Machamer James B, Vazquez-Cintron Edwin J, Stenslik Mallory J, Pagarigan Kathleen T, Bradford Aaron B, Ondeck Celinia A, McNutt Patrick M
BASF, Research Triangle Park, NC, United States.
United States Army Medical Research Institute of Chemical Defense, Gunpowder, MD, United States.
Front Cell Neurosci. 2023 Aug 15;17:1226194. doi: 10.3389/fncel.2023.1226194. eCollection 2023.
Botulinum neurotoxin (BoNT) causes neuroparalytic disease and death by blocking neuromuscular transmission. There are no specific therapies for clinical botulism and the only treatment option is supportive care until neuromuscular function spontaneously recovers, which can take weeks or months after exposure. The highly specialized neuromuscular junction (NMJ) between phrenic motor neurons and diaphragm muscle fibers is the main clinical target of BoNT. Due to the difficulty in eliciting respiratory paralysis without a high mortality rate, few studies have characterized the neurophysiological mechanisms involved in diaphragm recovery from intoxication. Here, we develop a mouse model of botulism that involves partial paralysis of respiratory muscles with low mortality rates, allowing for longitudinal analysis of recovery.
Mice challenged by systemic administration of 0.7 LD BoNT/A developed physiological signs of botulism, such as respiratory depression and reduced voluntary running activity, that persisted for an average of 8-12 d. Studies in isolated hemidiaphragm preparations from intoxicated mice revealed profound reductions in nerve-elicited, tetanic and twitch muscle contraction strengths that recovered to baseline 21 d after intoxication. Despite apparent functional recovery, neurophysiological parameters remained depressed for 28 d, including end plate potential (EPP) amplitude, EPP success rate, quantal content (QC), and miniature EPP (mEPP) frequency. However, QC recovered more quickly than mEPP frequency, which could explain the discrepancy between muscle function studies and neurophysiological recordings. Hypothesizing that differential modulation of voltage-gated calcium channels (VGCC) contributed to the uncoupling of QC from mEPP frequency, pharmacological inhibition studies were used to study the contributions of different VGCCs to neurophysiological function. We found that N-type VGCC and P/Q-type VGCC partially restored QC but not mEPP frequency during recovery from paralysis, potentially explaining the accelerated recovery of evoked release versus spontaneous release. We identified additional changes that presumably compensate for reduced acetylcholine release during recovery, including increased depolarization of muscle fiber resting membrane potential and increased quantal size.
In addition to identifying multiple forms of compensatory plasticity that occur in response to reduced NMJ function, it is expected that insights into the molecular mechanisms involved in recovery from neuromuscular paralysis will support new host-targeted treatments for multiple neuromuscular diseases.
肉毒杆菌神经毒素（BoNT）通过阻断神经肌肉传递导致神经麻痹疾病和死亡。临床肉毒中毒尚无特异性治疗方法，唯一的治疗选择是支持性护理，直至神经肌肉功能自发恢复，这可能在接触后数周或数月发生。膈运动神经元与膈肌纤维之间高度特化的神经肌肉接头（NMJ）是BoNT的主要临床靶点。由于在不导致高死亡率的情况下引发呼吸麻痹存在困难，很少有研究描述膈肌从中毒中恢复所涉及的神经生理机制。在此，我们建立了一种肉毒中毒小鼠模型，该模型涉及呼吸肌部分麻痹且死亡率低，允许对恢复情况进行纵向分析。
通过全身注射0.7 LD BoNT/A攻击的小鼠出现了肉毒中毒的生理体征，如呼吸抑制和自发运动活动减少，这些体征平均持续8 - 12天。对中毒小鼠分离的半膈肌制备物的研究表明，神经诱发的、强直和抽搐肌肉收缩强度显著降低，在中毒后21天恢复到基线水平。尽管功能明显恢复，但神经生理参数在28天内仍处于抑制状态，包括终板电位（EPP）幅度、EPP成功率、量子含量（QC）和微小EPP（mEPP）频率。然而，QC的恢复比mEPP频率更快，这可以解释肌肉功能研究与神经生理记录之间的差异。假设电压门控钙通道（VGCC）的差异调节导致了QC与mEPP频率的解偶联，药物抑制研究被用于研究不同VGCC对神经生理功能的贡献。我们发现，在从麻痹中恢复的过程中，N型VGCC和P/Q型VGCC部分恢复了结量子含量，但未恢复mEPP频率，这可能解释了诱发释放与自发释放的加速恢复。我们还确定了其他一些可能在恢复过程中补偿乙酰胆碱释放减少的变化，包括肌肉纤维静息膜电位去极化增加和量子大小增加。
除了识别因NMJ功能降低而出现的多种形式的代偿性可塑性外，预计对神经肌肉麻痹恢复所涉及分子机制的深入了解将支持针对多种神经肌肉疾病的新型宿主靶向治疗。
