Quantifying Demyelination in NK venom treated nerve using its electric circuit model

Abstract

Reduction of myelin in peripheral nerve causes critical demyelinating diseases such as chronic inflammatory demyelinating polyneuropathy, Guillain-Barre syndrome, etc. Clinical monitoring of these diseases requires rapid and non-invasive quantification of demyelination. Here we have developed formulation of nerve conduction velocity (NCV) in terms of demyelination considering electric circuit model of a nerve having bundle of axons for its quantification from NCV measurements. This approach has been validated and demonstrated with toad nerve model treated with crude Naja kaouthia (NK) venom and also shows the effect of Phospholipase A2 and three finger neurotoxin from NK-venom on peripheral nerve. This opens future scope for non-invasive clinical measurement of demyelination.

Figure 1. A demyelinated nerve consists of bundle of axons.

(a) a bundle of nerve denoting three nerves – nerve 1, nerve 2 and nerve 3 respectively where the Node of Ranvier of axons are misaligned by alignment factor A (where  ≤ A ≤ 1). A = 1 indicates that two axons are aligned exactly, and indicates that two axons are evenly staggered. “L” is the internodal length i.e., the distance between two internodes in the nerve. (b) Typical SEM image of normal nerve having bundle of axons in which axon 1 is surrounded by six axons. (c) Correspondingly to SEM image we consider axon 1 is surrounded by six axons of equal diameter. We consider only ephaptic interactions of six surrounded axons on neuro conduction in axon 1. (d) Electric circuit model corresponds to bundle of axons in a nerve shown in the figure. (e) Nerve conduction velocity (NCV in m/s) versus demyelinating factor is obtained by using equation (1). NCV is formulated by using electric circuit model of a nerve having bundle of axons misaligned by alignment factor, . The NCV of toad sciatic nerve decreases with increase of demyelination. The experimental points of black dots are obtained from conduction of action potential (latency between proximal and distal action potential) in sciatic nerve of frog model demyelinated by Naja kaouthia crude venom with concentration 0.1, 1.0 and 10 μg/ml. Similarly experimental points of triangle are obtained from sciatic nerves treated with NK-PLA₂ (purified from crude venom). The inset of Fig. 1(e) shows demyelination ΔD in μm versus concentration of crude venom/NK-PLA₂ (ΔD is the difference between normal nerve thickness and demyelinated nerve, obtained from SEM images shown in Fig. 2). The solid line and dashed line in inset figure are drawn by using experimental points with minimum deviation. The lines are almost close to each other proving NK-PLA₂ mainly responsible for demyelination of the nerve of toad model.

Figure 2. Effect of crude venom with different concentration on sciatic nerves of toad and their nerve conduction signals (proximal and distal action potential) with corresponding SEM images of their sciatic nerves.

We have repeated the experiments for six times and estimated results statistically (as shown in supplementary table S1 and supplementary Fig. S1(a)). (A) SEM image of normal sciatic nerves with myelin thickness of 1.79 ± 0.23 μm at γ = 0 (normal). (a) Corresponding neuro conduction signal obtained by AD instrument (Powelabs, Australia) consists of proximal and distal action potential. The NCV is determined by using distance between two electrodes and divided by latency between proximal and distal action potential (shift between peaks of proximal and distal action potential) as 32.1 ± 1.71 m/s, which is very close to normal value of toad nerve. (B) SEM image of toad sciatic nerve treated with 0.1 μg/ml crude NK-venom shows slight reduction of myelin sheath, the thickness measured is 1.22 ± 0.15 μm corresponding to demyelinating factor, γ = 0.32 (which represents 32% reduction of myelin thickness). (b) Corresponding neuro conduction signal provides NCV of 21.95 ± 0.99 m/s (determined from latency between proximal and distal action potential peaks) which is slight less than the normal value. (C) SEM image of toad sciatic nerve treated with 1 μg/ml crude NK-venom shows more reduction of myelin thickness of 1.00 ± 0.9 μm (i.e., more demyelination) with γ = 0.44 (which indicates 44% reduction of myelin thickness). (c) NCV is determined from neuro conduction signal as 18.33 ± 1.2 m/s which is less than normal value. (D) SEM image of toad sciatic nerve treated with 10 μg/ml crude NK-venom shows reduction of myelin sheath, the myelin thickness of which is found to be 0.91 ± 0.08 μm corresponding to demyelinating factor, γ = 0.49 (which shows 49% reduction of myelin thickness). (d) NCV is estimated from latency of neuro conduction signal as 14.28 ± 0.85 m/s which is far below normal value.

