Brain Advance Access originally published online on September 29, 2005
Brain 2005 128(12):2987-2996; doi:10.1093/brain/awh642
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Envenoming bites by kraits: the biological basis of treatment-resistant neuromuscular paralysis
School of Neurology, Neurobiology and Psychiatry, Faculty of Medical Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne, UK 1 Present address: Department of Biology, Naresuan University, Phitsanulok 65000, Thailand
Correspondence to: J. B. Harris, School of Neurology, Neurobiology and Psychiatry, Faculty of Medical Sciences, University of Newcastle upon Tyne, NE2 4HH, UK E-mail: j.b.harris{at}ncl.ac.uk
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ß-Bungarotoxin, a neurotoxic phospholipase A2 is a major fraction of the venom of kraits. The toxin was inoculated into one hind limb of young adult rats. The inoculated hind limb was paralysed within 3 h, and remained paralysed for 2 days. The paralysis was associated with the loss of synaptic vesicles from motor nerve terminal boutons, a decline in immunoreactivity of synaptophysin, SNAP-25 and syntaxin, a loss of muscle mass and the upregulation of NaV1.5 mRNA and protein. Between 3 and 6 h after the inoculation of toxin, some nerve terminal boutons exhibited clear signs of degeneration. Others appeared to be in the process of withdrawing from the synaptic cleft and some boutons were fully enwrapped in terminal Schwann cell processes. By 12 h all muscle fibres were denervated. Re-innervation began at 3 days with the appearance of regenerating nerve terminals, a return of neuromuscular function in some muscles and a progressive increase in the immunoreactivity of synaptophysin, SNAP-25 and syntaxin. Full recovery occurred at 7 days. The data were compared with recently published clinical data on envenoming bites by kraits and by extrapolation we suggest that the acute, reversible denervation caused by ß-bungarotoxin is a credible explanation for the clinically important, profound treatment-resistant neuromuscular paralysis seen in human subjects bitten by these animals.
Key Words: neuropharmacology; neuropathy; neuropathology; neuromuscular junction; neurobiology; snake-bite; krait
Abbreviations: NMJ = neuromuscular junction; PBS = phosphate-buffered saline.
Received April 28, 2005. Revised August 1, 2005. Accepted August 30, 2005.
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The kraits of Southeast Asia are elapid snakes represented by 12 species within the single genus Bungarus (Keogh, 1998
). They are generally secretive, unaggressive nocturnal animals that frequently enter rural dwellings in search of their prey. Envenoming bites on human subjects typically occur when the snakes are disturbed by the sleeping occupant (Kularatne, 2002). The clinical features of envenomed subjects have been recently defined in a major retrospective study of 210 subjects who had been bitten by the common krait, Bungarus caeruleus, in Sri Lanka (Kularatne, 2002). Most subjects exhibited ptosis, exophthalmoplegia, dysphagia, dyspnoea and neuromuscular weakness. A total of 101 subjects were severely poisoned (tidal volume <200 ml; neck flexor power <3; cyanosis; confusion; failing speech) and required mechanical ventilation. The time lag between bite and the initiation of ventilation ranged between 30 min and 50 h (mode 6 h) and the duration of ventilation ranged between 12 h and 29 days (mode 2 days). Mechanical ventilation was withdrawn on the basis of returning neck power and tidal volume but it was 8–9 days before neck power returned to normal. Neither polyvalent antivenom nor anticholinesterases provided any significant benefit in reversing the neuromuscular paralysis or reducing the duration of ventilatory support. Thirty-eight subjects exhibited delayed neurological signs such as nerve conduction deficits and one developed an ulnar nerve palsy. Electrophysiological studies have shown that envenoming bites by kraits are associated with a reduction in the compound muscle action potential and a decremental response to repetitive nerve stimulation (Singh et al., 1999). Other studies of definitive krait bites have also documented the severe respiratory paralysis, the need for prolonged ventilatory support and the difficulties associated with treatment of the human subject (Warrell et al., 1983; Sellahewa, 1987; Theakston et al., 1990; De Silva et al., 1993; Chan et al., 1995; Pe et al., 1997; Singh et al., 1999; Loathong and Sitprija, 2001).
The prolonged hospitalization required by many victims of envenoming bites by kraits places great demands on scarce resources in many parts of Southeast Asia. Antivenoms, which are costly, are ineffective but still used in the absence of any other pharmacological therapy. The possibility that some victims will express delayed neurological deficits (as described by Kularatne, 2002) has never been systematically studied but would be important in rural societies. The combined pressure on hospitals, economies and rural societies is immense, but no effective strategy for managing bites by these animals can emerge unless there is an understanding of the biological basis of the neurological features associated with the bites. The venom of kraits contains three major types of neurotoxin. 
