYLCHOLINESTERASEThis protein enzyme is secreted from the

muscle, but remain attached to it by thin stalks of collagen, attached to the basement membrane.

Acetylcholine molecules that don’t interact with receptors are released from the binding site & are destroyed almost immediately by acetylcholinesterase, in <1 ms, after its release into the junctional cleft.

PHYSICAL CHANNEL BLOCKADEVarious drugs can block the neuromuscular

junction and prevent depolarisation.

Blockade can occur in two modesa)Blocked when openb)Blocked when closed

OPEN CHANNEL BLOCKIn this, the drug molecule enters a channel

which has been opened by acetylcholine.

This is use dependent block

Physical blockade by a molecule of an open channel relies on the open configuration of the channel and the development of this is proportional to the frequency of channel opening.

This mechanism may explain the synergy that occurs with certain drugs such as local anaesthetic, antibiotics and muscle relaxants.

In addition, the difficulty in antagonizing profound neuromuscular blockade may be due to open channel block by the muscle relaxants

CLOSED CHANNEL BLOCKThe drugs occupy the mouth of the channel and

prevents ions from passing through the channel to depolarise the end plate.

Tricyclic drugs and naloxone may cause physical blockade of a closed by impending interaction of acetylcholine with the receptor.

For drugs interfering with the function of the acetylcholine receptor, without acting as an agonist or antagonist, the receptor lipid membrane interface may also be another site of action.

Eg: Volatile agents, Local anaesthetic and Ketamine

INTRODUCTIONNeuromuscular monitoring is based on two

important issues: 1. on the variable response to muscle relaxants and 2 because of the narrow therapeutic window.

There is no detectable block until 75 to 85% of receptors are occupied and paralysis is complete at 90 to 95% receptor occupancy.

Neuromuscular monitoring permit optimal surgical relaxation and reverses the block spontaneously or revesed quickly with antagonists.

Residual neuromuscular block is a major risk factor for many critical events in the immediate postoperative period such as ventilatory insufficiency, hypoxemia and pulmonary infections.

The most satisfactory method is by peripheral nerve stimulator and observation of evoked response in the muscle supplied.

FEATURES OF NEUROSTIMULATION

Key features of exogenous nerve stimulation are:• Nerve stimulator: A battery powered device that

delivers depolarizing current via the electrodes.

• Pulse width: Is the duration of the individual impulse delivered by the nerve stimulator.

• Each impulse should be <0.5msec and 0.1sec in duration to elicit nerve firing at a readily attainable current. Pulse width >0.5msec extends beyond the refractory period of the nerve resulting in repetitive firing.

• Current intensity: Is the amperage(mA) of the current delivered by the nerve stimulator. The current output of most stimulators can range from 0-80mA.

• Supramaximal current: Is approximately 10-20% higher intensity than the current required to depolarize all fibres in a particular nerve bundles.

• Submaximal current: A current intensity that induces firing of only a fraction fibres in a given nerve bundle and is less painful

• Stimulus frequency: The rate (Hz) at which each impulse is repeated in cycles per second (Hz).

• Single twitch is commonly repeated at 10 second intervals i.e. 0.1 Hz and

• Tetanic stimulation commonly consists of 50 impulses/ sec i.e. 50 Hz.

ELECTRODESSurface Electrodes: They contain gel conducting

surfaces for transmission of impulses to the nerves through the skin. With careful skin preparation the threshold for which response is generally <15mA.

Needle Electrodes: Subcutaneous needles deliver the impulse in the immediate vicinity of the nerve. These are highly effective because they bypass the tissue impedance so that the tissue impedance is typically <2000 Ohms.

Single twitch: This is the simplest form 0f neurostimulation entailing a single twitch at 0.1 to 0.12 msec.

Single twitch is delivered at a supramaximal current, it induces a single nerve action potential in each fiber of the nerve bundle.

During nondepolarizing block the response to single twitch stimulation is not reduced until atleast 75 to 80% of receptors are occupied and therefore does not detect block of less than 70%.

Train of four(TOF): This is a popular mode of stimulation for clinical

monitoring of neuromuscular junction first described by ali et al.

