Evidence of the Primary Afferent Tracts Undergoing Neurodegeneration in Horses With Equine Degenerative Myeloencephalopathy Based on Calretinin Immunohistochemical Localization

Abstract

Equine degenerative myeloencephalopathy (EDM) is characterized by a symmetric general proprioceptive ataxia in young horses, and is likely underdiagnosed for 2 reasons: first, clinical signs overlap those of cervical vertebral compressive myelopathy; second, histologic lesions--including axonal spheroids in specific tracts of the somatosensory and motor systems--may be subtle. The purpose of this study was (1) to utilize immunohistochemical (IHC) markers to trace axons in the spinocuneocerebellar, dorsal column-medial lemniscal, and dorsospinocerebellar tracts in healthy horses and (2) to determine the IHC staining characteristics of the neurons and degenerated axons along the somatosensory tracts in EDM-affected horses. Examination of brain, spinal cord, and nerves was performed on 2 age-matched control horses, 3 EDM-affected horses, and 2 age-matched disease-control horses via IHC for calbindin, vesicular glutamate transporter 2, parvalbumin, calretinin, glutamic acid decarboxylase, and glial fibrillary acidic protein. Primary afferent axons of the spinocuneocerebellar, dorsal column-medial lemniscal, and dorsospinocerebellar tracts were successfully traced with calretinin. Calretinin-positive cell bodies were identified in a subset of neurons in the dorsal root ganglia, suggesting that calretinin IHC could be used to trace axonal projections from these cell bodies. Calretinin-immunoreactive spheroids were present in EDM-affected horses within the nuclei cuneatus medialis, cuneatus lateralis, and thoracicus. Neurons within those nuclei were calretinin negative. Cell bodies of degenerated axons in EDM-affected horses are likely located in the dorsal root ganglia. These findings support the role of sensory axonal degeneration in the pathogenesis of EDM and provide a method to highlight tracts with axonal spheroids to aid in the diagnosis of this neurodegenerative disease.

Keywords: ataxia; calcium-binding proteins; horses; medulla oblongata neuroaxonal dystrophies; spinal cord.

Figure 1

Diagram of spinocuneocerebellar tract (pink), dorsal column–medial lemniscal tract (blue; forelimbs only depicted), and dorsal spinocerebellar tract (green). The locations of degenerative axons associated with equine degenerative myeloencephalopathy are denoted in red. The neuronal cell body of the primary afferent of the spinocuneocerebellar tract resides in the dorsal root ganglia (DRG), and the axon traverses through the dorsal funiculus at the level of C1 to C7 to synapse on the nucleus cuneatus lateralis. The secondary afferent synapses in the ipsilateral cerebellar cortex. Within the forelimbs, the neuronal cell body of the primary afferent of the dorsal column–medial lemniscal tract resides in the DRG, and the axon traverses through the fasciculus cuneatus at the level of C1 to C7 to synapse on the nucleus cuneatus medialis. The secondary afferent synapses on contralateral thalamic nuclei. The neuronal cell body of the primary afferent of the dorsal spinocerebellar tract resides in the DRG, and the axon traverses through the dorsal horn to synapse on the nucleus thoracicus at the level of T1-L1. The secondary afferent synapses in the ipsilateral cerebellar cortex. CF, fasciculus cuneatus; GF, fasciculus gracilis; GN, gracilis nucleus; LCN, nucleus cuneatus lateralis; MCN, nucleus cuneatus medialis; TN, nucleus thoracicus. Figures 2–6. Calretinin-positive pathways, control horse No. 5, immunohistochemistry for calretinin. Figure 2. Dorsal root ganglia at C7. Both calretinin-negative and calretinin-positive somata are present. The calretinin-positive somata are likely associated with the spinocuneocerebellar or dorsal column–medial lemniscal tracts. Figure 3. Fasciculus cuneatus at C7. Both calretinin-negative and a subpopulation of calretinin-positive axons (arrow) are present. Figure 4. Nucleus cuneatus lateralis. Calretinin-positive axons synapse on calretinin-negative soma (thin arrow). A few spheroids (thick arrows) representing mild axonal degeneration are present. Figure 5. Dorsal root ganglia at T11. Both calretinin-positive (thin arrow) and calretinin-negative (arrowhead) myelinated axons are present, as well as calretinin-positive and calretinin-negative somata. Figure 6. Thoracic nucleus at T11. Calretinin-positive axons synapse on calretinin-negative soma (thin arrow). A few axonal spheroids are present (thick arrows).

Figures 7–9

Calretinin-positive pathways, equine degenerative myeloencephalopathy–affected horse No. 1, immunohistochemistry for calretinin. Figure 7. Nucleus cuneatus lateralis. Axonal degeneration represented by large numbers of calretinin-positive spheroids is evident within the nucleus. Figure 8. Nucleus cuneatus lateralis. Varying sizes of calretinin-positive spheroids are present within the nucleus. Figure 9. Nucleus thoracicus at T15. Calretinin-positive spheroids are evident within the nucleus thoracicus. Figures 10–13. Equine degenerative myeloencephalopathy–affected horse No. 3, nucleus vestibularis lateralis. Figure 10. Calbindin-positive axons (presumed to be Purkinje cell axons) contain calbindin-positive spheroids (arrow) at end-terminal synapses with calbindin-negative soma. Immunohistochemistry for calbindin. Figure 11. Presumptive glutamic acid decarboxylase (GAD)–positive Purkinje cell axons contain GAD-positive spheroids (arrow) at end-terminal synapses with GAD-negative soma. Immunohistochemistry for GAD. Figure 12. Parvalbumin-positive spheroids (arrow) are present in axons that synapse on parvalbumin-negative soma. Immunohistochemistry for parvalbumin. Figure 13. Increased glial fibrillary acidic protein staining representing astrogliosis. Immunohistochemistry for glial fibrillary acidic protein.