-Bungarotoxins cause a failure of neuromuscular transmission by binding to post-synaptic nAChR at the neuromuscular junction (NMJ). Similar toxins are found in the venoms of all elapid snakes and their close relatives, the sea snakes (see Mebs and Claus, 1991). 
-Bungarotoxins are found exclusively in the venom of kraits. They are structurally similar to the -bungarotoxins, bind to neuronal nAChR but are minor components of the venom (Chiappinelli, 1991). The ß-bungarotoxins constitute >20% of the protein content of the venom and are the most toxic components of the venom. They are pre-synaptically active neurotoxic phospholipases A2. Exposure to these toxins in vivo and in vitro causes the failure of neuromuscular transmission for 2–3 h, and the depletion of synaptic vesicles from nerve terminal boutons is a primary pathological feature of toxicity (Chen and Lee, 1970; Dixon and Harris, 1999; Prasarnpun et al., 2004). Structural damage to the motor nerve terminal and terminal components of the motor axon follows rapidly and destruction of the nerve terminal is complete by 12–24 h (Chen and Lee, 1970; Chang et al., 1973; Abe et al., 1976; Dixon and Harris, 1999; Prasarnpun et al., 2004). It has been suggested that ß-bungarotoxin (alone or in combination with -bungarotoxin) is primarily responsible for the severe paralysis associated with envenoming bites by kraits. The underlying hypothesis is that the onset of paralysis is caused by the depletion of synaptic vesicles from the nerve terminal, the destruction of the terminal boutons explains the phase of profound treatment-resistant paralysis and the slow recovery of neuromuscular function reflects the regeneration of nerve terminals and the re-innervation of the denervated muscle fibres (Dixon and Harris, 1999; Singh et al., 1999; Prasarnpun et al., 2004). The problem with the hypothesis is that it relies on the interpretation of data collected from a number of unrelated studies, none of which had, as a primary consideration, the neurological features of envenoming bites by kraits. For ethical and practical reasons it is difficult to test these ideas directly in man because of the need for studies of neuromuscular pathology. We have, therefore, documented in detail the functional and morphological changes in the neuromuscular system of rats over the course of 7 days following a single inoculation of ß-bungarotoxin and we have compared the experimental data with relevant clinical reports. We conclude that the pharmacological activities of ß-bungarotoxin are sufficient to explain the treatment-resistant paralysis caused by envenoming bites by kraits.
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Materials and methods |
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Animals
Female outbred Wistar rats weighing 100–150 g were obtained from Bantin and Kingman, UK. They were maintained in full accord with the requirements of the Animals (Scientific Procedures) Act 1986, under the day-to-day care of a veterinarian. One week after delivery to the holding facility an inoculation of 0.2 ml of 0.9% w/v NaCl containing 2.0 µg of ß-bungarotoxin was made subcutaneously over the soleus muscle of the left hind limb (Harris and Johnson, 1978
). Buprenorphine (100 µg)—a veterinary analgesic—was administered concurrently by subcutaneous injection into the skin fold over the neck. All procedures had been formally approved by the local ethics committee and by the Home Office Inspectors. Animals were regularly examined for localized/systemic paralysis and evidence of discomfort (withdrawal, loss of exploratory behaviour, limb-biting, withdrawal of the inoculated limb during palpation).
Toxins and other reagents
ß-Bungarotoxin (cat. T5644) was purchased from Sigma Chemical Supplies. Fluorescein isothiocyanate (FITC)-conjugated -bungarotoxin (cat. F1176) was obtained from Molecular Probes Inc. Murine anti-synaptophysin monoclonal antibody (cat. S5768), anti-syntaxin monoclonal antibody (cat. S0664) and anti-SNAP-25 monoclonal antibody (cat. S9684) were purchased from Sigma Chemical Supplies. A monoclonal antibody recognizing a range of Na+ channels, including NaV1.4 and NaV1.5 was obtained from Sigma (cat. 58889; clone K58/35). A polyclonal antibody selective for NaV1.5 was obtained from Alomone (cat. ASC-005; lot AN-02). Primary anti-AChE antibody raised in rabbits against rat brain AChE was a gift from Dr A. Massoulie. Murine anti-neurofilament monoclonal antibody (RT97) was a gift from Dr N. Leigh. Rabbit anti-mouse rhodamine-conjugated polyclonal antibody (cat. R0270) and swine anti-rabbit rhodamine-conjugated polyclonal antibody (cat. R0156) were obtained from Dako. All secondary antibodies were incubated with rat serum and centrifuged to yield a clear supernatant before use. All other reagents were obtained from regular commercial sources and were routinely Analar grade.
