Purple Corn Anthocyanins Prevent the Progression of Multiple Sclerosis-Associated Trigeminal Pain

BACKGROUND AND AIMS Pain is one of the most disabling symptoms of multiple sclerosis (MS) [1]. Among MS-associated pain syndromes, trigeminal (TG) neuralgia and migraine often precede MS diagnosis and don’t correlate with disease severity [2]. Classic analgesics often fail to control pain in these patients, requiring the use of other drugs with severe side effects [3]. Therefore, new approaches based on the administration of nutraceutical supplements enriched in molecules with pharmacological activities could be a winning strategy. Anthocyanins (ACNs) from multiple sources can prevent chronic diseases through antioxidant, anti-inflammatory, and neuroprotective effects [4]. Our group already demonstrated the anti-allodynic effect of ACNs in an animal model of TG sensitization, through inhibition of microgliosis and production of anti-inflammatory mediators [5]. Here, we aimed to investigate if ACN-enriched purple corn can exert beneficial effects on the onset and progression of MS-associated TG pain. METHODS Relapsing-remitting experimental autoimmune encephalomyelitis (EAE) was induced in male Dark Agouti rats by single intradermal injection of MOG1-125 peptide in Incomplete Freund’s Adjuvant (IFA) and sodium acetate at the base of the tail, as described [6]. Eleven days before EAE induction rats were assigned to drink water, yellow corn (containing all classes of flavonoids except for ACNs) as control, or purple corn extracts as a preventive approach. Another group of animals began drinking purple corn extract from the onset of EAE motor symptoms, as a therapeutic strategy. From day post-immunization (DPI) 1 to 21 rats were weighed daily, the development of EAE was evaluated by a scale from 0 to 7, based on the degree of ascending paralysis, and spontaneous TG pain was measured by von Frey test. After sacrifice, CNS tissues (brainstem, lumbar and cervical spinal cord) were collected for subsequent analyses. RESULTS Preventive purple corn administration: 1) delays and reduces the development of EAE-associated TG pain; 2) to our surprise, facilitates the remission of EAE motor symptoms, protects against the development of relapses and improves body weight recovery; 3) limits glial cell activation; 4) ameliorates the ratio between anti- and pro-inflammatory mediators; 5) lowers the expression of glial purinergic P2X4, P2Y12 and A3 receptor subtypes involved in pain transmission; 6) activates autophagy; 7) reduces immune cells infiltration in the brainstem and lumbar spinal cord. Therapeutic purple corn administration does not affect EAE motor symptoms, only partially reduces the development of TG pain but maintains its ability to reduce neuroinflammation and glial cell activation and to promote autophagy. Moreover, therapeutic purple corn is only partially effective in reducing immune cell infiltration in the brainstem and completely loses its effect in the lumbar spinal cord. CONCLUSIONS In conclusion, our results show that the reduction of neuroinflammation and glial cell activation could be one of the mechanisms underlying the protective effect of ACNs against the development of EAE motor symptoms and associated TG sensitization when preventively administered. However, the only partial efficacy of the therapeutic administration of purple corn extract could rely upon its different ability to reduce immune cell infiltration in the brainstem and lumbar spinal cord with respect to the preventive administration. Nevertheless, further analyses are needed to set up a more effective therapeutic protocol of purple corn administration which could be later translated from the preclinical stage to MS patients, also in combination with current analgesics to either potentiate their effect or to reduce dosages and side effects. REFERENCES [1] Brola et al., Neurol Neurochir Pol 48:272-9, 2014 [2] Ceruti, Pain Manag 8:37-44, 2018 [3] Chisari et al., Expert Opin on Pharmacother 21:2249-63, 2020 [4] Magni et al., Biochem Pharmacol 205:1-19 2022 [5] Magni et al., Front Cell Neurosci 12:378, 2018 [6] Magni et al., Brain Behav Immun 89:268-280, 2020 ETHICAL PERMISSIONS We have strictly adhered to the current Italian and European rules on the use of laboratory animals in science; authorizations for the use of animals have been granted by the Italian Ministry of Health (authorization number 17/2021-PR). Consistently with Article 13, Paragraph 2 of the 26/2014 Italian Legislative Decree, we have employed procedures that require the minimum number of animals. Moreover, we made all possible efforts to apply the Reduce, Refine and Replace (3Rs) rule to our experiments. Housing of rats and all the procedures on living animals have been conducted by authorized and trained personnel only. RELEVANCE FOR PATIENTS CARE Results obtained from this study suggest a possible use of ACN-enriched purple corn extract as a preventive nutraceutical approach or as a safe adjuvant to be administered to MS patients suffering from TG pain in combination with current pharmacological therapies, in order to reduce their dosage and consequently the associated side effects and significantly improve their quality of life. In addition, since purple corn extract is derived from agricultural waste, it can be an economically viable source of ACNs.