Categorization and counts of NMJs following in-vivo two-color BTX experimentation

Mice were anesthetized via isoflurane inhalation and placed in a supine position. Neck hair was removed using a depilatory and the surgical site sterilized with povidone-iodine. An incision was made from chin to sternum and the skin retracted. The salivary glands were moved to the side to expose the STM. Surrounding connective tissue was gently removed and a non-saturating concentration (diluted 2 ug/mL in sterile lactated Ringers) of α-bungarotoxin conjugated to Alexa FluorTM 555 (ThermoFisher Scientific, Cat# 35451) applied to the surface of the muscles for 5 minutes (BTX-1). The neck cavity was then washed copiously with lactated Ringer’s ten times. For muscle injury experiments, superficial muscle fibers at areas close to the site of entry of the accessory nerve into the muscle were cut with a sterile #15 surgical scalpel. The contralateral muscle was also labeled with BTX-1, but not injured and used as an internal control (IC). For denervation experiments both STM muscles were labeled with BTX-1 and the accessory nerve of one muscle was severed near its insertion site into the muscle. The other muscle was spared and used as an IC. The salivary glands were moved back into place following labeling, washing, and injury. The skin was sutured with 8-0 silk and treated with 2% lidocaine hydrochloride jelly. Animals were allowed to convalesce for ten days post-surgery. Endpoints for the studies were P38, P66, P160, and P450. were counted and categorized using a Leica DMRX epifluorescence microscope and a 40X Oil objective (NA 1.4). Junctions were considered fragmented if they had 5 or more non-connected clusters of AChRs in close proximity to each other on the same myofiber. Junctions were categorized based of the robustness of a primary fluorescent bungarotoxin stain and a second label, as well as if they were continuous (< 5 AChR clusters), or fragmented (> 5 AChR clusters). They were classified as stable if BTX-1 and BTX-2 labels were equally robust, or as dynamic if BTX-1 label was mostly absent and BTX-2 label intense. In many cases dynamic junctions did not completely lose BTX-1, and small puncta of fluorescence was seen. It is likely that these puncta are BTX-1 labelled receptors tethered to the basal lamina and demarcate the initial synaptic site before myofiber degeneration. If both the BTX-1 and BTX-2 labels were faint, junctions were classified as lost, but this was rarely observed.

实验中，小鼠经异氟烷（isoflurane）吸入麻醉后取仰卧位。使用脱毛剂去除颈部毛发，以聚维酮碘（povidone-iodine）消毒手术区域。于下颌至胸骨处做切口，牵开皮肤。将唾液腺向外侧牵拉以暴露STM。轻柔剥离周围结缔组织，将Alexa Fluor™ 555（赛默飞世尔科技，ThermoFisher Scientific，货号：35451）标记的α-银环蛇毒素（α-bungarotoxin，以无菌乳酸林格液稀释至2 μg/mL的非饱和浓度）滴加至肌肉表面孵育5分钟，记为BTX-1组。随后用乳酸林格液充分冲洗颈部腔隙，共10次。

肌肉损伤实验部分：使用无菌15号手术刀片，于副神经（accessory nerve）进入肌肉的入口区域附近切断浅表肌纤维。对侧肌肉同样以BTX-1标记，但不施加损伤操作，作为内部对照（IC）。

去神经支配实验中，双侧STM肌肉均以BTX-1标记，切断一侧肌肉的副神经近肌肉附着点处；另一侧肌肉保留完整，作为内部对照。

标记、冲洗及损伤操作完成后，将唾液腺复位至原位。以8-0丝线缝合皮肤，并涂抹2%盐酸利多卡因凝胶。术后让小鼠恢复饲养10天。

本研究的观测时间点为P38、P66、P160及P450。采用徕卡DMRX落射荧光显微镜（epifluorescence microscope）搭配40倍油浸物镜（数值孔径NA=1.4）对样本进行计数与分类。

当同一肌纤维上存在5个及以上相互邻近的非连接性乙酰胆碱受体（AChRs, acetylcholine receptors）簇时，该神经肌肉接头被判定为碎片化。接头的分类依据包括：α-银环蛇毒素初级荧光染色的信号强度、次级标记信号强度，以及接头是否呈连续状态（<5个AChR簇）或碎片化状态（>5个AChR簇）。若BTX-1与BTX-2标记信号强度相当，则将接头归类为稳定型；若BTX-1信号基本消失而BTX-2信号强烈，则归类为动态型。部分动态型接头并未完全丧失BTX-1标记，仍可见少量荧光斑点，此类斑点大概率为锚定在基膜上的BTX-1标记受体，可指示肌纤维变性前的初始突触位点。若BTX-1与BTX-2标记信号均较弱，则将接头归类为丢失型，但该情况极少被观测到。