Analysis Tools
mortality/aging
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• 5% of mice die a few days after birth
• however, most mice are viable and develop into adulthood
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respiratory system
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• mice that die a few days after birth exhibit respiratory failure
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growth/size/body
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• mice that die a few days after birth are smaller than wild-type controls
• however, mice that develop into adulthood show no differences in the weight curve relative to wild-type controls
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• mice that die a few days after birth are smaller in weight relative to wild-type controls
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behavior/neurological
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• mice that die a few days after birth exhibit limb motor deficiency
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• mice exhibit reduced grip strength at all time points analyzed relative to wild-type controls (forelimb grip strength decrease: P20, 26%; P40, 23%; P60, 20%; P90, 30%; hindlimb grip strength decrease: P20, 37%; P40, 18%; P60, 23%; P90, 21%)
• postnatal treatment with lithium chloride significantly improves fore and hindlimb grip strength
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muscle
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• repeated tetanic nerve stimulation (60 Hz, 600 ms duration at 1 Hz) induces a more pronounced degree of fatigue than in wild-type muscles (fatigability increase: 30%)
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• mice progressively develop muscle weakness after 2 weeks of age; adult mice show signs of congenital myasthenia
• adult mice display poor motor performance from P20 to P90 as shown by a reduced latency to fall (latency decrease: P20, 62%; P40, 33%; P60, 64%; P90, 72%) in a rail-grip test
• postnatal treatment with lithium chloride significantly improves latencies to fall from the rail grip
• at P120, the strength of muscle twitch upon phrenic nerve stimulation is significantly reduced in isolated hemidiaphragms, both upon single and tetanic stimulations (strength decrease of muscle twitch: single twitch, 59%; T40 Hz, 54%; T60 Hz, 47%; T80 Hz, 44%; T100 Hz, 42%)
• the developed muscle specific force (i.e. muscle strength normalized to muscle weight) is significantly reduced, both upon single and tetanic (T100 Hz) stimulations by 52% and 24%, respectively
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nervous system
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• at E18.5, embryos show aberrant extension of motor axons, bypassing AChR clusters and growing excessively toward the periphery of the diaphragm muscle
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• mice show diaphragm innervation defects both during early muscle prepatterning (E14) and NMJ differentiation (E18.5) associated with a drastic deficit in AChR clusters and excessive growth of motor axons bypassing AChR clusters
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• at E14, mean neurite length is increased by 82% in whole-mount left hemidiaphragms
• at E18.5, the length of primary as well as secondary nerve branches is increased by 75% and 46%, respectively
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• NMJ formation is severely impaired during development as shown by reduced muscle prepatterning and neural AChR clusters and excessive neurite outgrowth in whole-mount left hemidiaphragms; similar defects are seen at P5
• at E14, neurites are increased in length by 82% and AChR clusters are distributed in a 2-fold wider diaphragm muscle area rather than being concentrated in the central zone of the muscle as in wild-type controls
• at E14, AChR cluster number, volume and intensity are reduced by 64%, 70% and 65%, respectively, while non-innervated AChR clusters are increased by 30%
• at E18.5, the synaptic endplate band width is 2-fold larger than normal while AChR cluster number, volume and intensity are reduced by 40%, 25% and 14%, respectively
• at E18.5, motor axons bypass AChR clusters and grow excessively toward the periphery of the diaphragm muscle; the length of primary as well as secondary nerve branches is increased by 75% and 46%, respectively
• at P20, most endplates in isolated muscle fibers from tibialis anterior (TA) muscles are ovoid and compact, unlike the perforated ones in wild-type controls, suggesting that NMJs are immature; AChR cluster number and nerve terminal area per NMJ are reduced by 49% and 58%, respectively
• at P60, 90% of NMJs in TA muscles show a discontinuous postsynaptic network, isolated AChR clusters, and a 4-fold increase in the number of AChRs fragments per NMJ; total AChR clusters area per NMJ is reduced by 40% while nerve terminal area is reduced by 39%; similar NMJ defects are noted in all muscles analyzed, including diaphragm, TA, soleus, and extensor digitorum longus
• at P120, TA muscles show a 67% decrease in synaptic vesicle density; on the postsynaptic side, ~30% of NMJs show highly disorganized or very few junctional folds (JFs); in some cases, the distance between JFs and muscle fibers is increased and mitochondria accumulate beneath the postsynaptic membrane
• treatment with lithium chloride from E12 to E18.5 almost fully rescues both presynaptic and postsynaptic defects seen in E18.5 left hemidiaphragms, with NMJs being indistinguishable from wild-type NMJs
• treatment with lithium chloride from P10 to P60 improves NMJ defects in isolated P40 TA muscle fibers and restores beta-catenin translocation to synaptic nuclei
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skeleton
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• mice develop abnormal spine curvature caused by the shrinkage of the pelvic and scapular region
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cellular
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• at E18.5, embryos show aberrant extension of motor axons, bypassing AChR clusters and growing excessively toward the periphery of the diaphragm muscle
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