(B-45) The use of Transcriptomics and Microscopy to investigate Volumetric Muscle Loss injuries

Thursday, October 12, 2023

9:30 AM – 10:30 AM PDT

Location: Exhibit Hall - Row B - Poster # 45

Presenting Author(s)

WQ

Wyatt Quinones

Undergraduate Researcher
University of Louisville and University of Arkansas
Louisville, Kentucky, United States

Co-Author(s)

CS

Chris Slavin

Graduate Student
University of Arkansas, United States

Primary Investigator(s)

JW

Jeffrey Wolchok

Professor
University of Arkansas, United States

Introduction:: Volumetric muscle loss (VML) is massive loss of skeletal muscle tissue due to trauma coming from events such as battlefield injuries, infections, and surgical procedures. In a mainly military setting, over fifty-percent of total war injuries have some type of muscle trauma. Unfortunately, therapeutic options to help recover or promote muscle regeneration are very limited at the current moment with no evidence proven options to recover a major portion of lost muscle. With the growing research accessibility to methods for generating big data, a possible way to examine the full complexity of transcriptional activity in a tissue sample is enabled. This opens up the avenue for a more thorough understanding of muscle healing, and its closely tied association with the immune response. In this study, we used a mouse model of a VML in the tibialis anterior to examine the transcriptome in a spatial context using Visium technology so we could identify possible irregularities that cause the poor healing outcomes which have been noted in this type of injury.

Materials and Methods:: Using a mouse model, a simulation of a VML injury was created using a biopsy punch. This injury site was at the center of the mouse’s tibialis anterior(TA) muscle with 25% of the TA’s total mass being removed. The left TA acted as the modified group while the right TA was kept uninjured to serve as a control group. The animals were sacrificed at 4 and 15 days post-injury and both the left and right TA muscles were harvested. Electrophysiology was performed on the 15 day time-point to verify there was decreased muscle function in the injured TA. The mass of the TA’s both normal and modified were recorded after each mentioned time point. After harvest, the samples were snap frozen with isopentane cooled by liquid nitrogen. The frozen samples were sectioned onto standard and 10x Visium slides using a cryostat. Standard slides were stained using H&E, LY6C/LY6G immunostaining for neutrophils, and Laminin staining for muscle fiber structure. Visium slides were used for spatial transcriptomic profiling. RNA samples were sent to the UC Davis Genomics core for sequencing, and Partek Flow’s analysis suite was used for the processing and analysis of spatial transcriptomic data.

Results, Conclusions, and Discussions::

From the results of the spatial transcriptomics, there was a wealth of transcriptional data to analyze (Figure 1). Each tissue section was split up into three parts for analysis: a distal end (near the foot), middle (injury site), and proximal end (near the knee). Based on the data from Partek Flow, the proximal and middle parts of the tissue contained the majority of gene expression, while the distal side had a very low activity in comparison. Interestingly, the distal part of the tissue also had a very high mitochondrial reads percentage, while the middle and proximal parts had a much lower one (Figure 2). A high percentage of mitochondrial reads is indicative of apoptosis/necrosis in the sample, and combined with the overall gene expression data, seems to point at high levels of cell deaths at the distal edge of the TA muscle.

A list of markers for myogenesis and angiogenesis were then constructed in order to analyze their spatial gene activity, with middle and proximal once again retaining a bulk of the listed markers (Figures 3-4). Based on these results, the directionality of healing appears to progress from proximal to distal. However, by 15 days post VML injury, this regeneration does not seem to have penetrated into the distal end of the TA muscle. Surprisingly, the middle part of the muscle, where the injury site was located, was very similar in terms of gene expression to the proximal end and there was no visually distinct wounded area (Figure 5). One possible explanation for this is that muscle tissue surrounding the biopsy site contracted into the VML deficit, which, along with some general atrophying of the muscle, resulted in the much narrower physical appearance of the left TAs compared to the uninjured right TAs (Figure 6). While these initial results point to some interesting developments that take place during the aftermath of VML, further examination at more time points needs to be conducted in order to fully understand the processes that lead to such poor outcomes following this type of injury.

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