Muscles
The soleus muscles were routinely used. The ipsilateral (toxin-treated) muscle was always processed alongside the contralateral (control) muscle. The muscles of humanely killed rats were surgically exposed, removed intact and weighed. For cryosectioning, a transverse, full width segment 5 mm long was cut from the belly of each muscle, mounted onto a strip of filter paper, supported with OCT embedding medium (BDH, Poole, Dorset, UK) and frozen in iso-pentane cooled in liquid nitrogen. Sections were cut at 6–10 µm and mounted on chrome alum/gelatine subbed slides. Muscles for electron microscopy were pinned out at 
1.2x resting length and fixed in Karnovsky's fluid (Karnovsky, 1965) for 1.5 h. The muscles were rinsed in phosphate buffer (pH 7.4) and teased into bundles of 10–15 fibres. End-plate AChE was localized by incubation in hexazotised paranosanilin and indoxyl acetate in citrate buffer (ice-cold, 1 h) as described by Strum and Hall-Craggs (1982). The resulting AChE reaction product formed a brick red/black deposit at end-plates. End-plate regions were cut out to form blocks 1 mm x 1 mm x 2 mm. The tissue blocks were post-fixed in OsO4 dehydrated in increasing strengths of acetone and embedded in TAAB resin (T032, TAAB Laboratory Equipment, UK). Ultra thin transverse sections were cut at 50–70 nm (gold/silver in colour), collected onto Athene Nero 300 grids, stained using uranyl acetate, hydrated and then counter stained in lead citrate.
NMJs were identified and photographed by one of us (J.W.) who was blind to the experimental status of the individual muscles. All junctions were photographed provided they were not obscured by a grid bar and were sitting within a deeply folded junctional trough. One hundred and sixty-two (162) junctions were examined; 38 controls, 14 at 3 h, 16 at 6 h, 11 at 12 h, 26 at 2 days, 23 at 3 days, 14 at 5 days and 20 at 7 days.
Neuromuscular function
Two methods were used to assess neuromuscular function. In the first, the limb/toe extension reflex was used. Animals were lifted by the tail before the inoculation of ß-bungarotoxin and again at 3, 6, 12 and 24 h, and then at 2, 3, 4, 5 and 7 days. The ability of the animal to extend the inoculated limb was arbitrarily scored on a scale of 0–4 where 0 = no extension and 4 = full extension. Function was also tested when muscles were surgically exposed prior to removal (see above). The exposed soleus nerves were electrically stimulated and the response of the muscle was classified as no response, weak response or powerful response.
Fluorescence labelling of AChE and nAChR
The primary anti AChE antibody (Ab) was diluted from stock 1:500 in 0.1 M phosphate-buffered saline (PBS), pH 7.4, containing 0.1 M lysine and 0.3% BSA. Tissue sections were exposed to the primary Ab for 1 h at room temperature and then washed 3 times in PBS over 30 min. Sections were then incubated with the rhodamine-conjugated secondary Ab and FITC-conjugated -bungarotoxin (1:200) in a moist chamber for 3 h. Slides were blotted dry, mounted in Vectashield and examined under the fluorescence microscope. To determine whether incubation in ß-bungarotoxin blocked nAChR the ‘labelling index’ was calculated. End-plates were recognized by the labelling of AChE. The number of those end-plates that co-labelled with FITC-conjugated -bungarotoxin (-BTX) was also counted. The calculation (-BTX/AChE) x 100 was termed the labelling index. Both the area and intensity of labelling by FITC-conjugated -bungarotoxin were measured using the software package Metamorph.
Fluorescence labelling of synaptic proteins
Tissue sections were permeabilized according to the primary antibody being used. For synaptophysin labelling, the sections were permeabilized in cold (–20°C) acetone for 10 min. For syntaxin and SNAP-25 labelling, the sections were pre-treated for 30 min at room temperature in 4% paraformaldehyde. They were then permeabilized in 0.l% Triton X-100 in PBS for 10 min at room temperature. After permeabilization, the sections to be labelled for syntaxin and SNAP-25 were rinsed in PBS 3 times over 15 min and the sections to be labelled for synaptophysin were air-dried. The relevant primary antibodies were diluted with 3% BSA and 0.1 M lysine in PBS to a final strength of 1/100, and were applied to the sections overnight in a closed moist chamber at 4°C. The following day, the sections were washed in PBS 3 times over 30 min, followed by incubation in secondary antibody (1:100 rabbit anti-mouse rhodamine-conjugated immunoglobulins) for 1 h at room temperature. Routinely, counterstaining of nAChRs was done by the inclusion of 1:200 FITC-conjugated -bungarotoxin in the secondary antibody solution. The sections were then washed 3 times in PBS over 30 min, and fixed in 1% paraformaldehyde for 30 min. The sections were again washed 3 times in PBS over 30 min before mounting in Vectashield. Some sections were treated in exactly the same way as the rest, but the primary antibody was omitted from the protocol. These internal ‘control sections’ were never labelled with the rhodamine-conjugated immunoglobulins.
