Skeletal muscle regeneration following acute injury is a multi-step process involving complex changes in tissue microenvironment. Macrophages (MPs) are one of the key cell types involved in orchestration and modulation of the repair process. Multiple studies highlight the essential role of MPs in the control of the myogenic program and inflammatory response during skeletal muscle regeneration. A variety of MP phenotypes have been identified and characterized in vitro as well as in vivo. As such, MPs hold great promise for cell-based therapies in the field of regenerative medicine. In this study we used bone-marrow derived in vitro LPS/IFN-y-induced M1 MPs to enhance functional muscle recovery after tourniquet-induced ischemia/reperfusion injury (TK-I/R). We detected a 15% improvement in specific tension and force normalized to mass after M1 (LPS/IFN-γ) MP transplantation 24 hours post-reperfusion. Interestingly, we found that M0 bone marrow-derived unpolarized MPs significantly impaired muscle function highlighting the complexity of temporally coordinated skeletal muscle regenerative program. Furthermore, we show that delivery of M1 (LPS/IFN-γ) MPs early in regeneration accelerates myofiber repair, decreases fibrotic tissue deposition and increases whole muscle IGF-I expression.

/pdf/the-development-of-macrophage-mediated-cell-therapy-to-41buntoz9k.pdf

The Development of Macrophage-Mediated Cell Therapy to Improve Skeletal Muscle Function after Injury

Effects of macrophage on the responses of soleus fiber size to hind limb unloading and reloading were studied in osteopetrotic homozygous (op/op) mice with inactivated mutation of macrophage colony...

https://www.physiology.org/doi/pdf/10.1152/ajpcell.00348.2014

Macrophage deficiency in osteopetrotic (op/op) mice inhibits activation of satellite cells and prevents hypertrophy in single soleus fibers

White blood cells (WBCs) are the most diverse cell types observed in the healing process of injured skeletal muscles. In the course of healing, WBCs exhibit dynamic cellular response and undergo multiple protein expression changes. The progress of healing can be analyzed by quantifying the number of WBCs or the amount of specific proteins in light microscopic images obtained at different time points after injury. In this paper, we propose an automated quantifying and analysis framework to analyze WBCs using light microscopic images of uninjured and injured muscles. The proposed framework is based on the Localized Iterative Otsu's threshold method with muscle edge detection and region of interest extraction. Compared with the threshold methods used in ImageJ, the LI Otsu's threshold method has high resistance to no-object area and achieves better accuracy. The CD68-positive cell results are presented for demonstrating the effectiveness of the proposed work.

/pdf/automated-quantification-of-white-blood-cells-in-light-23sqgde18o.pdf

Automated quantification of white blood cells in light microscopic images of injured skeletal muscle

White blood cells (WBCs) play an important role in the muscle recovery process. Detection and quantification of WBC expressions in light microscopy images captured at different time points after injury deliver valuable information about underlying processes. In this paper, an optimized CNN architecture is designed for classifying CD68 macrophages in 10x light microscopy images of injured muscle cross-sections. Based on the CNN classification results, hybrid masks are generated to post-process the segmentation results obtained by the LIOtsu thresholding method as a step towards extracting and quantifying CD68-positive macrophages. The segmentation is completed by the earlier designed LIOtsu thresholding method. The experimental results confirm that a high accuracy of classification is achieved by the proposed CNN architecture and high performance of quantification of CD68-positive macrophages is achieved by the LIOtsu thresholding method, augmented by CNN.

Automated Quantification of White Blood Cells in Light Microscopy Muscle Images: Segmentation Augmented by CNN

Skeletal muscle injury is one of the common injuries caused by high-intensity sports activities, military related works and natural disasters. In order to discover better therapies, it is important to study muscle regeneration process. Muscle regeneration process tracking is the act of monitoring injured tissue section over time, noting white blood cell behavior and cell-fiber relations. A large number of microscopic images are taken for tracking muscle regeneration process over multiple time instances. Currently, manual approach is widely used to analyze a microscopic image of muscle cross section, which is time consuming, tedious and buggy. Automation of this research methodology is essential to process big amount of data. The objective of this thesis is to develop a framework to track the regeneration process automatically. The framework includes dynamic thresholding, morphological processing, and feature extraction.Based on the clinical assumptions, the threshold is calculated using standard deviation and mean of probable single cells. After thresholding, six parameters are calculated including average size, cell count, cell area density, cell count on the basis of size, the number of cytoplasmic and membrane cells as well as the average distance between cellular objects. All these parameters are estimated over the time, which helped to note the pattern of change in leukocytes (White blood cells) presence. Based on these results, a clear pattern of leukocyte movement is observed. Our future work includes deriving the mathematical equations using regression model on the data set of increased time points.

/pdf/a-study-on-automated-process-for-extracting-white-blood-32nzm0bd1e.pdf

A Study on Automated Process for Extracting White Blood Cellular Data from Microscopic Digital Injured Skeletal Muscle Images

Background Unaccustomed exercise, high-intensity dynamic sports activities, or the resumption of normal weight-bearing after a period of disuse can induce skeletal muscle injury, which activates an inflammatory response followed by muscle regeneration. Specific subsets of macrophages are involved in muscle regeneration. But the exact role of macrophage differentiation during muscle regeneration remains to be elucidated. Objective The objective of the study was to examine the effect of macrophage colony stimulating factor (M-CSF)-differentiated, lipopolysaccharides (LPS)-stimulated-macrophage-conditioned medium on muscle-cell proliferation, fusion, and elongation, which are key events during muscle regeneration and myogenesis. Method Murine C2C12 myoblasts were cultured in conditioned medium obtained from PU5-1R macrophages that were (a) undifferentiated, unstimulated; (b) M-CSF-differentiated, unstimulated; (c) undifferentiated, LPS-stimulated; or (d) M-CSF-differentiated, LPS-stimulated. Myoblast proliferation ratio, nuclei number, and length were measured. Results C2C12 cells cultured in conditioned medium from M-CSF-differentiated, LPS-stimulated macrophages had significantly more nuclei and greater length than cells cultured in conditioned medium from undifferentiated, LPS-stimulated macrophages. Dilution and denaturization of the M-CSF-differentiated, LPS-stimulated-macrophage medium prevented a marked increase in C2C12 nuclei number and length. However, the C2C12 myoblast proliferation ratio was significantly greater in conditioned medium from undifferentiated, LPS-stimulated macrophages than in conditioned medium from M-CSF-differentiated, LPS-stimulated macrophages. Conclusions M-CSF-differentiated, LPS-stimulated macrophages may influence myogenesis and the early and terminal stages of muscle regeneration. This knowledge may aid in developing therapies that will directly expedite muscle repair and lead to faster rehabilitation and reduced rehabilitation costs.

Macrophage colony stimulating factor-induced macrophage differentiation influences myotube elongation.

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Macrophage colony stimulating factor-induced macrophage differentiation influences myotube elongation.