Four successive stimuli are delivered at 2 Hz (every 0.5sec). In the presence of non depolarizing relaxants, the margin of safety is decreased such that some end plates in train of four progressively fade.

In the absence of non depolarizing block, the T4/T1 ratio is approximately one.

For complete recovery T4/T1 ratio should be more than 0.9

ADVANTAGES OF TOF STIMULATIONThis pattern of stimulation can be applied at

anytime during the neuromuscular block and can provide quantification of depth of block without the need for control measurement before relaxant administration.

It is more sensitive to lesser degree of receptor occupancy than single twitch.

The relatively low frequency allows response to be evaluated manually or visibly.

There is no post tetanic facilitation therefore can be repeated every 10 to 12 sec.

It may be delivered at sub maximal current which is less painful and is associated with same degree of fade.

TETANIC STIMULATIONHigh frequency stimulation (50Hz or more)

results in sustained or tetanic contraction of the muscle during normal neuromuscular transmission despite decrement in acetylcholine release.

During tetanus, progressive depletion of acetylcholine output is balanced by increased synthesis and transfer of transmitter from its mobilization stores.

The presence of nondepolarizing muscle relaxants reduces the margin of safety by reducing the number of free cholinergic receptors and also by impairing the mobilization of acetylcholine within the nerve terminal there by contributing to the fade in the response to tetanic and TOF stimulation.

A frequency of 50Hz is physiological as it is similar to that generated during maximal voluntary effort. Fade is first noted at 70% receptor occupancy.

It has been shown that tetanic response to 50 Hz for five sec is sustained when TOF ratio is greater than 0.7.

DISADVANTAGES

• Is post tetanic facilitation which depends on frequency and duration of block

• It is very painful and therefore not suitable for unanaesthetised patients.

DOUBLE BURST STIMULATIONTOF ratio of less than 0.2 to 0.3 is difficult to

detect even by trained observers. To improve the detection rate, a new mode of

stimulation which consist of two short tetani, separated by a interval long enough to allow relaxation, evaluating the ratio of second to first response has been proposed.

Many patterns have been suggested but the most promising one consists of two train of three impulse of 50 Hz separated by 750msec.

POST- TETANIC COUNT (PTC)Tetanus at 50 Hz for five seconds is applied

followed 3 sec later by single twitch stimulation at 1 Hz.

The number of evoked post-tetanic twitches detected is called the post-tetanic count (PTC).

PTC is a prejunctional event, the response can vary with the nondepolarising muscle relaxant used.

A PTC of 8 to 9 indicated imminent return of TOF.

APPLICATION OF PTCEvaluating the degree of neuromuscular

blockade when there is no reaction to single twich or TOF as after administration of large dose of nondepolarizing muscle relaxant.

PTC can also be used whenever sudden movement must be eliminated (Ophthalmic Surgery). Elimination of responses to tracheobronchial stimulation requires intense neuromuscular blockade of zero PTC.

PTC can be used during continuous infusion of intermediate nondepolarizing muscle relaxant as a guidance to intensity of neuromuscular blockade.

PTC predicts time to reappearance of first response to TOF stimulation

MONITORING SITES

The specific nerve-muscle site utilized for monitoring has drawn interest in the recent years because of the variability among muscle groups in sensitivity and onset time

Relative sensitivities of muscle groups to nondepolarizing muscle relaxants

MUSCLES SENSITIVITY

Vocal cord Most Resistant

Diaphragm

Orbicularis oculi

Abdominal rectus

Adductor pollicis

Masseter

Pharyngeal

Extraocular Most sensitive

ULNAR NERVEThe nerve is most commonly used for neuromuscular

monitoring in the perioperative period.

The ulnar nerve innervates the adductor pollicis, abductor digiti mimimi, abductor pollicis brevis and dorsal interosseous muscles.

One stimulating electrode is typically placed more than 2cm proximally on the volar forearm or over the olecranon groove.

The recording electrodes are placed over the

appropriated muscle.

FACIAL NERVE:

The response to the stimulation is monitored commonly at the orbicularis oculi (contraction of eyebrow) and orbicularis oris(contraction of the lip)

NERVES OF THE FOOTThe posterior tibial nerve may be stimulated

as it comes behind the medial malleolus, caused plantar flexion of the great toe and foot.

