At birth, all affected pups exhibited multiple contractures of axial and appendicular joints. Examination did not reveal airway blockage. Severing the phrenic nerve caused immediate spasmodic contraction of the ipsilateral diaphragm in normal littermates sacrificed at birth, but not in affected pups, suggesting that failure to inspire was due to lack of innervation or intrinsic dysfunction of respiratory muscles. Despite a wide range in each group, the mean birth weight of affected puppies (193 ± 36 g, mean ± SD, range 124-288 g; n = 26) was less (p<2×10-5) than that of normal littermates (237 ± 38 g, range 149-316 g; n = 38). Total lung wet weight was significantly less in the affected pups (3.8 ± 0.9 g vs. 8.6 ± 1.7 g; p<8×10-11) even when normalized to body weight (0.021 ± 0.005 vs. 0.036 ± 0.007; p<7×10-9), indicative of pulmonary hypoplasia. The difference in lung weight was far greater than could be attributed to increased blood content likely present in the normal pups' lungs.
Gross morphologic abnormalities of other internal organs were confined to the CNS. Cerebral hemispheres of affected pups showed mild generalized volume reduction compared to normal littermates euthanized at birth, but otherwise demonstrated no malformation or encephaloclastic lesions. Serial coronal sections of the cerebral hemispheres showed well-developed gray and white matter structures in both affected and control pups, and the extent of myelination was similar. The weight of the formalin-fixed cerebrum with diencephalon of affected pups was ~ 75% of that of normal littermates sacrificed at birth (5.2 ± 0.74 g, n = 19 vs. 6.8 ± 1.1 g, n = 10; p<0.002). The brainstem of affected animals was similarly reduced in weight (~75%) but also without focal lesions or gross evidence of malformation. Strikingly, however, the cerebellum in affected dogs was markedly reduced in size compared to those in control animals (, panels A and C), with a rudimentary folial pattern in both the cerebellar vermis and lateral cerebellar hemispheres. Weight of the formalin-fixed cerebellum of affected pups was less than 50% of littermate controls (0.15 ± 0.02 g, n = 19 vs. 0.32 ± 0.06 g, n = 10; p<10-5). Throughout its length, the cross-sectional area of spinal cord in affected pups was ~ 35% that of normal controls, and the reduction in area affected white and grey matter nearly equally (, panels B and D). Dorsal and ventral spinal roots and peripheral nerves of affected pups were reduced in diameter and more translucent than those of control pups.
Figure 2 Cerebellar and spinal cord hypoplasia. Panels A and C show the left lateral view of cerebellum and brainstem of a newborn normal pup and an affected littermate, respectively. In each panel the fat arrow indicates the caudal colliculus, and the arrowhead (more ...)
Histological abnormalities of the CNS were apparent by light microscopy of routinely stained sections, but lesions were confined to specific nerve tracts and nuclei. Specifically, no microscopic differences between affected pups and controls were detected in the cerebral hemispheres. The neuronal layers were formed similarly in both groups, including the placement of large pyramidal neurons. In frontal lobe coronal sections, the frontal neocortex, cingulated gyrus, corona radiata, caudate, and putamen showed intact neuronal populations. More posteriorly, the cerebral neocortex, hypothalamus, optic nerves, mamillothalamic tract, crus of fornix, hippocampus, parahippocampal gyrus, habenular nucleus, pituitary, subthalamic nucleus, and zona incerta were anatomically intact and devoid of pathological lesions in affected and control pups. Active myelination (“myelination gliosis”) was present within the white matter, and neither qualitative nor quantitative differences in glial acidic fibrillary protein (GFAP) immunoreactivity were detected in the cerebral hemispheres of affected pups and controls.
In contrast to the above, there was extensive pathology in the cerebellum, brainstem, spinal cord and peripheral nerves. Brainstem sections in affected animals were remarkable for widespread neurodegeneration and the presence of swollen axons/spheroids (neuroaxonal dystrophy) in nuclei and tracts of the extrapyramidal components of the motor system (). In longitudinal section, axonal swellings observed in the CNS were 5-12 μm in diameter, tapered at both ends, and 20-120 μm long. They were eosinophilic on H&E, blue/grey on cresyl violet, blue on Klüver-Barrera, grey/black on Holmes' silver, red on PAS stains before and after diastase digestion, and were almost unstained on the hematoxylin counterstain used during immunohistochemical staining. In cross-section, dystrophic axons were round, and they were variously homogeneous, palely vacuolated, or more intensely stained centrally on H&E, PAS, and Holmes' silver stained sections. The identity of spheroids as swollen axons was confirmed by intense immunostaining for neuron-specific enolase (NSE, , panel E) and absence of staining for GFAP (, panel F). Some, but not all, dystrophic axons in the brainstem and spinal cord stained intensely for phosphorylated neurofilaments (, panel D). Electron microscopy of dystrophic brainstem axons revealed axon-filling accumulations of small, pleomorphic, membrane-bound vesicles containing variously electron-dense fragments of organelles and amorphous material (, panels G and H). Some mitochondrial fragments were contained in double membrane vesicles and were in various stages of degeneration, suggestive of ongoing mitophagy. There were whorls of disorganized intermediate filaments among the vesicles in some spheroids, consonant with the variable neurofilament immunostaining indicated above.