Fluorescence labelling of NaV1 Na+ channels
Frozen transverse sections of ipsilateral and contralateral muscles, 6–10 µm thick were cut as described above and mounted on chrome-alum subbed slides. Sections were exposed to Triton (0.1%, room temperature, 10 min), washed and then incubated overnight at 4°C with either anti-NaV1 (dilution 1:100), or anti-NaV1.5 (dilution 1:200). Following incubation with the primary antibody, slides were incubated with the rhodamine-conjugated secondary antibody (diluted 1:100) containing FITC-conjugated -bungarotoxin (see above) for 1 h. The slides were then washed and processed as described above.
Confocal microscopy
A dual channel Bio-Rad laser confocal system (MRC 600) mounted on an Olympus upright microscope (BH2) and equipped with an Ar–Kr laser, was used to prepare images of labelled NMJs on tissue sections prepared as above. The 488-nm line of the laser was used to excite the fluorescein-labelled nAChRs at the end plates, and the 547-nm line to excite the rhodamine-labelled proteins. The emissions were collected separately into the two channels of the confocal system using 515- and 576-nm barrier filters. The optical section thickness to be scanned was set at 0.5 µm. The intensity threshold for control images was set at 100–250 Gy. Intensity thresholds were set using control sections and those thresholds were not changed for the duration of the examination of sections of toxin-treated muscles. Typically, single sections were randomly chosen from each of 3–4 pairs of muscles. Sections contained between 11 and 61 junctional profiles. All junctions seen in a single section were examined and the respective fluorescence intensities were measured using Cosmos Version 7 software. The average intensity of labelling on the ipsilateral section was expressed as a percentage of the average intensity of labelling on the contralateral section.
In situ hybridization
Anti-sense probes specific for NaV1.4 and NaV1.5 isoforms of NaV1 Na+ channels were used to study mRNA expression at the NMJs of muscles exposed in vivo to ß-bungarotoxin. The probes were prepared and used as described in detail by Awad et al. (2001). The experiments were made on frozen muscle sections 6 µm thick. Briefly, pGEM-T easy plasmids were linearized with Nco1 and Spe1 (NaV1.4) or Pst1 and Apa1 (NaV1.5) restriction enzymes and used as templates for phage T7 and SP6-driven IVT to produce either sense-specific or antisense-specific probes, depending on the orientation of the insert. Transcripts were double-labelled with 35S-CTP and 35S-UTP (>1000 Ci/mmol; Amersham Pharmacia Biotech). Sections were hybridized overnight with 104–105 c.p.m./µl 35S-labelled probes in 40 µl of hybridization buffer and washed as described previously (Young et al., 1998).
Autoradiography and analysis
Hybridized sections were air-dried, dipped in Kodak NTB2 emulsion (Anachem Scotlab, Luton, UK), exposed at 4°C for 7–10 days, developed for 3 min at 17°C in Kodak Dektol developer, and counterstained with 0.25% toluidine blue in 1% borax.
Labelled sections were viewed with a Leica (Nusslock, Germany) DMRA microscope, and images were recorded using both bright-field and dark-field optics, with a cooled CCD camera with 12 bit intensity resolution (SPOT 2; Diagnostic Instruments, Sterling Heights, MI). The distribution and intensity of labelling were determined from dark-field images, as described by Vater et al. (1998) using NIH Image software. The spatial distribution of labelling was determined from the mean signal intensity, expressed as grey levels per second exposure time per square micrometre (gl/s/µm2), in concentric annuli centred on the cluster being analysed. At most clusters the intensity of labelling declined to 30% of maximum at 12 µm from the centre of the grain cluster, as reported previously for utrophin mRNA (Vater et al., 1998). For subsequent comparisons of the intensity of labelling at different grain clusters, we used the total signal intensity within a circle of 12 µm radius, centred on the cluster.
The signal intensity of grain clusters at NMJs depended on the concentration of the radioactive probe used in the hybridization reactions. At 20 x 103 c.p.m./µl, the labelling intensity with the NaV1.4 probe was well above background but still only approximately two-thirds of that with 100 x 103 c.p.m./µl (data not shown). Thus, at the lower concentration, the emulsion and detection system were not saturated. A concentration of 20 x 103 c.p.m./µl was, therefore, used in all subsequent experiments with the NaV1.4 probe. Similarly, optimum labelling with the NaV1.5 probe was obtained at 30 x 103 c.p.m./µl, and this concentration was used in all experiments described.
Axon counts
Soleus nerves were ligated proximally where they joined the main branch of the sciatic nerve. They were then dissected distally and sectioned at the point of entry into the soleus muscle. The removed nerves were mounted under light tension onto dental wax, fixed in 3% glutaraldehyde in 0.1 M phosphate buffered saline and embedded in Araldite. Transverse sections 1 µm thick were made of the distal stumps and stained with toluidine blue. Photographs of the sections were used to count the numbers and measure diameters of all myelinated axons in the soleus nerve.
