Pathological findings in a Dachshund-cross dog with neuroaxonal dystrophy

Two Dachshund-cross breed puppies belonging to the same litter developed progressive
difficult walking since they were a few weeks old. One puppy was euthanized without
post mortem examination, while the other puppy was presented at 1 year of age for
clinical examination. Neurological examination reveled hypermetria, proprioceptive
positioning deficits and irreversible ataxia, particularly of the hind limbs. Muscular
tone, bulk and strength were normal and higher mental functions appeared integral.
The dog was humanely euthanatized because of the progressive incurable nature of the
disease.

At necropsy, brain, spinal cord, tracts of the sciatic nerve, as well as samples from
a number of forelimb muscles, liver, and kidney were collected and promptly fixed
in 10 % neutral buffered formalin. Coronal sections were obtained at six different
levels of the encephalon (basal nuclei, thalamus, mesencephalon, pons, medulla oblongata,
cerebellum), as well as at the levels of each emergence of the nerves of the spinal
cord and then routinely processed and embedded in paraffin wax. Sections were cut
serially from paraffin blocks at 5 µm and stained using hematoxylin and eosin, modified
Period Acid Shiff Picro Indigocarmine Morel–Maronger (PAS-M.M.), Klüver-Barrera Luxol
fast Blue (KB), and Perl’s stain. Furthermore, in-depth immunohistochemical investigations
were performed on selected tissue sections using specific primary antibodies for the
following markers of axonal transport: neurofilaments light chain (NF-Ls,) and tau,
cytoskeletal proteins; ubiquitin, a heat shock protein; synaptophysin, a synapse-associated
protein; glial fibrillary acidic protein (GFAP), the specific marker of astrocytes
(Table 1). To investigate the possible role of autophagic mechanisms in the formation of spheroids,
a specific primary antibody against the autophagosomal microtubule associated protein
1A/1B-light chain 3 B (LC3B) was used (Table 1). Primary antibodies were always incubated overnight at 4 °C and immunoreactivity
(IR) was detected via the biotin-avidin-peroxidase method, using the 3,3?-diaminobenzidine
as a chromogen, Additional technical details are shown in Table 1. Transversal and longitudinal sections of the sciatic nerve as well as sections from
muscles, liver and kidney were also embedded in paraffin, cut serially from the paraffin
blocks at 5 ?m and stained using hematoxylin and eosin. Finally, sections of the sciatic
nerve were also stained immunohistochemically for GFAP, and histochemically for myelin
by using KB technique. Immunohistochemically negative controls were obtained by omitting
the primary antibody and using normal brain tissues of two crossbreed dogs aged 6 months
and 1 year, respectively.

Table 1. Technical details of the immunohistochemical examinations

At necroscopy, no gross changes were observed. Histological examination throughout
the central nervous system (CNS) revealed a bilateral symmetric neuroaxonal dystrophy,
which was uniquely at the level of the ventral posterior lateral nucleus of the thalamus,
medial lemniscus, gracilis nucleus, and medial cuneatus nucleus in the brain. The
same change in the spinal cord was found in the gracilis and cuneatus fasciculi, particularly
in the thoracic tract (Fig. 1). In the transversal sections, neuroaxonal changes were characterized by 3–50 µm
sized axonal spheroids, round to ovoid in shape and stained slight to intensely eosinophilic
(Fig. 2a).

Fig. 1. Distribution of the axonal spheroids. Illustration of the encephalic and spinal sections
indicating the fasciculi and nuclei with spheroids throughout the central nervous
system. Brain section: Red dotted circle: ventral posterior lateral nucleus of the thalamus; yellow dotted circle: medial lemniscus; turquoise dotted circle: gracilis nucleus; blue dotted circle: medial cuneatus nucleus. Spinal cord sections: turquoise dotted circle: funiculis gracilis; blue dotted circle: funiculis cuneatus

Fig. 2. Photomicrographs of the histological patterns. Section of the medulla oblongata showing
the medial cuneatus nuclei (a). Presence of numerous 3–50 µm sized eosinophilic axonal spheroids (arrow head), some of them are vacuolized (arrow). Thoracic tract the spinal cord (b). Longitudinal section at level of gracilis and cuneatus fasciculi displays digestion
chambers with axonal debris and scattered macrophages (asterisk). HE. Bar = 100 µm

In longitudinal sections at the level of the gracilis and cuneatus fasciculi, aspects
of Wallerian-like degeneration with different digestion chambers containing sporadic
macrophage and axonal debris were found (Fig. 2b). Occasionally, spheroids were found in the dorsal horn of the spinal cord.

Histochemical staining revealed that some spheroids contained PAS-M.M positive material
and KB staining evidenced relative preserved myelin. Perl’s stain did not highlight
the presence of iron in any sections of the encephalon.

In all CNS sections, spheroids showed immunoreactivity for NF-Ls, tau, synaptophysin
and ubiquitin with the signal being more intense at the level of the spinal cord (Fig. 3). In addition, immunohistochemistry for LC3B protein detected an intense autophagosome
accumulation within spheroids (Fig. 3).

Fig. 3. Photomicrographs showing immunohistochemical reactivity of axonal spheroids. Transversal
sections of thoracic spinal cord (gracilis and cuneatus fasciculi) show numerous spheroid
immunohistochemically reactive (IR) for neurofilaments (a), tau (c), synaptophysin (e), ubiquitin (g), and LC3B (i) of thoracic tract of the spinal cord the NAD-affected puppy. As expected, single
spheroids were identified in the healthy dog (b, darrow head), while LC3B IR is observed in the glial cells of the affected and control dogs (i, j). Synapthophysin was also evident in the neuropil of the control (f). Avidin–biotin-peroxidase complex method with Mayer’s hematoxylin counterstain.
Bar = 100 µm

Finally, the immunohistochemistry for GFAP confirmed the presence of evident astrogliosis
and astrocytosis in the nuclei displaying spheroids. The sections from muscles, liver,
kidney and sciatic nerve were histologically normal. As in the control, the affected
dog showed weak GFAP immunoreactivity with a linear pattern in the longitudinal sections
of the sciatic nerve (Fig. 4). In the same nerve, no demyelization was observed by using KB stain.

Fig. 4. Photomicrographs of the immunohistochemical reactivity of the sciatic nerve. Sciatic
nerve from NAD-affected dog (a) and healthy control dog (b). Longitudinal section of sciatic nerve shows weak linear immunohistochemical reactivity
for GFAP both in affected (a) and healthy dog (b). Avidin–biotin-peroxidase complex method with Mayer’s hematoxylin counterstain.
Bar = 100 µm

On the basis of these clinical, histopathological and immunohistochemical features,
a NAD disorder was diagnosed.