Fig 1: Accelerated recovery of compound muscle action potential from the paralysis caused by Bungarus venoms induced by NUCC‐390. (A) Compound muscle action potential (CMAP) values recorded on gastrocnemius muscles 96 h after mock treatment or injection with B. caeruleus, B. candidus, or B. multicinctus venoms, w/o NUCC‐390 daily local administration, using concentration as in Figure 2. The venom affects all the muscles around the site of the injection in mice. Data are expressed as CMAP amplitude (mV) ± SEM. (B. caeruleus) **p = .0049, (B. candidus) **p = .0028, (B. multicinctus) **p = .005. **p < .01. (B) Representative immunostaining of intoxicated neuromuscular junctions (NMJs), performed on the same muscles used for CMAP analysis. Motor neurons axon terminals are identified by vesicle‐associated membrane protein isoform‐1 (VAMP‐1) immunostaining (green), post‐synaptic nicotinic acetyl choline receptors (nAChRs) by fluorescent α‐BTX (red) and the axon by neurofilaments (NF) staining (chicken polyclonal, Abcam, cat# Ab4680) (white). Scale bars: 50 μm
Fig 2: Faster recovery of lung ventilation in mice treated with the Bungarus caeruleus venom. A ventilation index was estimated by recording mediastinum pressure variations by placing a plastic feeding tube carrying a pressure sensor placed in the lower third of the esophagus, at the level of mediastinum. (A) Representative traces of mediastinum pressure variations in untreated mouse or (B) in a mouse after 24/96/168 h after injection of B. caeruleus venom (36 ng/gr) (upper traces), or in a mouse envenomed with the B. caeruleus venom and daily i.p. injected with NUCC‐390, (lower traces of panel B). (C) Quantitative assay performed by measuring the peak area of 20 consecutive peaks of the traces (±SEM) obtained from a group of N = 4 mice. (D) Representative immunostaining of intoxicated neuromuscular junction (NMJ), performed on the diaphragm muscle 96 h after the intoxication with B. caeruleus not treated (upper panels) or i.p. treated with NUCC‐390 daily (lower panels). Motor neurons axon terminals are identified by vesicle‐associated membrane protein isoform‐1 (VAMP‐1) immunostaining (green), post‐synaptic muscle membrane by nicotinic acetyl choline receptor (nAChR) staining with fluorescent α‐BTX (red). Axon is identified by neurofilaments (NF) staining (chicken polyclonal, Abcam, cat# Ab4680) (white). Scale bars: 50 μm
Fig 3: CXCR4 receptor expression on mouse neuronal axons after i.m. injection of Bungarus caeruleus venom, β‐ and α‐bungarotoxins. 36 ng/g B. caeruleus venom (2nd, 3rd and 4th rows), 1 ng/g of β‐bungarotoxin (5th, 6th and 7th rows) and 1 ng/g of α‐bungarotoxin (bottom row) were injected i.m. in the hind limb of young mice, and the soleus muscle was dissected and stained with antibodies specific for the following antigens. Left set of panels: first column: vesicle‐associated membrane protein isoform‐1 (VAMP‐1), a marker of motor axon terminals; second column: nicotinic acetyl choline receptor (nAChR), a marker of the post‐synaptic muscle membrane, was stained with fluorescent alpha‐bungarotoxin; third column: merging with the staining obtained with an anti‐neurofilaments (chicken polyclonal, Abcam, cat# Ab4680) in grey. The right set of panels shows samples of soleus muscle, poisoned as above, stained with: first column: anti CXCR4 receptor (rabbit monoclonal, Abcam, cat# Ab124824) (red); second column: anti‐neurofilaments (NF, green), third column, merging with Schwann cells expressing GFP (light blue) showing that the CXCR4 staining is concentrated on nerve axon stumps close to the perisynaptic Schwann cells. Symbols of the rows: Samples taken from non‐injected control muscles (ctr), or venom treated muscles after 24 h, 96 h and 168 h from i.m. injection of Bungarus caeruleus venom in the hind limb. Notice that the degeneration of the motor axon terminal at the neuromuscular junction with time is complete after 24 h from poisoning as shown by the loss of VAMP‐1 staining. Degeneration is accompanied by the expression of the CXCR4 receptor on the remaining motor axon, and this receptor is still present 4 days after poisoning. A lower magnification was used (scale bar 50 μm) in most panels to provide a view of several neuromuscular junctions in the same field, whilst a higher one (scale bar 20 μm) was used in other panels to better show the expression of the CXCR4 receptor
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