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Results |
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Animals inoculated with ß-bungarotoxin tolerated the toxin without loss of exploratory behaviour, mobility or general awareness. They did not object to being handled or to having the inoculated limb palpated. They ate and drank normally. There was no mortality. The only adverse effect was the paralysis of the inoculated limb.
Ultrastructural studies
Images of end-plates typically comprised 1–4 terminal boutons, each sitting in a clearly defined, deeply folded post-synaptic trough in the muscle fibre membrane. The terminal boutons of contralateral NMJs were filled with synaptic vesicles and mitochondria and were capped by terminal Schwann cells (Fig. 1A). Three to six hours after the inoculation of ß-bungarotoxin, terminal boutons on ipsilateral muscle fibres were depleted of synaptic vesicles and they exhibited significant mitochondrial damage. Many boutons appeared to be degenerating. Others were isolated from the post-synaptic membrane by Schwann cell processes invading the cleft between terminal bouton and post-synaptic membrane. Some boutons were shrunken, densely stained and appeared to be withdrawing from the synaptic cleft. (Typical images are shown in Fig. 1B and C.) These forms of nerve terminal pathology could often be seen together when sections of single NMJs cut through more than one terminal bouton (data not shown). By 12 h all muscle fibres examined were structurally denervated and synaptic troughs were either filled with debris or were empty and collapsed. Retraction bulbs were commonly identified in the vicinity of the synaptic region (Fig. 1D). Forty-eight hours after the inoculation of ß-bungarotoxin pseudopodial-like structures, devoid of any definitive sub-cellular organization, appeared in the empty synaptic troughs (Fig. 2A). These processes possessed no basal lamina, so are not interpreted as growth cones or immature axons. By 3 days, the invading ‘pseudopodia’ were more common and immature nerve terminals, invested with a basal lamina, could be identified at 25% of synapses (Fig. 2B). It was at 3 days that there was the first clear evidence of attachments forming between immature nerve terminals and post-synaptic structures (Fig. 2B). By 5 days nerve terminals, always small but filled with increasingly densely packed synaptic vesicles, were identified at 60% of synapses (Fig. 2C). By 7 days all synaptic troughs were occupied by small terminal boutons.
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nAChR, synaptophysin, SNAP-25 and syntaxin
At no stage was the labelling of nAChR affected by exposure to ß-bungarotoxin. The labelling index was between 93 and 100% at NMJs of contralateral and ipsilateral muscles and pixel intensity remained constant.
The pixel intensity of labelling of synaptophysin in ipsilateral muscles fell rapidly to 30% of that in contralateral muscles by 3 h and to 20% by 2 days. The loss of labelling of syntaxin and SNAP-25 was similar to that of synaptophysin, except that the rate of loss of labelling intensity was slightly slower. It was of some interest that at no time was there a total loss of immunoreactivity to the synaptic proteins synaptophysin, syntaxin and SNAP-25 even at 2 days when terminal boutons were missing from synaptic troughs. We suspect that terminal debris and the distal stumps of terminal axons contain remnants of immunoreative protein. There would be no physical evidence of this in the ultrastructural data but the presence of the protein would be picked up during the confocal fluorescence imaging of thicker sections at the level of light microscopy. During the recovery of neuromuscular function the intensity of labelling of synaptophysin increased so that by 7 days it was >75% of that in contralateral muscles. Recovery of labelling of SNAP-25 and syntaxin was slightly faster than that of synaptophysin. Typical images of labelling are shown in Fig. 3 and the fully analysed data are expressed graphically in Fig. 4.
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Muscle function and pathology
Within 3 h of the inoculation of ß-bungarotoxin there was a flaccid paralysis of the ipsilateral hind limb and the limb and toe-extension reflex was abolished. Exposed muscles failed to twitch when the soleus nerve was electrically stimulated in situ in freshly killed animals. The response of the contralateral limb was unaffected. Limb and toe extension returned in the ipsilateral limb at 3 days in 1 out of 12 animals tested. The extension response was weak (grade = 1). By 5 days there was a grade 3–4 response in all animals and from 7 days the response in the ipsilateral limb was indistinguishable from the response in the contralateral limb. Similarly, at 3 days, there was a weak twitch response in the indirectly stimulated ipsilateral soleus muscle in 1 out of 4 animals tested. By 5 days 3 out of 4 muscles responded and from 7 days all muscles responded (Fig. 4).
