Methods for In situ Quantification of Mitochondrial Morphology in Muscle and Terminal Schwann Cells of Mice

Our research investigates mitochondrial morphology of terminal Schwann cells at the neuromuscular junction for the first time. Our approach enables quantification of mitochondrial morphology in otherwise hard-to-obtain tissues, such as capillary and lymphatic beds. To begin, obtain a wild type and a D2-mdx mouse.

After confirming the anesthetic depth by toe-pinch reflex, insert the needle at the distal tendon toward the right knee. Inject 50 microliters of two-micromolar far red fluorescent mitochondrial stain on the tibialis interior, or the TA, muscle while withdrawing the needle along the injection path, and incubate the dye in vivo for one hour. While maintaining the animal under anesthesia, use scissors and forceps to dissect and isolate the TA muscle prior to euthanasia.

Place the isolated TA muscle in 4%paraformaldehyde at room temperature for 15 minutes. Then, under a stereo microscope, gently separate the fibers by teasing. Add cell-permeant nucleic acid stain to the 4%paraformaldehyde to obtain a final concentration of five to 10 micrograms per milliliter for nuclear labeling.

Then, wash the TA muscle three times in PBS for five minutes each to remove excess dye. Dehydrate the muscle sequentially in 50%75%and 100%ethanol for five minutes each. Then, incubate the sample sequentially in tissue-clearing solutions one and two for 30 minutes each, ensuring they are fully submerged.

Place the muscle on a coverslip. Use a 2.5-by-2-by-1-centimeter glass block weighing 7.8 grams to gently flatten it. Obtain an S100 beta mouse.

After confirming the anesthetic depth by toe-pinch reflex, make a small skin incision above the gluteus maximus, or GM, muscle. Inject 75 microliters of two-micromolar far red fluorescent mitochondrial stain beneath the GM muscle. Close the wound and let the dye incubate for 45 minutes to selectively label terminal Schwann cells.

While maintaining the animal under anesthesia, dissect the GM muscle within 10 minutes, and immediately place it in chilled 0.9%sterile saline. Only if using D2-mdx mice, add fluorescent acetylcholine receptor dye at a one-to-500 dilution to the microdissection bath, and incubate the tissue for 30 minutes. Trim the excess fat and connective tissue, and transfer the GM muscle onto a coverslip with the ventral side facing down for inverted microscopy.

Add two microliters of general mounting medium onto the dorsal surface of the GM, and flatten the tissue using glass block before imaging. In the software, set the excitation wavelength to 646 nanometers. After adding two drops of clearing solution two, select the 63X objective lens to image the TA muscle, and set the Z stack depth close to 100 micrometers.

Then, select deconvolution mode, and apply system optimized parameters. Set the step size to one micrometer and acquire images. To acquire terminal Schwann cells'images, select the 20 times magnification objective lens, set the Z stack depth to approximately 17 micrometers, and acquire images.

Then, select the 63 times magnification objective lens and acquire images of individual terminal Schwann cells. Import individual Z slices to train the software to recognize the mitochondrial signal. And conduct quantitative analysis using AI-driven software.

Develop image masks for investigator review. Separate 3D image stacks into individual optical sections following algorithm optimization. Select every fifth Z slice from each 3D image, and calculate the average per sample.

Standardize imaging parameters across all samples, and quantify mitochondrial morphology. Perform immunohistochemistry and statistical analysis to assess group differences and correlations. Mitochondrial connectivity was reduced in mdx muscle compared to the wild type mice.

A greater number of small mitochondrial fragments and a reduced number of large interconnected mitochondrial networks were observed in mdx mice. In wild type mice, mitochondria in terminal Schwann cells exhibited interconnected loop structures near bifurcations. In contrast, in mdx mice, mitochondria appeared fragmented and lacked loop formations near bifurcations.

Confocal and electron microscopy confirmed similar donut-shaped mitochondrial structures in terminal Schwann cells. Postsynaptic analysis showed sub-sarcolemmal mitochondria clustered around acetylcholine receptors, lacking the characteristic pretzel-like structure. Image analysis revealed that terminal Schwann cell mitochondria in mdx muscle showed increased area and fragmentation compared to wild type muscle, with no difference in relative particle size.

Muscle force measurements showed reduced twitch with greater variability and decreased peak force in mdx muscle. Force production across increasing stimulation frequencies was also significantly reduced in mdx muscles. The mdx muscle showed a leftward shift in fiber size, indicating approximately 60%smaller myofibers compared to wild type.

This protocol allows researchers to study and quantify mitochondrial morphology in skeletal muscle and terminal Schwann cells. Future studies can adapt this approach to investigate mitochondrial morphology in micro-circulation and lymphatic vessels.