Hemidiaphragm

Phrenic-nerve hemidiaphragm preparations were preincubated in modified Krebs solution containing 1.0 x 10 6m [methyl-%]choline (specific activity 8 Ci/mmole) and 0.5 mM eserine salicylate for 30 min, followed by a brief rinse in eserine-containing modified Krebs solution but no label. Preparations were then incubated at 37°C in modified Krebs solution containing eserine for 120 min, taking duplicate 0.1 ml samples of the supernatant fluid at 10 min intervals. 

Figure 1. The effect of toxin T17 administration on the release of [ Hjcholine ( ) and [ Hjacetylcholine ( ) from rat phrenicnerve hemidiaphragm preparations.

Curare-like activity, lowered blood pressure (dogs) antagonized phenylephrine-induced contractions of isolated rabbit aorta blocked neuromuscular transmission in rat hemidiaphragm preparation 440... 

A comparison between the chemiluminescence and the radioenzymatic assay methods that are used for the measurement of acetylcholine released from a rat phrenic nerve hemidiaphragm preparation was reported by Ehler et al. [57]. The comparison demonstrated quantitative equivalency and limits of detection for different analytes as 2 pmol. 

Advantages of the Mouse Hemidiaphragm Assay Current approaehes to the inhibition of BoNT activity involve a number of strategies, eaeh with potential advantages and disadvantages. Ultimately, model test systems that can incorporate eaeh of these potential approaches are needed to evaluate the relative merit of potential therapeutic compounds. Since the presynaptic terminal is the primary target for BoNTs, a test system based on toxin action at... 

FIGURE 35.1. Representative profiles of the activity of the AChE molecular forms in soleus, EDL, and hemidiaphragm muscles. Profiles at the top of each column are from untreated muscles followed by profiles of activity of AChE molecular forms of muscles 24 h and 7 days, respectively, after receiving an acute dose of soman (100 pg/kg, s.c.). The AChE activity scale is in arbitrary units based on the pmole substrate hydrolyzed/min by the enzyme activity in each fraction. The sedimentation values of the AChE molecular forms are given in the profiles of untreated muscles above the associated peaks. Sedimentation values were determined by the location of the added sedimentation standards, P-galactosidase (16.0 S), catalase (11.1 S), and alkaline phosphatase (6.1 S), following velocity sedimentation of the gradients. 

FIGURE 35.3. Representative profiles of the activity of the AChE molecular forms in EDL, soleus, and hemidiaphragm muscles fi om rats following an acute suhlethal injection of VX (12 Hg/kg, S.C.). For further details, see legend to Figure 35.1. 

Thiermann, H., Worek, F., Szinicz, L., Eyer, P. (2005). Effects of oximes on muscle force and acetylcholinesterase activity in isolated mouse hemidiaphragm exposed to Paraoxon. Toxicology 214 190-7. 

Singh YN, Harvey AL, Marshall IG. Antibiotic-induced paralysis of the mouse phrenic nerve-hemidiaphragm preparation, and reversibihty by calcium and by neostigmine. Anesthesiology 1978 48(6) 418-24. 

Amaki Y, Nagashima H, Radnay PA, Foldes FF. Ketamine interaction with neuromuscular blocking agents in the phrenic nerve-hemidiaphragm preparation of the rat. Anesth Analg 1978 57(2) 238 3. 

The first report in this area compared the actions of botulinum neurotoxin and botulinum binary toxin on transmission in the phrenic nerve-hemidiaphragm preparation (Simpson, 1982). There was the expected finding that neurotoxin blocked transmission by blocking acetylcholine release from nerve terminals, but the binary toxin had no effect. Apart from showing that C2 toxin did not block exocytosis, this study showed that the toxin did not act on the diaphragm to block muscle twitch. This observation is in keeping with the fact that the predominant form of actin in striated muscle is not that which is ADP-ribosylated by C2 toxin. 

Spontaneous release of acetylcholine, as measured electrophysiologically as increased miniature endplate potential (MEPP) frequency, was determined in myofibers from rat hemidiaphragms exposed to 2,4-DNP and/or methylmercury (Levesque and Atchison 1987). Tissues exposed to... 

Internalization affords the next opportunity to ameliorate the toxic actions of BoNT. A number of pharmacological agents have been examined for inhibition of this process with various degrees of success. Simpson (1983) demonstrated that pretreatment of phrenic nerve-hemidiaphragm preparations with the lysosomotropic agents ammonium chloride or methylamine hydrochloride delayed the time-to-block of nerve-evoked muscle contractions after exposure to BoNT serotypes A, B, Cl, and TeNT. Incubation of nerve-muscle preparations with ammonium chloride and methylamine hydrochloride was effective if applied before, concurrently, or up to 20 min after toxin exposure. The efficacy of the lysosomotropic agents was reduced rapidly with further delays, such that no effect was observed if they were administered 30-35 min after toxin exposure. At optimal concentrations, these compounds produced a twofold delay in the time-to-block (Simpson, 1983). 

FIGURE 16.1 The effect of BoNT/A on muscle tension in isolated mouse phrenic nerve-hemidiaphragm preparations. Muscles were exposed to BoNT/A at concentrations ranging from 1 pM to 1 nM and the time to paralysis was monitored. Toxins were added to the muscle bath at 0 time from concentrated stock solutions each concentration was tested on a separate hemidiaphragm muscle. Twitch tensions were elicited by supramaximal stimulation of the phrenic nerve at 30 s intervals. Temperature, 37°C. 

Adler, M., Scovill, J., Parker, G., Lebeda, F.J., Piotrowski, J., and Deshpande, S.S. 1995. Antagonism of botulinum toxin-induced muscle weakness by 3,4-diaminopyridine in rat phrenic nerve-hemidiaphragm preparations. Toxicon 33 527-537. 

Simpson LL. 1973. The interaction between divalent cations and botulinum toxin type A in the paralysis of the rat phrenic nerve-hemidiaphragm preparation. Neuropharmacology 12(2) 165-176. 

Inhibitons of AChE. such as OPs. are fast acting and lethal it i.s therefore a surprise that a knockout animal for AChE can survive for almost 1 year. The animal must therefore have developed maximal tolerance to a high concentration of ACh. Investigation of the peripheral nerv-ous system using the phrenic nerve-hemidiaphragm. showed that the knockout animal had developed markedly greater twist tensions and slower rise and relaxation time. To compensate for the absence of AChE activity, the size of the endplate was reduced. This may provide some compensation because less ACh is provided. The juctional folds of the endplate and the number of nAChRs were heavily reduced. This allowed. smaller responses to ACh and also more ACh to diffuse out... 

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