There was a small transient loss of wet weight of the inoculated soleus muscle. The loss was maximal at 2 days [mean wet weight of inoculated muscles 80 ± 5% of the contralateral muscles; difference significant at 
<0.05 (n = 4)] and was fully reversed by 7 days. At no point was there histological evidence of muscle fibre necrosis. There was also a transient upregulation of the tetrodotoxin-resistant voltage gated Na+ channel (NaV1.5) in the muscle fibres of the ipsilateral soleus muscle. NaV1.5 mRNA was first detected at junctional regions by in situ hybridization 6 h after the inoculation of ß-bungarotoxin (Fig. 5) and this was followed at 3 days by the appearance of NaV1.5 protein at junctional regions (Fig. 6). By 8 days the level of NaV1.5 mRNA was greatly reduced and by 10 days after the inoculation of ß-bungarotoxin NaV1.5 protein was undetectable. The changes were selective for the NaV1.5 isoform as there were no changes in the expression of NaV1.4 mRNA (Fig. 6).
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Axonal loss
Counts of myelinated axons in the soleus nerve trunk at the point of entry into the soleus muscles were made in contralateral and ipsilateral nerves at 2 days and 6 months. At 2 days the number of myelinated axons in the nerve trunk (178 ± 6; n = 4) was similar to that in contralateral nerve trunks (180 ± 4; n = 8). At 6 months, the number of axons in the ipsilateral nerves had fallen significantly to 146 ± 6 axons (P < 0.05; n = 4). The loss of myelinated axons appeared to involve axons of all diameters as the distribution of axon diameter did not change (data not shown).
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Discussion |
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The venom of kraits contains two classes of toxin that could be implicated in the onset of severe and potentially fatal neuromuscular weakness, the post-synaptically active
-bungarotoxins and the pre-synaptically active ß-bungarotoxins. -Bungarotoxins bind rapidly and with high affinity to the -sub-unit of nAChR and cause a neuromuscular paralysis of rapid onset. Both experimental and clinical studies suggest that neuromuscular paralysis caused by -bungarotoxin and related toxins is rapidly reversed by appropriate antivenoms and anticholinesterases (Watt et al., 1986; Gatineau et al., 1988). This rapid reversal of neuromuscular paralysis is inconsistent with clinical reports that envenomed patients bitten by kraits exhibit a neuromuscular paralysis that is unresponsive to treatment. Thus, we conclude that -bungaratoxin is not primarily responsible for these effects of the bites. Neuromuscular paralysis caused by ß-bungarotoxin is poorly reversible by either antivenoms or anticholinesterases (S.P. and J.B.H., unpublished data). It seems reasonable to argue that the ß-bungarotoxin-induced depletion of synaptic vesicles is primarily responsible for the onset of the severe, treatment-resistant paralysis associated with envenoming bites by kraits.
Twelve hours after exposure to ß-bungarotoxin, the muscles exposed to ß-bungarotoxin were denervated. The denervation of the skeletal muscle was first reported by Abe et al. (1976) but its clinical significance was ignored until Dixon and Harris (1999) suggested that it might be the explanation for the lack of success of the treatment strategies commonly used for managing neurotoxic problems at the NMJ. We have now shown that, in the living rat, the denervation involves NMJs in all inoculated soleus muscle fibres and lasts for between 3 and 5 days before the appearance of the first signs of re-innervation. The upregulation of NaV1.5, a well-documented response of skeletal muscle to denervation and inactivity (Kallen et al., 1990; Yang et al., 1991; Grampp et al., 1992; Pasino et al., 1996) was entirely consistent with the denervation caused by ß-bungarotoxin.
At least three mechanisms appeared to be involved in the process of denervation. Necrotic degeneration of the terminal boutons (defined as the depletion of synaptic vesicles, degeneration of the mitochondria and fragmentation of the plasma membrane of the nerve terminal was common and has been described in detail in previous work (Dixon and Harris, 1999; Prarapun et al., 2002). There were also numerous examples of shrunken nerve terminals that appeared to have become detached from the post-synaptic muscle sarcolemma. We consider these terminals to be withdrawing from the synaptic trough. Empty synaptic troughs, sometimes containing debris or Schwann cell profiles, were the ultimate expression of the denervation. At many of these denervated NMJs the empty synaptic troughs were decorated with structures we consider to be retraction bulbs. This is a contentious description because the bulbs were devoid of synaptic vesicles and mitochondria, and only possible remnants of neurofilament could be identified visually. This is not inconsistent with the known actions of ß-bungarotoxin, which include synaptic vesicle depletion, mitochondrial damage and neurofilament breakdown (Dixon and Harris, 1999), but we concede that our interpretation is not secure. Finally, many examples were seen of terminal boutons completely enwrapped in Schwann cell processes and, thus, physically isolated from the post-synaptic muscle sarcolemma. Whether these represent the ‘pinched-off’ nerve terminals described by Gillingwater and Ribchester (2003) is unclear for we have no morphological evidence from these experiments that there was any discontinuity between the terminal axon and the boutons. These various forms of nerve terminal pathology—necrosis, withdrawal and isolation of terminal boutons—could be seen in all combinations at single NMJs where more than one terminal bouton could be identified, confirming that ß-bungarotoxin-induced denervation involves several processes occurring simultaneously. Similarly, many other toxins that target the NMJ such as anticholinesterases and acrylamide cause a denervation that involves the loss of synaptic vesicles, necrosis and withdrawal (Duxson and Vrbova, 1985; Hudson et al., 1985; Kawabuchi et al., 1991; Tsujihata et al., 1994; LoPachin et al., 2002). This is a very different situation from that of development where the evidence in favour of nerve terminal necrosis as a factor involved in the loss of supernumery innervation (Rosenthal and Taraskevitch, 1977) has not been generally accepted and the prevailing view is that the elimination of unnecessary innervation is achieved exclusively by the withdrawal of the unwanted axon and its eventual resorption into the parent axon [see Gillingwater and Ribchester (2003) for a recent discussion]. It seems that the loss of synaptic connectivity at the NMJ can occur by a variety of mechanisms and signalling systems that converge to eliminate a synaptic connection (Eaton and Davis, 2003).
