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A pharmacological examination of Pseudechis SPP. venoms on muscle and nerve.
thesisposted on 09.02.2017, 02:45 by Hart, Andrew James
The current study examined venoms from black snakes (Family Elapidae: Pseudechis spp.) found within the Australo-Papuan region. The focus of the study was an examination of the effects of the venoms at the skeletal neuromuscular junction (i.e. nerve tenninal and skeletal muscle). Envenoming by Australian black snakes can produce a range of clinical manifestations with the major effects being myotoxicity, anticoagulant coagulopathy and haemolysis. Acute renal failure is a rare complication of myotoxicity. Neurotoxicity is not seen clinically, with the exception of envenoming by P. papuanus, yet it is clearly evident in vitro in avian preparations. This is an interesting discrepancy and an aspect that we wished to investigate to gain a better understanding of these venoms and potential species differences. P. colletti (Collett's snake) venom was used to examine the ability of postsynaptic (a) neurotoxins to bind to the skeletal muscle nicotinic ACh receptor (nAChR). A previous in vitro study that indicated neurotoxic activity of P. colletti venom used the chick biventer cervicis muscle preparation, so we utilised this muscle as well as frozen human skeletal muscle in an immunohistochemical examination. P. colletti venom (2.1 -210 Ilg/ml) caused a concentration-dependent block of fluorescently labelled a-bungarotoxin at the nAChR of the chick skeletal muscle, but had little effect on labelling at the human nAChR. This indicated that the venom contains components capable of blocking the nAChR, but that human tissue appears relatively resistant to the effects of the venom. Following this, a more intensive investigation of P. colletti venom at the neuromuscular junction and skeletal muscle was initiated. Rats were injected with venom (20 Ilg, s.c.) into their left hindlimb. Six hours after inoculation there was clear evidence of hypercontraction and muscle degeneration (20-35% of total population) close to the injection site, but no haemorrhage or other vascular damage was observed. After 4 days muscle regeneration had begun to occur. Fluorescent labelling of various nerve proteins indicated partial damage at the neuromuscular junction, but was not suggestive of any presynaptic neurotoxic activity. It was also found that P. colletti venom (2.1 -210 ~g/ml) caused an inconsistent reduction in fluorescent labelling of the rat nAChR, suggesting some post¬synaptic toxin activity. The above studies indicated that chick nAChRs were more sensitive to toxins in P. colletti venom than the nAChRs of rats and humans. The next study showed this functionally using the chick isolated biventer cervicis nerve-muscle preparation and the rat isolated phrenic nerve-diaphragm preparation. We also investigated if this phenomenon was common across the Pseudechis genus. Venom from the six main species (P. australis, P. porphyriacus, P. colletti, P. guttatus, P. butleri and P. papuanus) was used in this study. It was found that nerve-mediated twitches of the chick preparation were inhibited significantly faster than nerve-mediated twitches in the rat preparation, except in the case of P. colletti venom. It is apparent that there is a marked difference in species sensitivity to postsynaptic neurotoxins found in Pseudechis spp. venoms. We subsequently identified, isolated and characterised a short-chain a-neurotoxin from P. colletti and P. porphyriacus venoms, which we named a-elapitoxin-Pc1 and a-elapitoxin-Pprl, respectively. Based on their amino acid sequences, the calculated molecular weight of a-elapitoxin-Pcl and a-elapitoxin-Pprl was 6759.6 Da and 6746.5 Da, respectively, and they share 97% sequence homology. It was confirmed that these toxins exhibited a more potent inhibitory activity in the avian skeletal muscle preparation than in the mammalian skeletal muscle preparation. The final component of this thesis was the use of an anesthetised rat preparation to examine the pharmacokinetic and pharmacodynamic properties of a myotoxic venom (i.e, P. australis venom) in vivo. This model was used successfully to measure plasma creatine kinase (CK) levels, which is an accepted biochemical marker indicating myotoxicity, plasma creatinine levels, which is an indicator for renal dysfunction, and serum venom levels. An increase in creatinine was only seen when venom (100 Ilglkg) was administered i.v., while CK levels increased when venom was administered i.v., i.m. or s.d. Antivenom administration was tested prophylactically, 1 h or 6 h after venom administration and in each case stopped the CK level from continuing to increase. Pharmacokinetic analysis found that after i.m. and s.d. venom administration, rapid absorption of the venom into the central compartment occurred until approximately 30 min, after which a plateau was reached. Intravenous administration saw an exponential movement out of the central compartment with a half-life of 16 min. This thesis has successfully investigated the venom from members of the Pseudechis genus. The results will assist with future interpretation of in vitro experimentation and may aid the extrapolation of experimental findings to clinical settings. In addition, the in vivo investigation may help assist researchers in future screening of venoms with myotoxic activity and assist in the development of treatment for snake bite.