The histochemical periodic acid-Schiff (PAS) method is less quantitatively precise than the chemical data above, but adds considerably to recognition of regional differences and the cell types affected. The major pathological process in the nervous system of the 6neo/6neo Pompe model mouse is intracellular accumulation of glycogen in cells of all types, but with significant regional differences in amount and temporal progression. No evidence was found of developmental abnormality, neuronal loss, cellular infiltration or myelin breakdown. Brain shapes were normal; ventricles in the brains of knockout mice were not enlarged compared to matched controls; pyknotic nuclei were not seen; perivascular inflammatory cell cuffs were absent; macrophage and microglial cell numbers stained by F4/80 were minimal and not different than in control sections; and no myelin breakdown was seen by electron microscopy. Only at the oldest ages (i.e. 15 and 22 months) was there evidence of some astrogliosis, particularly in white matter tracts, as seen in GFAP-stained sections (data not shown).
Glycogen, visualized by the red color imparted by the PAS stain and absent from control sections pretreated with salivary amylase, was below the threshold for detection in the normal mouse nervous system but was dramatically increased in the knockout mice. The striatum () was representative of regions reacting for glycogen progressively as a function of age, reaching only moderate intensity at this site even by the relatively advanced age of 15 months. Slight glycogen accumulation in neurons, glia, pericytes and choroid plexus was seen starting at 3 months of age and progressively increased over time. Similar results were seen in the thalamus, although there was considerable variation among thalamic nuclei (data not shown).
Figure 2 Glycogen accumulation in each CNS region is progressive. Sagittal sections of paraffin-embedded tissue, sectioned at 7 μm and stained with PAS-hematoxylin show a portion of the lateral ventricle containing choroid plexus (upper left of each field) (more ...)
Large neuron cell bodies in multiple brainstem motor and sensory relay nuclei contained no detectable glycogen in control mice, but some large neurons in the knockout mice, such as the facial nucleus in the ventral medulla, were already packed with glycogen-rich enlarged lysosomes at one month of age. Both the total amount and concentration of glycogen per cell body increase progressively, so that the neuron cell bodies became correspondingly enlarged (). By contrast, adjacent fascicles of white matter in the knockouts contained relatively little glycogen at corresponding early ages, although the amount increased with age.
Figure 3 Glycol methacrylate-embedded specimen, sectioned at 2 μm, stained with the PAS method and the Richardson counterstain, showing cells in the facial nucleus in the ventral pons. (A) Wild-type 3-month-old control mouse. (B–D) Panels are from (more ...)
As demonstrated in , most parts of the cerebral cortex stained weakly to moderately for glycogen, even at ages greater than one year. An exception was the glomerular layer of the olfactory bulb in which granule cell neurons within and around the glomeruli were distinctly positive for glycogen at one month, and then gained glycogen dramatically and progressively, as illustrated at 3 and 15 months ().
Figure 4 Olfactory bulb glomerular layer, paraffin, 7-μm section, PAS-hematoxylin stain. (A) Wild-type 6-month control. (B–D) Knockouts at 1, 3, and 15 months, respectively. Glycogen increases progressively in periglomerular granule cell neurons (more ...)
The rostral migratory stream (RMS), delineated distinctively by its strong glycogen staining, carried stem cells from the subventricular zone () in the RMS () all the way to the olfactory bulb (). The RMS widened as its cells were about to enter the deep (internal granular layer) and superficial (glomerular layer) parts of the olfactory bulb. The ependymal cells contained lipid droplets but were almost glycogen-free (), whereas the majority of the large and medium-sized subventricular astrocytic cells in the adult stem cell niche (35
) () and RMS (36
) () were intensely glycogen-positive, and were thus distinguished from CNS astrocytes in general, which stained relatively lightly or moderately for glycogen.
Figure 5 (A, B) Glycol methacrylate-embedded 3-μm sections of 4% paraformaldehyde-fixed, post-osmicated tissue stained with the periodic acid-Schiff method for glycogen, counterstained with Richardson’s stain, through the adult stem cell niche (more ...)
Similar to the cerebral cortex, striatum, and thalamus, the hippocampal formation (, center of the field) became moderately rich in glycogen by 15 months, but the prominent bands of granule cell neurons of the dentate gyrus and the pyramidal neurons of the hippocampus itself contained less glycogen than the interstitial, presumably GABAergic neurons. Note that the pyramidal neurons in hippocampal area CA1, between the two bars, are almost entirely free of glycogen. Likewise, most of the projection neurons of the cerebral cortex contained little glycogen, in contrast to the heavily stained penetrating blood vessels. Thus, glycogen accumulation was present in multiple cell types including neurons, glia and pericytes, but some cells were more severely involved than others. Accumulation of glycogen, however, was not the sole abnormality; the inset at the lower right corner displays a band of CA1 pyramidal neurons oriented like the cell band between the two bars in the main part of the figure stained with PAS/hematoxylin, whereas the inset displays a non-fluorescent background (black) and superimposed fluorescence (white) due to binding of the fluorescent molecule, filipin, to lysosomal cholesterol which is known to accumulate non-specifically in the abnormal lysosomes in several of the lysosomal storage diseases.