We can make only tentative suggestions on the molecular toxicology of ß-bungarotoxin. Denervation by necrosis of the terminal bouton is explicable because ß-bungarotoxin is a potent phospholipase A2. The toxin binds to specific, but poorly characterized, binding sites on neuronal tissue to initiate the hydrolysis of membrane phospholipids. This leads to a loss of ion homeostasis, depolarization and a rise in the internal Ca2+, an accelerated exocytosis and damage to mitochondria (Wernicke et al., 1975; Howard and Wu, 1976; Nicholls et al., 1985; Jambrina et al., 2003; Gulbins et al., 2003; Prasarnpun et al., 2004). Homologous myotoxic phospholipases A2 act in a precisely similar fashion to initiate the necrotic degeneration of skeletal muscle (Dixon and Harris, 1996). The withdrawal of nerve terminals and their parent axons in development is a complex process that involves a number of interactive factors including a loss of synaptic efficiency of either pre-synaptic or post-synaptic origin, the loss of nerve or muscle derived trophic factors and the release of synaptotoxic factors or signals from the muscle fibre. In our experiments, there was a loss of synaptic activity and the hydrolytic properties of ß-bungarotoxin are well known but how the effects of ß-bungarotoxin promote active withdrawal of terminal boutons is unclear, and current knowledge of the molecular pathology of the peripheral nervous system offers no guidelines. Our lack of understanding of the molecular toxicology of the activity of ß-bungarotoxin is complicated by the fact that although it is possible to block the loss of synaptic vesicles by inhibiting Ca2+ entry via voltage-gated Ca2+ channels in the motor nerve terminal or by treatment with botulinum toxin C (Prasarnpun et al., 2004) no intervention has yet been found to prevent nerve terminal damage in either isolated preparations or in the living animal.
The return of neuromuscular function was associated with a rise in immunoreactivity of SNAP-25 and syntaxin that began between 3 and 5 days and a slightly slower rise in the immunoreativity of synaptophysin. The appearance of small, differentiated nerve terminals in the surviving synaptic trough of some junctions at 3 days and the return of neuromuscular function at the same time was entirely consistent with the morphological data. Full recovery of neuromuscular function occurred 7 days after the inoculation of ß-bungarotoxin, although junctions at this stage were still morphologically immature. The speed of regeneration and re-innervation once axonal growth commences is now well recognized. It results from the upregulation of the production and transport of signalling proteins and the proteins of the axonal cytoskeleton by the axotomized cell body and the concentration of a wide range of synapse-specific proteins and synaptic vesicles in the growth cones of regenerating axons (Ahmari et al., 2000; Roos and Kelly, 2000; Friedman et al., 2000; Shapira et al., 2003).
