Analysis of neurofilament concentration in healthy adult horses and utility in the diagnosis of equine protozoal myeloencephalitis and equine motor neuron disease.

Neurofilaments (NFs) are structural proteins of neurons that are released in significant quantities in the cerebrospinal fluid and blood as a result of neuronal degeneration or axonal damage. Therefore, NFs have potential as biomarkers for neurologic disorders. Neural degeneration increases with age and has the potential to confound the utility of NFs as biomarkers in the diagnosis of neurologic disorders. We investigated this relationship in horses with and without neurological diagnosis. While controlling for horse type (draft, pleasure, and racing), we evaluated the relationship between serum heavy-chain phosphorylated neurofilaments (pNF-H) and age, sex, and serum vitamin E concentrations. Serum pNF-H concentrations increased by 0.002 ng/ml for each year increase in age. There were significant differences in the serum pNF-H concentration among the type of activity performed by the horse. The highest serum pNF-H concentration was found in horses performing heavy work activity (racehorse) and with lower serum pNF-H concentration found among light (pleasure riding) and moderate (draft) activity. There was no significant association between the pNF-H concentration and sex or vitamin E concentration. Serum pNF-H concentration was elevated among horses afflicted with EMND and EPM when compared with control horses without evidence of neurologic disorders. Accordingly, serum pNF-H concentration can serve as a useful biomarker to complement the existing diagnostic work-up of horses suspected of having EPM or EMND.

Effects of various antiplatelet drugs on ex vivo platelet activation induced by equine herpesvirus type 1.

OBJECTIVE To evaluate the effects of treatment of horses with standard platelet inhibitors on ex vivo inhibition of platelet activation by equine herpesvirus type I (EHV-I). ANIMALS II healthy adult horses. PROCEDURES In a double-blinded, placebo-controlled crossover study, horses were treated orally for 5 days with theophylline (5 mg/kg, q 12 h), pentoxifylline (10 mg/kg, q 12 h), clopidogrel bisulfate (4 mg/kg, q 24 h), acetylsalicylic acid (20 mg/kg, q 24 h), or placebo. Horses received all treatments, each separated by a 3-week washout period. Platelet-rich plasma was prepared from citrated blood samples obtained before each treatment session and 4 hours after each final drug dose. Platelets were exposed to 2 EHV-I strains (at I plaque forming units/cell) or positive (thrombin-convulxin) and negative control substances for 10 minutes, then platelet activation was assessed by determining the percentages of P-selectin-positive platelets and platelet-derived microparticles (PDMPs; small events positive for annexin V) with flow cytometry. Platelet aggregation in response to 10µM ADP was also assessed. RESULTS No significant differences in median percentages of P-selectin-positive platelets and PDMPs in EHV-I-exposed platelets were identified between measurement points (before and after treatment) for all drugs, nor were differences identified among drugs at each measurement point. Only clopidogrel significantly inhibited platelet aggregation in response to ADP in platelet-rich plasma samples obtained after that treatment session. CONCLUSIONS AND CLINICAL RELEVANCE Treatment of horses with standard platelet inhibitors had no effect on EHV-I-induced platelet α-granule exteriorization or microvesiculation and release of PDMPs ex vivo, suggesting these drugs will not prevent platelet activation induced directly by EHV-I in vivo.

Effects of clopidogrel on horses with experimentally induced endotoxemia.

OBJECTIVE: To evaluate the effects of clopidogrel on clinical and clinicopathologic variables in healthy horses with experimentally induced endotoxemia. ANIMALS: 12 adult mares. Procedures-Horses were assigned with a randomization procedure to receive clopidogrel (4 mg/kg, once, then 2 mg/kg, q 24 h; n = 6) or a placebo (6) through a nasogastric tube. After 72 hours of treatment, horses received lipopolysaccharide (LPS; 30 ng/kg, IV). Heart rate, respiratory rate, rectal temperature, CBC variables, plasma fibrinogen concentration, serum tumor necrosis factor-α concentration, plasma von Willebrand factor concentration, and measures of platelet activation (including ADP- and collagen-induced platelet aggregation and closure times, thrombelastography variables, and results of flow cytometric detection of platelet membrane P-selectin, phosphatidylserine, and microparticles) were determined at various times before and after LPS administration by investigators unaware of the treatment groups. Statistical analyses were performed with repeated-measures ANOVA. RESULTS: 4 of 6 clopidogrel-treated horses had significant decreases in ADP-induced platelet aggregation before and after LPS administration. Heart rate increased significantly after LPS administration only for the placebo group. No significant differences were detected between groups for CBC variables, closure time, and plasma concentration of fibrinogen or serum concentration of tumor necrosis factor-α, and no clinically relevant differences were detected for other hemostatic variables. CONCLUSIONS AND CLINICAL RELEVANCE: In this study, administration of LPS did not induce platelet hyperreactivity in horses on the basis of measures of platelet adhesion, aggregation, degranulation, and procoagulant activity. Administration of clopidogrel was associated with variable platelet antiaggregatory activity and attenuated some clinical signs of endotoxemia.

Effects of clopidogrel on the platelet activation response in horses.

OBJECTIVE: To evaluate the platelet activation response before and after treatment with clopidogrel in horses. ANIMALS: 12 healthy adult mares. PROCEDURES: In a masked study, horses (6/group) were randomly allocated to alternately receive placebo or clopidogrel via nasogastric tube at a loading dose of 4 mg/kg followed by 2 mg/kg every 24 hours. Blood samples were collected before and 72 hours after initiation of treatment for ADP- and collagen-induced light transmission aggregometry; determination of closure time in collagen-ADP cartridges; modified thrombelastography for comparison of maximal amplitudes generated by kaolin, reptilase, and reptilase plus ADP activation; and flow cytometric tests to detect platelet fibrinogen binding, P-selectin expression, and phosphatidylserine externalization before and after ex vivo stimulation with thrombin, convulxin, thrombin with convulxin, and calcium ionophore. RESULTS: Clopidogrel administration induced a significant decrease in mean aggregation response to 5 µM and 10 µM ADP stimulation; however, 2 horses had resistance to clopidogrel's inhibitory action. Significant differences after clopidogrel treatment were not found in any other tests of platelet function. CONCLUSIONS AND CLINICAL RELEVANCE: Assays using commercially available reagents were configured to measure different variables of the platelet activation response; however, clopidogrel's platelet inhibitory action was only detected by ADP-induced light transmission aggregometry. Results also suggested that horses, like humans, have interindividual variability in response to clopidogrel that may influence the drug's clinical efficacy as an antiplatelet agent.