Figure 3 Staining, antigenic, and morphologic characteristics of dystrophic axons. Axonal swellings stained pink with H&E (panel A, red nucleus) and PAS (panel B, rostral cerebellar peduncle shown, stained after diastase digestion), and grey to black with (more ...)
Neuroaxonal dystrophy was prominent throughout the mesencephalic, pontine, and medullary tegmentum. Swollen axons were observed in lateral and medial ventral thalamic, red, and caudal olivary nuclei; the cerebellorubral tract, the rubrospinal tract, the mesencephalic tract of the trigeminal nerve, and rostral and caudal cerebellar peduncles; and diffusely distributed in the reticular formation (). They were particularly obvious at midline decussations of the rostral and caudal cerebellar peduncles and the myencephalic reticular formation. The medullary reticular formation was conspicuously devoid of intact large neuron perikarya of the gigantocellular tegmental field in affected pups, with only remnants of cells remaining in normal positions. There were signs of neuronal degeneration and dystrophic axons in cranial nerve (CN) nuclei and tracts, respectively, of CN V, VII, IX, X, XI, and XII. These lesions were not observed in the cerebellar white matter, crus cerebri, pontine corticospinal tract, colliculi, pyramidal tracts, rostral olives, pontine nuclei, or CN III, IV, and VI. Iron deposition was not observed in the brainstem or any other part of the CNS.
Figure 4 Brainstem neuroaxonal dystrophy and astrocytic reaction. Transverse sections through the red nucleus and nucleus of CN III (panels A and B), genu of CN VII nerve tract and nucleus of CN VI (panels C and D), nucleus and ascending fibers of CN VII (panels (more ...)
In comparison to controls, the affected pup brainstem sections exhibited variably increased GFAP staining and astrocyte hypertrophy in the areas of the red nucleus, the decussation of the cerebellorubral tract, the transverse pontine fibers, throughout the medullary tegmentum, ascending and decussating fibers and the septae of the caudal olives, all along the course of CN VII, and throughout the nucleus ambiguous (). Areas that were consistently spared included the pyramids, superior olives, and the pontine nuclei. Despite widespread morphologic evidence of apoptotic cells (condensed, fragmented nuclei and darkly eosinophilic cytoplasm), there was very little activated caspase-3 staining of brainstem neurons and no more so in affected than in control pup sections.
Sections of cerebellar cortex, including vermis and hemispheres, from affected pups were remarkable for reduced foliation, decreased neuronal precursor populations in the external granular cell layer, and decreased Purkinje cells and internal granular neurons (). Purkinje cell loss was patchy; many were degenerating or absent, but a subset of residual Purkinje cells were present in their usual position between the external and internal granular cell layers. Calbindin immunostain also revealed a dearth of Purkinje cell axons in the arbor vitae. None of the routine or immunohistochemical stains applied revealed swollen axons/spheroids in the cerebellum. The external granular cell layer varied in thickness from complete absence in some areas up to 35 microns in others, while the external granular layer in control pups was of uniform thickness throughout, at approximately 40 microns. Affected and normal pups showed approximately equal numbers of mitotic figures in the external and internal granular layers and of activated caspase-3 stained cells in the internal granular layer and deeper in the arbor vitae. Similarly, in both affected and normal pups there was exuberant GFAP staining of astrocytes throughout the arbor vitae with fine extensions through the internal granular, Purkinje, and external granular cell layers. In contrast, only affected pups showed morphologic or activated caspase-3 evidence of increased Purkinje cell apoptosis, and only in affected pups were there patches of astrocyte hypertrophy in the Purkinje and external granular layers (, panels F and H). Positions of neurons of (deep) cerebellar nuclei were often observed as holes in the neuropil or mere remnants of cells (, panel J). Remaining neurons in these nuclei exhibited pyknotic nuclear fragmentation and deeply eosinophilic cytoplasm on H&E stained sections. Some, but only a small subset of cells with such morphologic evidence of apoptosis also stained for activated caspase-3. GFAP staining was attenuated in the deep nuclei of normal pups relative to the surrounding neuropil but was somewhat increased in the deep nuclei of affected pups.
Figure 5 Cerebellar histopathology. Tissue sections derived at birth of normal littermates (panels A, C, E, G) and FNAD affected pups (panels B, D, F, H, I, and J) are shown. Panels A-H and I show near-midline sagittal sections of cerebellum with the dorsal surface (more ...)