The peroneal nerve and lateral popliteal nerve elicit dorsi flexion of the foot

ASSESSMENT OF EVOKED RESPONSE TO NEUROMUSCULAR STIMULATIONVisual or tactile assessment: Visual or tactile

methods of evaluation of the evoked response to stimulation is the simplest means of assessment

During recovery of neuromuscular function all responses of TOF can be felt.

An estimation of TOF ratio may be attempted but the method is not sensitive enough to exclude possibility of residual neuromuscular blockade.

Fade is usually undetected until TOF ratio values are less than 0.5

Greater sensitivity for fade detection is achieved with DBS

RECORDING DEVICES FOR MEASURING NEUROMUSCULAR FUNCTIONCompound muscle action potential: It is the

cumulative electrical signal generated by the individual action potentials of the individual muscle fibres.

Electromyogram(EMG): It records the compound MAP via recording electrodes place near the mid portion or motor point of the muscle.

The latency of the compound MAP is the interval between stimulus artifact and evolved muscle response

The amplitude of the compound MAP is proportional to the number of muscle units that generate an MAP within the designated time interval (epoch) and this correlates with the evoked mechanical responses.

This method is used mostly for experimental studies

ACCELEROMYOGRAPHYThis technique used a miniature piezoelectric

transducer to determine the rate of angular acceleration.

This is a nonisometric measurement and there are less stringent requirements for immobilization of arm, fingers and thumb and also no preload is necessary.

However recording of tetanic responses is not possible as the movement is an essential component of accelerography.

It is a simple method useful in operating room and in the intensive care unit.

CLINICAL APPLICATIONTo differentiate depolarising block and Non-

depolarising blockTo see efficacy of depolarising block after

administration of drug and to judge whether the patient is fully relaxed

To see whether the pt is out of effect of block of depolarising muscle relaxed

As a guide to administer first dose of non-depolarising muscle relaxant

As a guide to see whether completeness of non-depolarising neuromuscular block

As a guide to for starting of reversal of non-depolarising block

To see a completeness of recoveryAs a guide for incremental doses administration

of non-depolarising muscle relaxantTo differentiate respiratory paralysis i.e.

central or peripheral due to nueromuscular block

To diagnose overdose of sedatives cerebral depressants or muscle relaxants

To diagnose phase II block after suxamethonium

To diagnose various neuromuscular disordersTo diagnose site of nerve injuryTo diagnose electrolyte imbalance or

disturbances affecting NM TransmissionAs a guide in diagnosis of prolonged apnea or

recovery after balanced anaesthesia

WHICH PATIENT SHOULD BE MONITOREDBy the foregoing discussion, it would seem

prudent to monitor NMJ in all pts receiving NMBs.

Such monitoring is advisible particularly in conditions where the pharmacokinetics and pharmacodynamics of NMBs are altered significantly as listed below:

Several renal, liver diseaseNeuromuscular disorders such as myasthenia

gravis, myopathies , and upper and lower motor neuron lesions

Pts with severe pulmonary disease or marked obesity to ensure adequate recovery of skeletal muscle function.

Neuromuscular blockade achieved with continuous infusion of NMBs

Pts receiving long-acting NMBs

Pts undergoing lengthy surgical procedures

LIMITATIONS OF NEUROMUSCULAR MONITORINGDespite the important role of NMJ monitoring in

anaesthesia practice, it is necessary to use a multifactorial approach for the following reasons:

Neuromuscular responses may appear normal despite persistence of receptor occupancy y NMBs. T4:T1 ratio is 1 even when 40-50% of the receptors are occupied.

Because of wide individual variability in evoked responses some pts may exhibit weakness at TOF ratio as high as high as 0.8 to 0.9

The established cut-off values for adequate recovery do not guarantee adequate ventilatory function or airway protection

Increased skin impedence resulting from perioperative hypothermia limits the appropriate interpretation of evoked responses

REFERENCES:1. Millers text book of anesthesia,7th ed.2. Clinical anesthesia,Barasch3. Morgan`s principles of anesthesia4. Textbook of physiology,Ganong.5. Snell`s Textbook of anatomy.6. Guyton & Hall Textbook of physiology.7. RACE 2005 & 20028. ISACON 2009

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