Case Studies in Neuroscience: Neuropathology and diaphragm dysfunction in ventilatory failure from late-onset Pompe disease

Abstract

Pompe disease (PD) is a neuromuscular disorder caused by a mutation in the acid alpha-glucosidase (GAA) gene. Patients with late-onset PD retain some GAA activity and present symptoms later in life, with fatality mainly associated with respiratory failure. This case study presents diaphragm electrophysiology and a histological analysis of the brainstem, spinal cord, and diaphragm, from a male PD patient diagnosed with late-onset PD at age 35. The patient was wheelchair dependent by age 38, required nocturnal ventilation at age 40, 24-h noninvasive ventilation by age 43, and passed away from respiratory failure at age 54. Diaphragm electromyography recorded using indwelling "pacing" wires showed asynchronous bursting between the left and right diaphragm during brief periods of independent breathing. The synchrony declined over a 4-yr period preceding respiratory failure. Histological assessment indicated motoneuron atrophy in the medulla and rostral spinal cord. Hypoglossal (soma size: 421 ± 159 µm²) and cervical motoneurons (soma size: 487 ± 189 µm²) had an atrophied, elongated appearance. In contrast, lumbar (soma size: 1,363 ± 677 µm²) and sacral motoneurons (soma size: 1,411 ± 633 µm²) had the ballooned morphology typical of early-onset PD. Diaphragm histology indicated loss of myofibers. These results are consistent with neuromuscular degeneration and the concept that effective PD therapy will need to target the central nervous system, in addition to skeletal and cardiac muscle.NEW & NOTEWORTHY This case study offered a unique opportunity to investigate longitudinal changes in phrenic neurophysiology in an individual with severe, ventilator-dependent, late-onset Pompe disease. Additional diaphragm and neural tissue histology upon autopsy confirmed significant neuromuscular degeneration, and it provided novel insights regarding rostral to caudal variability in the neuropathology. These findings suggest that a successful treatment approach for ventilator-dependent Pompe disease should target the central nervous system, in addition to skeletal muscle.

Keywords: Pompe disease; diaphragm; neuropathology; respiratory; spinal cord.

Graphical abstract

Figure 1.

Timeline of relevant case events. DC, discontinued; dx, disease; EMG, electromyogram; MV, mechanical ventilation; NIV, noninvasive ventilation.

Figure 2.

Representative diaphragm EMG recordings over a 3-yr period. EMG was recorded while the patient was spontaneously breathing without ventilator support (left) and during ventilator assistance (right). The traces show bilateral diaphragm EMG activity (purple is left side, blue is right) and respiratory efforts (green; recorded via chest band or pneumotachography) 6-wk, 2-yr, and 4-yr postimplant. In all panels, the scaling (arbitrary units, y-axis) is the same between traces. A shows EMG recordings obtained while the patient was breathing independently, without mechanical ventilator support. Clear inspiratory bursting can be seen, as well as additional asynchronous patterns between the left vs. right diaphragm. Six weeks postimplant, the left diaphragm showed small bursts of activity during expiratory period; two years later, a similar observation was noted in the left diaphragm. Four years postimplant, tonic discharge occurred in the left diaphragm throughout the respiratory cycle. The plots at the right of A show the correlation between left and right peak diaphragm inspiratory burst amplitude. Note that the left and right hemidiaphragm burst amplitude became less correlated over the next four years. The recordings shown in B were obtained while the patient was receiving mechanical ventilator support and confirm that the diaphragm was less active during these conditions. The low activity enabled discrimination of individual motor unit activity. The unit traces on the right of the figure show a histogram of burst rate overlaid with the respiratory cycle, and an average of the unit waveform. Both phasic and tonic motor units were detected in the left hemidiaphragm but were otherwise unremarkable in appearance. a.u., arbitrary units; EMG, electromyogram.

Figure 3.

Histological sections stained with cresyl violet and IBA-1 antibodies. Immunohistochemistry to recognize IBA-1 was performed to provide a marker for microglia; tissues were counterstained using cresyl violet to enable visualization of neuronal morphology. The boxed areas in the left (indicated by i and ii) are shown at a higher magnification in the middle and right. Within those panels, additional higher magnification views are provided in the insets. The circle in Ai indicates the approximate location of the hypoglossal motor nucleus. Neuronal soma were larger in the lumber spinal cord as compared to the more rostral locations. IBA-1 staining was prominent in white matter in the medulla, cervical, and thoracic cord (e.g., arrows in Aii and Bii; inset panels in Bii and Cii). Calibration bars: A–D left: 1 mm, middle (i): 500 µm, inset: 100 µm; right (ii): 500 µm, inset: 100 µm.

Figure 4.

Motoneuron soma size was smaller in the medulla, cervical, and thoracic spinal cord compared to the lumbar and sacral spinal cord. Sample sizes: medulla, n = 49; cervical, n = 50; thoracic, n = 23; lumbar, n = 63; sacral, n = 31. One-way ANOVA on ranks indicated a statistical difference across the neuraxis: lumbar and sacral soma size were different than medulla, cervical, and thoracic (P < 0.001). Cerv., cervical; Thor., thoracic.

Figure 5.

PAS staining indicates neuronal lipofuscin accumulation. A shows putative motoneurons from the anterior horn of the thoracic spinal cord. B shows putative motoneurons from the lumbar spinal cord. Neuronal lipofuscin accumulation was prevalent (arrows). Note also the striking difference in the size of motoneurons between the thoracic and lumbar spinal cord. Scale bars = 100 µm. PAS, periodic acid-Schiff.

Figure 6.

Additional histological examples from the mid cervical spinal cord. Tissues shown in A were stained with H&E and luxol fast blue. The dorsal root can be clearly observed (i), as well as ventral rootlets (ii). The relative small size of the ventral rootlets may indicate atrophy and/or loss of motoneuron axons. In B, tissues were incubated with anti-GFAP antibodies to provide a marker for astrocytes. There is a greater density of GFAP staining in dorsal and ventral lateral white matter. Higher magnification views of gray matter (right) show GFAP-positive cells in the immediate vicinity of putative motoneurons. Calibration bars: A, left: 1 mm, right i and ii: 250 µm; B, left: 1 mm, right: 50 µm. GFAP, glial fibrillary acidic protein; H&E, hematoxylin-eosin.

Figure 7.

Diaphragm histology indicates profound pathology with apparent loss of myofiber protein. The tissues in A were stained for Myosin-1 (red) and Myosin-2 (brown). The most striking observation is the complete lack of stain in many putative myofibers. These “ghost fibers” are seen to have a cell membrane but do not stain for Myosin. B shows the results of PAS staining to recognize glycogen. Calibration bars: A, left: 100 µm; right: 50 µm; B, left: 200 µm; right: 50 µm. PAS, periodic acid-Schiff.