Diagnosing noninfectious neurologic diseases in horses, from cervical vertebral compressive myelopathy (CVCM) to shivers, can be challenging. Conditions can mimic one another, and certain diseases can only be confirmed on necropsy examination, as is the case with equine neuroaxonal dystrophy (eNAD)/ equine degenerative myeloencephalopathy (EDM). So, researchers are keen to identify markers in blood and cerebrospinal fluid (CSF) that could provide more diagnostic information.

Lisa Edwards, DVM, a fellow in equine emergency and critical care at the University of California, Davis, and her collaborators Carrie Finno, DVM, PhD, Dipl. ACVIM, Callum Donnelly, BVSc, Dipl. ACT, Dipl. ACVIM, and Munashe Chigerwe, DVM, PhD, MPH, MsC, at UC Davis; Stephen Reed, DVM, Dipl. ACVIM, at Rood & Riddle Equine Hospital; Stephanie Valberg, DVM, PhD, Dipl. ACVIM, ACVSMR, at Michigan State University; and Amy Johnson, DVM, Dipl. ACVIM, at the University of Pennsylvania, recently examined the ability of phosphorylated neurofilament heavy (pNfH), a protein unique to neurons, to serve as a biomarker for equine neurodegenerative diseases.

“Neurofilaments are components of the neuronal cytoskeleton,” said Edwards. “They play a role in structural support, regulation of axon (the long threadlike part of a nerve cell that conducts impulses to other body cells) diameter, and transmission of nerve impulses. There are three major subunits, including heavy, medium, and light chains. These are all synthesized in the cell body, and they travel along the axon.

“Neurofilaments are specific to neurons and particularly concentrated in axons,” she added. “Abnormal accumulations are known to occur in many neurodegenerative diseases like ALS (Lou Gehrig’s disease) in people.”

When axons get injured, they release neurofilaments into the CSF and, to a lesser extent, the blood.

Before this study, researchers had shown that plasma pNfH isn’t increased in foals with hypoxic-ischemic encephalopathy (dummy foals) or horses with acute equine grass sickness. In a recent study serum and CSF pNfH concentrations were increased in some horses with CVCM and equine protozoal myeloencephalitis (EPM, another neurologic disease, though caused by an infectious agent). In another study serum pNfH concentrations were also increased in some horses with equine motor neuron disease (EMND).

Edwards and her co-authors used a commercial test to measure serum and CSF pNfH concentrations in samples from 51 neurologically normal horses and horses with the following diseases:

Equine neuroaxonal dystrophy (eNAD, 64 horses, confirmed on necropsy) This causes symmetric ataxia (incoordination) by 6-12 months of age, often displayed as an abnormal stance at rest. It develops in genetically predisposed horses that experience vitamin E deficiency early in life. Equine degenerative myeloencephalopathy is its more severe variant, with lesions extending to the white matter of the spinal cord. Veterinary pathologists detect degenerative lesions, such as spheroids (axonal swelling), during microscopic examination of brainstem and spinal cord tissue samples from eNAD and EDM horses.

Cervical vertebral compressive myelopathy (CVCM, 26 horses, also confirmed on necropsy) A common cause of ataxia in horses due to compression of the spinal cord, CVCM is often seen in young, well-fed, rapidly growing animals, said Edwards, and researchers have reported a higher incidence in males, Thoroughbreds, Tennessee Walking Horses, and Warmbloods.

Shivers (nine horses; five necropsy confirmed, four clinically phenotyped by Dr. Stephanie Valberg) This chronic, gradually progressive movement disorder, which researchers most frequently see in tall male horses, causes muscle tremors and abduction and hypertonic flexion or extension of the pelvic limb when the horse is asked to back or lift the leg.

Edwards and her co-authors also sought to determine the effects of age, breed, and sex on pNfH concentrations in CSF and serum and assess the inter-assay reliability of the test (its plate-to-plate consistency).

Results

The research team found that serum pNfH concentrations >1 ng/ml were significantly associated with an eNAD/EDM diagnosis (P=0.01). Serum pNfH concentrations were <1 ng/ml in neurologically normal, CVCM, and some eNAD/EDM and shivers horses. CSF pNfH concentrations of >3 ng/ml were significantly associated with axonal degeneration due to either eNAD/EDM or CVCM (P=0.0001) but not shivers. Values below these levels did not rule out neurologic disease.

“At this time, it does not appear that varying magnitudes of pNfH correlate with a specific disease but simply represent axonal degeneration overall,” she explained, also noting that “pNfH concentrations can provide useful antemortem (in the live horse) diagnostic information in cases of equine neurologic disease.

“It is quite possible that we need more sensitive assays to detect pNfH in equine samples,” she added. “Certainly, in human medicine they’ve moved on to more sensitive assays like electrochemiluminescence when detecting serum and plasma pNfH.”

They saw no effect of the horses’ sex on serum or CSF pNfH levels but noted that age was a significant variable among eNAD/EDM and CVCM groups, with the predictive effect of a high CSF pNfH concentration for axonal degeneration having higher sensitivity in horses <5 years of age.

“The association between age and axonal degeneration in young horses was an interesting finding and may implicate an interaction between age and disease, especially considering that eNAD/EDM and CVCM are often diagnosed in young horses as they begin work,” Edwards said.

She noted that the number of horses <1 year of age and shivers horses in the study were limited, so it’s important to interpret serum and CSF pNfH concentrations in these groups cautiously.

The full scientific manuscript regarding this study is currently in review at the Equine Veterinary Journal.

Find information on serum and CSF pNfH testing here.