**Downregulation of CD73/A2AR-Mediated Adenosine Signaling as a Potential Mechanism of Neuroprotective Effects of Theta-Burst Transcranial Magnetic Stimulation in Acute Experimental Autoimmune Encephalomyelitis**

**Milorad Dragi´c 1, \* , Milica Zeljkovi´c 1 , Ivana Stevanovi´c 2,3 , Marija Adži´c 1 , Andjela Steki´c 1 , Katarina Mihajlovi´c 1 , Ivana Grkovi´c 4 , Nela Ili´c 5,6 , Tihomir V. Ili´c 3 , Nadežda Nedeljkovi´c <sup>1</sup> and Milica Ninkovi´c 2,3**


**Abstract:** Multiple sclerosis (MS) is a chronic neurodegenerative disease caused by autoimmunemediated inflammation in the central nervous system. Purinergic signaling is critically involved in MS-associated neuroinflammation and its most widely applied animal model—experimental autoimmune encephalomyelitis (EAE). A promising but poorly understood approach in the treatment of MS is repetitive transcranial magnetic stimulation. In the present study, we aimed to investigate the effect of continuous theta-burst stimulation (CTBS), applied over frontal cranial bone, on the adenosine-mediated signaling system in EAE, particularly on CD73/A2AR/A1R in the context of neuroinflammatory activation of glial cells. EAE was induced in two-month-old female DA rats and in the disease peak treated with CTBS protocol for ten consecutive days. Lumbosacral spinal cord was analyzed immunohistochemically for adenosine-mediated signaling components and pro- and anti-inflammatory factors. We found downregulated IL-1β and NF- κB-*ir* and upregulated IL-10 pointing towards a reduction in the neuroinflammatory process in EAE animals after CTBS treatment. Furthermore, CTBS attenuated EAE-induced glial eN/CD73 expression and activity, while inducing a shift in A2AR expression from glia to neurons, contrary to EAE, where tight coupling of eN/CD73 and A2AR on glial cells is observed. Finally, increased glial A1R expression following CTBS supports anti-inflammatory adenosine actions and potentially contributes to the overall neuroprotective effect observed in EAE animals after CTBS treatment.

**Keywords:** CD73; adenosine; A2AR; A1R; neuroinflammation; theta-burst stimulation; rTMS; purinergic signaling