In all respects our data on the effects of a single inoculation of ß-bungarotoxin on neuromuscular function in rats are similar to clinical data on the pathophysiology of neuromuscular function in human subjects receiving envenoming bites by kraits. In these experiments the latent period between the inoculation of ß-bungarotoxin and the onset of severe paralysis was 3 h, the duration of severe paralysis was 2 days and functional recovery took 7 days. In human subjects, the time between a bite and the onset of paralysis was 6 h, the duration of severe paralysis (i.e. of severity sufficient to require assisted ventilation) was 2 days and recovery occurred by 8–9 days (all modal values). We suggest that there is substantial evidence for the hypothesis that the onset of paralysis in victims of envenoming bites by kraits is primarily caused by the depletion of synaptic vesicles from the nerve-terminals, that the period of treatment-resistant paralysis represents the period of denervation and the recovery period represents the restoration of motor innervation. We conclude that the neurological consequences of the bites result primarily from the presence of ß-bungarotoxin in the venom. This conclusion would have greater validity if we could show that the circulating plasma levels of ß-bungarotoxin in the experimental animals equated to the levels in the envenomed subject. Unfortunately relevant data do not exist. We have, therefore, tried to calculate the amount of ß-bungarotoxin the ‘average’ krait would inoculate into the ‘average’ Southeast Asian adult male during an envenoming bite. We have used the following guidelines. A typical bite occurs on the limbs. The fangs of a krait are 3 mm in length so venom will be inoculated into a superficial site. The average venom yield from a krait is 60 mg dry weight (Minton and Minton, 1969; Chanholme, personal communication, 2005); protein content is 90% (Tu, 1977);ß-bungaratoxin comprises 20% of total protein (Chang and Lee, 1963); the proportion of total venom inoculated during a bite is 50% (a best guess based on the observation that snakes can make multiple successive envenoming bites); the average body weight of a Southeast Asian adult male is 60 kg. Thus it can be calculated that an envenoming bite will result in the inoculation of 90 µg of toxin per kilogram body weight into the human subject. This level of envenoming causes a severe, potentially fatal neuromuscular paralysis. In the animal experiments 2.0 µg of ß-bungarotoxin was inoculated s.c. into the hind limb of rats weighing 100 g, equating to 20 µg/kg. Personal observations (unpublished data) are that 100 µg/kg (s.c.) is fatal. Given the caveats involved in this kind of calculation we consider our experimental paradigm to represent the clinical condition to an acceptable degree.
We now consider the clinical implications of our studies. The immediate need must be to find a way to neutralize ß-bungarotoxin bound to its binding site on the nerve terminal. The binding process is complete within 5 min (Prasarnpun et al., 2004) and bound toxin is inaccessible to antivenoms (Simpson et al., 1993). Thus, although antivenoms may neutralize toxic components of the venom as they are released into the circulation from the bite site, they will not prevent nerve terminal degeneration. No available pharmacological agents will enhance transmitter release from nerve terminals that contain no synaptic vesicles and are destined to degenerate. The only obvious strategy is to develop a new range of selective anti-toxins that will target bound toxin and induce a conformational change that will lead to the dissociation of toxins and binding site. In the absence of such anti-toxins, best practice would be to administer anti-venoms to neutralize circulating toxic compounds of the venom, supportive nursing and ventilation as required. Axon damage caused by the inoculation in vivo of ß-bungarotoxin was first reported in detail by Dixon and Harris (1999). They showed that the degeneration of the motor nerve terminal was followed by such extensive damage to intramuscular components of motor axons. The acute degenerative process does not appear to extend beyond the intramuscular compartment because we have now shown that at 2 days the number of myelinated axons in the ipsilateral soleus nerve at the point of entry into the muscle was similar to normal. We also showed that there was a loss of myelinated axons 6 months after inoculation. Is it possible that the initial attack by ß-bungarotoxin on the nerve terminal can initiate a delayed and slowly developing degeneration of the motor axon? Might the sensory nerves also be involved? Could such mechanisms explain the long-term neurological signs reported by Kularatne (2002)? Could long-term axonal damage lead to the eventual loss of the motor neuron? These possibilities are not usually considered seriously by neuropathologists but there is evidence that long-term consequences might follow nerve terminal damage caused by other toxins such as anticholinesterases and acrylamide (Kawabuchi et al., 1991; LoPachin et al., 2002) and the possibility is an emerging topic of discussion amongst neurobiologists (see, e.g. Gillingwater and Ribchester, 2003). It may be relevant that ß-bungarotoxin is known to be toxic to neurons in a number of experimental situations (see, e.g. Hirokawa, 1977; Pittman et al., 1978; Olek, 1980; Rehm and Betz, 1982; Herkert et al., 2001; Shakhman et al., 2003). There is a clear need for a much better understanding of the long-term consequences of severe toxicological damage to peripheral synapses.
Although this study has concentrated on envenoming bites by kraits, similar considerations probably apply to other envenomings. Envenoming bites by taipans (large Australasian snakes of genus Oxyuranus) cause a severe treatment-resistant neuromuscular paralysis (Connolly et al., 1995; Trevett et al., 1995a, b). The venom of taipans is rich in taipoxin, a pre-synaptically active neurotoxic phospholipase (Harris et al., 2000), and Connolly et al. (1995) pointed out that the pathophysiologically significant signs of abnormality in neuromuscular function in victims of taipan bites are consistent with the known features of taipoxin-induced neuromuscular toxicity.
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We thank Carol Young, Julie Coaker and Tracey Scott-Davey, for help and advice on fluorescence microscopy, Trevor Booth for help with confocal microscopy, Judy Preece for help with graphics, and to Jamie Stogden and Maria Bale for handling numerous drafts of this manuscript.
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