Figure 3. Effect of purified NK-PLA₂ with different concentration on sciatic nerves of toad and their nerve conduction signals (proximal and distal action potential) with corresponding SEM images of their sciatic nerves.

We have performed the experiments repeatedly and the myelin thickness with its demyelinating factor is estimated statistically (as shown in supplementary table S1 and supplementary Fig. S1(b)). (A) SEM image of sciatic nerve with 0.1 μg/ml NK-PLA₂ with myelin thickness of 1.4 ± 0.29 μm with γ = 0.21 (which shows 21% reduction of myelin thickness). (a) Corresponding neuro conduction signal obtained by AD instrument and NCV is obtained from distance between two electrodes divided by latency between proximal and distal action potential (shifts between peaks of proximal and distal action potential). (B) SEM image of toad sciatic nerve treated with 1 μg/ml NK-PLA₂ shows a reduced thickness of myelin of 1.12 ± 0.34 μm corresponding to demyelinating factor, γ = 0.37 (which represents 37% reduction of myelin thickness). (b) Corresponding neuro conduction signal. (C) SEM image of toad sciatic nerve treated with NK-PLA₂ concentration of 10 μg/ml with more demyelination, the myelin thickness being 0.89 ± 0.15 μm, the demyelinating factor being γ = 0.5(which indicates 50% reduction of myelin thickness). (c) Corresponding neuro conduction signal at 10 μg/ml of NK-PLA₂. The SEM images shows increase of demyelination with increase in purified NK-PLA₂ concentration, while the neuro conduction signals confirms the reduction of NCV as the concentration of purified NK-PLA₂ increases.

Figure 4. Mechanism of channel blocking in cell membrane.

We have performed channel blocking of sciatic nerve using 3FTx neurotoxin for 5–6 times and the results obtained from repeated experiments are almost same. We have shown the results which are estimated statistically. (a) transportation of Na⁺ into cell through cell membrane and K⁺ transportation leaving from cell through membrane and provides saltatory movement of the nerve impulse (action potential). (b) Shows less blocking of Na⁺ and K⁺ channel by NK crude venom in which 3FT weak neurotoxin binds with acetylcholine receptors (AchR) and then blocks channel. Number of blocking is small if crude venom concentration is less. (c) Represents more blocking if crude venom concentration becomes more because of presence of more 3FT neurotoxin. (d) Represents most of channels are blocked if crude venom concentration is very high (more than 10 μg/ml). (e) Neuro conduction signal (proximal and distal action potential) obtained by using AD instruments. There is a reduction of peak of distal action potential with respect to proximal action potential showing Na⁺ and K⁺ channel blocking. (f) Neuro conduction signal of 1 μg/ml 3FTx-weak neurotoxin treated sciatic nerve shows a sharp reduction of peak of action potential. (g) Neuro conduction signal of 10 μg/ml 3FTx neurotoxin shows almost same sharp reduction of distal action potential peak. As in 1 μg/ml 3FTx treated sciatic nerve, most of channels are blocked (Fig. 4(d)), same reduction of distal action potential was observed even after treatment of 10% 3FTx treated sciatic nerve. (h) SEM image of toad sciatic nerve treated with 10 μg/ml 3FTx shows no reduction of myelin sheath even at high concentration. The myelin thickness of which is found to be 1.74 ± 0.18 μm, which is similar to normal sciatic nerve.

Figure 5. A schematic picture of the recording set up.

AD Instrument consists of a dual Bio Amp/stimulator and a nerve chamber. The dual Bio Amp/stimulator is used to obtain and record CAP and NCV. The nerve chamber is equipped with 15 stainless steel electrodes and the dissected nerve is mounted on it filled with Ringer’s solution. The setup was comprised of two male BNC (Bayonet Neill–Concelman) connectors to three micro-hooks constructed of gold-plated beryllium copper which was used to stimulate the nerve. CAP was analyzed by SCOPE (Powerlabs, Australia).