Activated platelets: a potential new diagnostic and therapeutic target in preclinical experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis

2017-02-21T00:13:26Z (GMT) by Nair, Ashish Kumar Narayan
Multiple sclerosis (MS) is a chronic autoimmune disease wherein the central nervous system (CNS) becomes the target of the host’s immune system, resulting in demyelination, neuroinflammation and axonal injury; and thereby causing a progressive loss of neuronal function. Platelets are known for their pro-inflammatory role in cardiovascular disease but their role in neuroinflammatory diseases such as MS and Alzheimer’s disease has also come under greater scrutiny in recent years. Recent studies have shown significant upregulation of platelet microparticles as well as biomarkers such as CD41 and Cd42b in MS patients as well as in murine experimental autoimmune encephalomyelitis (EAE), the animal model of MS. Additional observations include the increased presence of platelets in the CNS tissue in EAE mice and the success of platelet depletion in reducing EAE disease progression. However there is very little information on platelet roles in the preclinical, asymptomatic disease phase just prior to clinical disease onset. Thus the objectives of this project were as follows- 1) To establish a possible link between platelet burden and disease progression in EAE induced mice prior to the manifestation of clinical symptoms. 2) To investigate the possibility of using activated platelets as a potential predictive candidate for live imaging of disease progress in EAE mice during the asymptomatic phase of the disease. 3) To study the functional role of platelets in preclinical EAE with a view to exploring them as potential therapeutic targets. NOD/lt mice were induced with EAE using MOG35-55 peptide emulsion and subsequent pertussis toxin injections. Balb/c mice were induced with EAE using PLP peptide injection and pertussis toxin injections. Brains, optic nerves and spinal cords were harvested from NOD/lt EAE-induced mice at 8, 10, 12 and 14 days post induction (dpi). Optic nerves and brain tissue were harvested from the EAE induced Balb/c mice at 7 and 14 dpi. Immunohistochemistry was carried out with anti-platelet antibodies to determine and map platelet presence in each tissue. This was followed by double staining, using both platelet specific anti-CD41 and anti-CD42b to confirm the specificity of the staining. Finally brains and optic nerves from EAE induced NOD/lt mice were co-incubated with cocktails of anti-CD41 and anti-MBP and anti-CD41 and anti-iba1 to correlate platelet presence with other neuroinflammatory hallmarks such as demyelination and microglial activation. We show that platelet accumulation and distribution in the CNS in both the preclinical phase and the acute disease phase in EAE-induced Balb/c mice and NOD/lt mice followed a specific, non-uniform recurring pattern. Additionally platelet appearance in the CNS precedes cellular infiltration as well as the appearance of clinical symptoms in both EAE models, with platelets first appearing in the optic nerves of the EAE- induced NOD/lt mice a good 24-48 hours before clinical disease onset as measured by clinical score. Platelet presence in the CNS was intimately associated with inflammation as evidenced by the lack of platelet staining in the non-inflamed optic nerves of EAE-induced Balb/c mice and the correlation of platelet staining with the concomitant appearance of other inflammatory hallmarks such as increasing demyelination and increased microglial reactivity in EAE-induced NOD mice. Importantly, platelet presence in the CNS peaked over a very short time frame in the preclinical phase before gradually reducing in frequency, establishing them primarily as preclinical participants in EAE. We next conjugated a highly specific single-chain antibody raised against the activated conformation of platelet-specific GPIIb/IIIa to radioactive 64Cu and injected it into EAE-induced NOD/lt mice. These mice were then scanned using a PET/CT machine. We were able to show a) a significant difference in signal between sham induced and EAE induced animals, b) a strong signal prior to the first attack in EAE induced NOD/lt mice at the preclinical timepoint of 10 dpi and c) the co-relation of the PET/CT signal with the immunohistochemical presence of platelets in CNS tissue in mice at the same timepoints in the disease. Lastly, we carried out platelet depletion studies in EAE induced NOD/lt mice. We demonstrate that even limited platelet depletion in the preclinical disease phase was enough to delay clinical disease onset in EAE-induced mice. We submit therefore that platelets are important and early participants in preclinical events leading upto clinical disease onset. We further show that activated platelets can be used, in combination with our single-chain antibodies and non-invasive imaging modalities, to monitor disease progression in EAE even before the appearance of external clinical symptoms; thereby being potentially indicative of early, clinically silent disease processes. Finally, we demonstrate that platelets have potential as a target for relatively safe and effective therapeutic intervention.