Figure 6 Paraffin section, 7 μm, PAS-hematoxylin. The hippocampal formation (dentate gyrus, hippocampus CA1, Ca2, and CA3, and subiculum) occupies the center of the field, and a sector of cerebral neocortex lies in the left part of the field of a 15-month-old (more ...)
The extent of the glycogen accumulation and the increase with age varied among brain regions and the cell types involved. For example, in thalamus and striatum, glycogen increased earlier and staining was more intense in small blood vessels and glial cells than in small- to medium-sized neurons (, ). By contrast, in spinal cord and lower brainstem the most prominent abnormality was the massive and early increase of glycogen in large neurons, especially motor neurons (, ), but also in some other sites such as the medial vestibular nucleus, the ventral nucleus of the posterior colliculus, and the mesencephalic nucleus of the trigeminal (data not shown).
Figure 10 (A) Paraffin-embedded 7-μm section stained with the PAS method and counterstained with Richardson’s stain shows that many, but not all, dorsal root ganglion (DRG) neuron cell bodies are filled with glycogen and contain vacuoles at sites (more ...)
In contrast to the consistent observation of marked glycogen accumulation in large neurons of the ventral horn of the spinal cord and in the brainstem, the Purkinje neurons, which are among the largest cells in the brain, contained very little glycogen and only slight vacuolation (, white arrows). By contrast, the adjacent specialized astrocytes, called Golgi epithelial cells, appear to bear the brunt of the disease process, as they had intensely vacuolated cell bodies (, black arrows) and stained intensely for glycogen (, black arrows). Even at 15 months of age, the Purkinje neurons were relatively spared, but the adjacent Golgi epithelial astrocyte cell bodies and their radially oriented cytoplasmic processes (Bergman fibers) crossing the molecular layer were filled with glycogen (). At this late stage, the entire molecular layer at this late stage was diffusely glycogen-positive and there was abundant glycogen in the granular layer (, in clumps adjacent to the small granule cell bodies [perhaps in mossy fibers—incoming axon terminals contacting granule cell dendrites], in glial cells of the folial white matter (, right lower quadrant), and in the entire nuclear region deep in the cerebellum (data not shown). The final point is that Purkinje neurons, although almost devoid of glycogen, did fluoresce intensely based on binding of the fluorescent reagent, filipin, to cholesterol in the apical cytoplasm of these cells (, white arrows). Loss of Purkinje and granule cell neurons was not recognized at any age examined and myelin appeared normal by the methods used.
Figure 7 Cerebellar cortex, 15-month-old knockout mice. (A) Glycol methacrylate-embedded tissue sectioned at 3 μm, stained with Richardson’s stain. Purkinje neuron cell bodies (white arrows) are mildly vacuolated (and almost free of glycogen, as (more ...)
A striking finding was the presence of remarkably enlarged degenerating axons and especially axon terminals in the lower brainstem and spinal cord. The affected axons arise in sensory neurons of the PNS, in dorsal root ganglia (DRGs) and in the trigeminal ganglion. Axons of DRGs run up the spinal cord within the dorsal columns and terminate in the nuclei gracilis and cuneatus of the lower medulla; axons arising outside the brain in the trigeminal ganglion course caudally through the pons and medulla in the spinal tract of the trigeminal nerve and terminate in contact with second order neurons in the spinal nucleus of the trigeminal tract. Swollen degenerating axonal shafts were seen within the spinal cord in sections stained with Fluoro-Jade C (). Progressive evidence of degeneration was seen starting at 6 months of age. Degenerating axon terminals were most vividly demonstrated with silver degeneration staining methods (). Conclusive evidence that the silver images reliably demonstrate abnormal axons and axon terminals was obtained by staining adjacent sections with hematoxylin and eosin, which showed relatively uniformly stained eosinophilic structures of the same positions and sizes as the silver images (). The diameters of the degenerating terminals ranged from approximately five to more than 50 μm (). Trigeminal axon terminals () were less markedly swollen than dorsal root ganglion axons (), but were still far larger than their normal counterparts, which are not resolvable by standard light microscopy. Axonal profiles of comparable large size are never seen in spinal cords or brains of normal rodents.
