Intravital microscopy: An innovative approach to track real-time muscle integrity and pathology in Pompe disease

Abstract

The recent article “Intravital imaging of muscle damage and response to therapy in a model of Pompe disease” by Meena et.al published in Clinical and Translational Medicine brings forth crucial insights into the utility of intravital microscopy (IVM) of muscle especially tongue muscle as a possible imaging technique that can be used for monitoring Pompe disease (PD).

PD is an autosomal recessive disease characterized by intralysosomal glycogen accumulation due to the deficiency of acid alpha-glucosidase enzyme. Based on the age of onset and clinical presentation, PD can be broadly divided into two categories: infantile-onset PD (IOPD) and late-onset PD (LOPD). Symptom onset in IOPD is usually before 12 months of age, with progressive hypertrophic cardiomyopathy, hypotonia, axial myopathy, and respiratory failure. LOPD, on the other hand, has a variable age of onset with usual clinical presentation after 12 months, primarily skeletal muscle myopathy with limb-girdle, axial, and respiratory muscle weakness. Lingual weakness resulting in dysarthria and dysphagia have been described as characteristic but commonly overlooked signs of PD.^{1, 2}

Meena et al. have introduced high-resolution IVM as a novel approach to visualize muscle damage in PD and monitor treatment responses. Utilizing a reporter mouse model expressing green fluorescent protein (GFP) fused to autophagosomal marker LC3, (noted as GFP-LC3: KO), researchers demonstrated autophagic buildup in muscle fibres, a hallmark of the disease, using this method. IVM allowed for real-time imaging of muscle tissues, revealing the effectiveness of gene therapy in reversing pathology in both limb and tongue muscles. Further, they combined the GFP signal with NAD(P)H fluorescence signal excited by two-photon microscopy, to measure mitochondrial function and subcellular metabolic activity in live animals. This non-invasive imaging technique offers insights into disease progression and treatment efficacy, presenting a promising tool for assessing emerging therapies for PD.

IVM is a powerful method for visualizing individual cells within intact tissues in near physiological conditions. It is increasingly used in preclinical studies to evaluate dynamic cellular processes underlying disease pathology and response to therapy. Moreover, in combination with the surgical implantation of imaging windows, this technique is being used to facilitate repeated imaging over a prolonged period of time in the same animal.³ It has also been utilized to understand cellular dynamics and interactions in neurodegenerative conditions such as multiple sclerosis at the lesion site in vivo.⁴ Another application would be simultaneous imaging of different cellular and intracellular structures in the muscles, such as neuromuscular junctions and sarcomeres in skeletal muscles in myotubular myopathy disease models.⁵

The quest for disease-specific biomarkers in PD that may aid in predicting a patient's phenotype, disease progression, and response to the current medical management continues to be an area of research. This has become increasingly critical after the implementation of newborn screening, resulting in the early diagnosis of a presymptomatic LOPD cohort. The specificity of the currently used clinical biomarkers, that is, creatine kinase, glucose tetrasaccharide and aspartate aminotransferase, are questionable in infants and children with LOPD since in some cases clinical findings have been found despite normal biomarkers. Therefore, there is an unmet need for non-invasive, sensitive, and specific diagnostic and prognostic biomarkers for PD that could be used in quantifying disease burden, monitoring disease progression, and treatment outcomes.

Recent studies have shown that muscle imaging techniques, such as magnetic resonance imaging (MRI) and ultrasound, can visualize the structural and functional changes in muscles in PD. These changes include muscle atrophy, fatty infiltration, and impaired muscle contractility, which are associated with disease progression. Therefore, muscle imaging could provide a valuable tool for diagnosing and monitoring PD, especially for the non-classic presentations or presymptomatic cohort. However, obtaining a regular MRI is not feasible in the pediatric population due to the need for sedation and the prolonged duration of imaging. Therefore, it is crucial to evaluate the utility of IVM as a possible diagnostic and monitoring tool for PD in the clinical realm. The initial muscles affected in PD include the proximal and paraspinal trunk muscles. Due to the variable amount of subcutaneous adipose tissue between the muscle facia, IVM of skeletal muscle would be invasive, questioning its practicality in the clinical world.

IVM of the lingual musculature, as described by Meena et al., is, however, less invasive.⁶ Early involvement of the tongue muscle in PD has been previously described.² Additionally, the detection of tongue involvement in the form of reduced tongue strength and muscle thickness in PD has been suggested as a valuable marker in differentiating PD from other myopathies.¹ Nonetheless, how early this finding could be detected, the correlation of glycogen deposition in the tongue when compared to the skeletal muscle, and its response to treatment in comparison with skeletal muscle are all areas that should be further explored. Additional animal and human studies should be pursued to evaluate the utility of IVM in the diagnosis and monitoring of PD.

In addition to its utility in human research, as highlighted by Meena et al., IVM is valuable in animal research since it provides a less invasive method by minimizing euthanasia and tissue harvesting for evaluating the efficacy of different therapeutic interventions. This approach reduces the need for a large cohort of animals required for preclinical studies, thereby adhering to ethical guidelines and reducing research costs associated with animal husbandry and experimentation. Furthermore, real-time imaging allows for longitudinal studies within the same animal over time, providing richer data and reducing inter-animal variability. Future directions should involve further animal and human studies exploring the utility of IVM as a tool for comprehensive diagnosis, measuring disease progression and therapeutic efficacy in PD.