Pompe disease is an autosomal recessive lysosomal storage disorder caused by a deficiency of the lysosomal
enzyme acid alpha-glucosidase, responsible for the degradation of lysosomal
glycogen. Absent or low levels of the
enzyme leads to lysosomal
glycogen accumulation in cardiac and skeletal muscle cells, resulting in progressive
muscle weakness and death from cardiac or
respiratory failure. Recombinant
enzyme replacement and gene therapy are now being investigated as treatment modalities for this disease. A knockout mouse model for
Pompe disease, induced by the disruption of exon 6 within the
acid alpha-glucosidase gene, mimics the human disease and has been used to evaluate the efficacy of treatment modalities for clearing
glycogen. However, for accurate histopathological assessment of
glycogen clearance, maximal preservation of in situ lysosomal
glycogen is essential. To improve retention of
glycogen in Pompe tissues, several fixation and embedding regimens were evaluated. The best
glycogen preservation was obtained when tissues fixed with 3%
glutaraldehyde and postfixed with 1%
osmium tetroxide were processed into
epon-
araldite. Preservation was confirmed by staining with the
Periodic acid-Schiff's reaction and by electron microscopy. This methodology resulted in high-resolution light microscopy (HRLM) sections suitable for digital quantification of
glycogen content in heart and skeletal muscle. Combining this method of tissue fixation with computer-assisted histomorphometry has provided us with what we believe is the most objective and reproducible means of evaluating histological
glycogen load in
Pompe disease.