Figure 8 (A, B, and b′) From a transverse section through the cervical spinal cord of a 6-month-old knockout mouse stained with the fluorescent dye, Fluoro-Jade C, showing swollen axons that cross the dorsal gray matter (A, arrowheads in the boxed area, (more ...)
The one additional site in which degenerating axon terminals consistently occurred was in the mossy fiber terminals on pyramidal neuron dendrites in hippocampal segments CA2 and CA3 of knockout mouse brains. These axons originate from granule neuron cell bodies in the dentate gyrus. The strongest reaction, beginning at about one month of age, was close to the CA2–CA3 boundary. In the dorsal part of the rostral hippocampus in coronal sections, the exact position of this boundary is uncertain in Nissl-stained sections, but the silver reactivity was always stronger on the boundary’s CA2 side (, black arrow) than on the CA3 side (, white arrow), and was minimally detectable in the hilus and dorsal limb of the dentate gyrus (). More caudally in coronal sections (), the hippocampal formation curves down toward the ventral surface of the brain, and the CA2 (black arrow) and CA3 (white arrow) segments in this area are distinctly separated from each other. Here it was clear that the stronger reactivity was in CA2 (, black arrow). The CA2 region is functionally distinct from CA1 and CA3 (37
Figure 9 (A) Rostral part of the hippocampal formation in a coronal section from a 7-month-old knockout mouse. Staining intensity is strongest on the CA2 side of the fuzzy boundary between segments CA2 (black arrow) and CA3 (white arrow). The pyramidal cell body (more ...)
Many neurons in the PNS, like those in the CNS, also developed cytoplasmic vacuoles and accumulate glycogen, as illustrated in a typical DRG (, arrowheads). Excessive glycogen was also seen in Schwann cells (, arrows) and less obviously in myelinating glia (data not shown). Also, at all levels of the spinal cord, accumulation of glycogen in neurons of various sizes was prominent, for example, in the thoracic spinal cord of an 8-month-old knockout mouse (). This site was chosen for illustration because Pompe patients typically develop respiratory signs, as well as “floppy baby” motor signs and at older ages, debilitating fatigue (38
). These manifestations might be the consequence purely of the skeletal and cardiac muscle abnormalities, but could possibly also result from spinal cord neuron malfunction. Prominent neuronal storage was found in the ventral horns and in all motor nuclei of the brainstem at 3 month of age. The glycogen in large neurons such as those illustrated in and was predominantly in lysosomal organelles, as shown by electron microscopy ().
Figure 11 Electron micrograph of part of the cell body of a spinal cord motor neuron. The cell nucleus occupies the middle right part of the field, and is surrounded by cytoplasm in which more than 50% of the volume is occupied by enlarged lysosomes. Magnification: (more ...)
Another site of glycogen accumulation was in columnar ependymal cells lining the spinal canal (). By contrast, the cuboidal ependymal cells lining the ventricles throughout the mutant brain were almost free of glycogen that could be detected by the methods used in this study ().
In sections doubly stained with PAS and GFAP immunohistochemistry, glycogen was increased particularly in astrocyte cell bodies, but also was evident in astrocytic cytoplasmic processes extending several cell body diameters outward (data not shown). Glycogen storage in glial cells was widespread but was most pronounced in the white matter of the cerebral hemispheres and spinal tracts. Evidence of moderate gliosis was seen only at 15 and 22 months of age, and was mainly present in white matter tracts and brainstem. Glycogen accumulation was not, however, limited to neurons and glial cells since meningeal cells and blood vessels also accumulated very large amounts of glycogen. The specialized large flat meningeal cells arranged in a monocellular sheet on the external surface of the CNS became filled with glycogen by 15 months of age (), even though cells of peripheral nerve in the immediate vicinity (cranial nerve IV, asterisk) were glycogen-free. Likewise, pericytes and smooth muscle cells in the walls of arteries on the external surface of the brain were rich in glycogen, though the thin endothelial cells lining the same arteries and adjacent veins were glycogen-free (). Arteries can also be affected in the human disease (41
Figure 12 (A–C) Paraffin-embedded 7-μm section stained with the PAS method and counterstained with hematoxylin, showing meningeal cells and large blood vessels at the dorsal surface of the midbrain of a 15-month-old knockout mouse. (A) Large flat (more ...)
The extent of intracellular glycogen accumulation in various nervous system regions as a function of mouse age is summarized in the . These results are consistent with the regional distributions in a 6-month human infant listed in Table 1 in Martin et al (14
Qualitative Evaluation of Increases in Cellular Glycogen