In spinal cord, the affected pups had fewer intact neurons in the lateral and ventral horns and dorsal root ganglia at all levels of the cord when compared to littermate controls (, panels A and B). The neurons that remained were in various stages of degeneration as evidenced by chromatolysis with eccentric nuclei or overall light staining (ghost cells), occasional apoptotic morphology, and some neurons that were surrounded by gliotic tissue. Swollen axons and spheroids similar to those in the brainstem were seen at all levels of the spinal cord white matter and occasionally in gray matter. In the cranial cord, these were in all funiculi but most consistently observed in the fasciculi gracilis and cunteatus and the spinal tract of the trigeminal nerve. More caudally, spheroids were most often observed in the lateral and ventral funiculi. Compared to controls, there were astrocyte hypertrophy and increased GFAP-immunoreactive cell processes throughout the lateral and ventral horns of spinal cord gray matter (, panels C and D). Neurons exhibiting activated caspase-3 staining were rare in affected pup sections of cord but were not observed at all in normal littermate sections.
Figure 6 Spinal cord histopathology. Transverse sections of spinal cord segment C6 of a normal littermate (panels A and C) and an FNAD affected pup (panels B and D) at birth are shown with Klüver-Barrera stain (panels A and B) or GFAP immunostain (panels (more ...)
In order to observe earlier stages of disease progression in the CNS, two pregnancies were interrupted by cesarean section at 53 and 60 days of gestation, respectively, and fetal tissues were examined. The same cerebellar and brainstem abnormalities observed in full-term affected pups were seen in affected fetuses, although there were fewer swollen axons and less neuronal degeneration. Delayed cerebellar development was already evident at 53 days of gestation, but it appeared that the folia had developed further in the full term affected pups. In these, the youngest affected fetuses examined, neurons of the deep cerebellar nuclei were present, but signs of neurodegeneration were already evident. The spinal cord of affected pups was already reduced in diameter to ~70 % of normal controls at 53 days of gestation. The large neuronal perikarya of the ventral horn were in appropriately placed groups but already showed some mild degenerative changes, and there was increased astrocytosis in the lateral and ventral gray matter.
Dorsal and ventral spinal nerve roots in affected pups examined at birth were remarkable for the paucity of myelinated axons (, panels E-H). For instance, at the L5 segment, an affected pup dorsal root had fewer than 50% of the myelinated axons of a normal littermate (1806 vs. 3793), and the number of ventral root axons was reduced to ~ 60% of normal (3156 vs. 5305). Numbers and morphology of Schwann cell nuclei appeared normal, and myelin sheaths had normal appearance. Dystrophic axans were present in spinal roots of affected pups as well (, panels F, I, and J). Electron microscopy of dorsal roots demonstrated some hugely dilated axons (5-15 μm diam. vs. 1-3 μm diam. of surrounding axons in affected pups, as well as in normal littermates). They had intra-axonal accumulation of disordered neurofilaments, degenerating mitochondria, and small vacuoles containing amorphous material, but the myelin sheaths appeared normal.
Spheroids were also observed in peripheral nerves and intramuscular nerve branches of affected pups (). In the peroneal nerve, there were fewer myelinated axons overall (1235 ± 75 vs. 3051 ± 273), and the average diameter of remaining axons (excluding the obviously swollen axons with diameters > 6 μm) was less than that of normal pups (1.5 ± 0.5 μm vs. 2.8 ± 1.1 μm). In the phrenic nerve of affected pups, myelinated axon number was better preserved (1053 ± 65 vs. 1308 ± 27), but average axon diameters were again reduced (0.9 ± 0.3 μm vs. 1.5 ± 0.5 μm) (, panels A and B). Myelin sheaths appeared normal in the peripheral nerves examined.
Figure 7 Peripheral nerve and skeletal muscle histopathology. Transverse sections of phrenic (panels A and B) and sciatic (panels C and D) nerves and semimembranosus muscle (panels E-H) of a normal littermate (panels A, C, E, and G) and an FNAD affected pup (panels (more ...)
In skeletal muscle of both affected and normal pups observed at birth, most muscle cells were small, though in some areas they varied in size with occasional hypertrophic cells, and some had central nuclei. In both, there was a mixture of myotubes and myofibers, and there were appropriate numbers of muscle spindles. Enzymatic histochemical fiber typing revealed almost entirely type II fibers, as previously described in newborn dog muscle (Braund and Lincoln 1981
; Shelton et al., 1988
). In affected pup muscle, however, there was increased space between fibers, sporadic or groups of small fibers, and increased numbers of fibers showing pyknotic and fragmented nuclei, suggestive of apoptosis (, panels G and H). This was confirmed by observation of increased numbers of myocytes in affected pup muscle staining positively for activated caspase-3 (7.4 ± 3.4/high power field vs. none observed in normal littermates) (, panels E and F).
Microscopic examination of the stifle in affected pups revealed multiple abnormalities previously described as secondary to joint immobilization (Akeson et al., 1987
). The patellar tendon was 50-60 % the thickness of that in normal littermates, and femoral and tibial cortices were thinned. The origin of the patellar tendon exhibited disorganization of collagenous fibers, and its insertion on the tibia was compromised by loss of Sharpey's fibers and osteoclastic resorption of bone. There was early-stage proliferation of fibrofatty connective tissue within the joint space and among tissues caudal to the joint.