Abstract
Despite the medical urgency presented by cubozoan envenomations, ineffective and contradictory first-aid management recommendations persist. A critical barrier to progress has been the lack of readily available and reproducible envenomation assays that (1) recapitulate live-tentacle stings; (2) allow quantitation and imaging of cnidae discharge; (3) allow primary quantitation of venom toxicity; and (4) employ rigorous controls. We report the implementation of an integrated array of three experimental approaches designed to meet the above-stated criteria. Mechanistically overlapping, yet distinct, the three approaches comprised (1) direct application of test solutions on live tentacles (termed tentacle solution assay, or TSA) with single image- and video-microscopy; (2) spontaneous stinging assay using freshly excised tentacles overlaid on substrate of live human red blood cells suspended in agarose (tentacle blood agarose assays, or TBAA); and (3) a “skin” covered adaptation of TBAA (tentacle skin blood agarose assay, or TSBAA). We report the use and results of these assays to evaluate the efficacy of topical first-aid approaches to inhibit tentacle firing and venom activity. TSA results included the potent stimulation of massive cnidae discharge by alcohols but only moderate induction by urine, freshwater, and “cola” (carbonated soft drink). Although vinegar, the 40-year field standard of first aid for the removal of adherent tentacles, completely inhibited cnidae firing in TSA and TSBAA ex vivo models, the most striking inhibition of both tentacle firing and subsequent venom-induced hemolysis was observed using newly-developed proprietary formulations (Sting No More™) containing copper gluconate, magnesium sulfate, and urea.
Highlights
Jellyfish envenomations, which have been increasing worldwide, result in myriad clinical outcomes, ranging from sting-site pain and inflammation to life-threatening sequelae and death [1].In response to heightened public health concerns and lay inquiries, clinicians and emergency-care personnel often rely on tertiary Internet resources, such as Medscape, eMedicine Health, or Mayo ClinicOnline [2,3,4]
The Tentacle Skin Blood Agarose Assay (TSBAA) satisfied the four requirements of a good envenomation model: (1) it utilized the natural stinging activity of live tentacles rather than electrically-forced or chemically-induced discharge and, more accurately reflects the amount of venom discharge during a natural sting event; (2) it allowed for visualization of cnidae discharge through microscopic examination; (3) it used hemolysis, a well-categorized venom activity, to directly evaluate sting outcomes; and (4) it allowed rigorous controls, including withholding of treatment as well as inert treatments, such as seawater or the application of heat or cold
Jellyfish Squish® (Coastal Solutions Inc., Savannah, GA, USA) led to an approximate 25% reduction in hemolysis, and Sting No MoreTM (Alatalab Solutions, LLC, Honolulu, HI, USA) Spray treatment reduced hemolysis by more than 80% (Figure 8)
Summary
Jellyfish envenomations, which have been increasing worldwide, result in myriad clinical outcomes, ranging from sting-site pain and inflammation to life-threatening sequelae and death [1]. Activities of jellyfish venoms have been well-studied, and hemolytic units (specific activity per venom mass) for different species have been calculated [20,21] Such biochemical baselines provide clear standards for envenomation models. It is critical to investigate the effects of comparative controls including null treatment or the application of a control “mock” solution such as seawater to exclude experimental artifacts, including direct physical manipulation effects of adding solutions in treatment activities Such controls are especially important given that there is no careful study to determine what percentage of cnidae discharge during natural sting events; far less than 100% of the cnidae present on a tentacle may fire. Apart from satisfying these criteria, the model is simple and inexpensive and, could be employed in laboratories with minimal budgets and limited equipment
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