Immunomodulatory approaches after experimental ischemic stroke

Ischemic stroke is one of the leading causes of death and disability worldwide but the treatment options remain limited. Ischemic stroke, or cerebral infarct, occurs when blood flow to a focal brain region is restricted due to arterial blockage. Lack of oxygen and energy leads to rapid neuronal death in the ischemic region and to an inflammatory response via activation of brainresident immune cells, microglia, and infiltration of peripheral leukocytes after ischemia-induced blood-brain barrier damage. Acute neuroprotective strategies need to be executed within a few hours after ischemia induction to be effective and have not proven successful in clinical trials. However, inflammation persists in the post-stroke brain and modulation of post-stroke inflammation could provide a therapeutic strategy with a large time window. Inflammation has both beneficial and harmful effects on injury progression but our understanding of many aspects of post-stroke inflammation remains incomplete. We characterized the neuroinflammatory response in the rat distal middle cerebral artery occlusion (dMCAo) model that was used to induce cortical infarcts in this thesis work. We found long-lasting inflammation and presence of phagocytic cells for up to 4 months after dMCAo, especially in the ipsilateral thalamus. We also found delayed neuronal loss occurring in the ipsilateral thalamus between 1-2 weeks after dMCAo due to connecting projection pathways between the cortex and the thalamus. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an 18 kDa endoplasmic reticulum luminal protein that is neuroprotective in experimental ischemic stroke models and has been associated with immunomodulatory properties. However, the knowledge of MANF’s recoverypromoting effects, mechanism of action, and endogenous expression pattern after cerebral ischemia are still limited. Thus, we characterized the endogenous MANF protein expression pattern in the dMCAo model and in ischemic stroke patient brains. Notably, we found that MANF protein expression is strongly induced in activated immune cells in the infarcted rodent and human brains. We also studied intracerebral post-stroke MANF therapy via viral delivery and recombinant protein injection and found that MANF promotes functional recovery when administered into the brain 2 days post-stroke as an adeno-associated viral (AAV) vector or as a recombinant protein starting 3-7 days post-stroke. Post-stroke MANF treatment did not alter the infarct size but the AAV-MANF therapy induced a transient increase in the number of phagocytic cells and innate immunity-related transcript levels in the peri-infarct area. In addition, we conducted a proof-of-concept study using intranasal MANF delivery to explore alternative delivery routes for administering the blood-brain barrier impermeable MANF protein. Pre-stroke intranasal MANF therapy decreased infarct volume and behavioral deficits. These data suggest a theoretical potential for intranasal MANF therapy, but bioavailability requires further improvement. As another approach, we investigated the efficacy of repeated post-stroke intranasal (+)-naloxone delivery in the dMCAo model. (-)-Naloxone is a small molecule drug which has been in clinical use for opioid overdose for decades and studied in the acute treatment of ischemic stroke because of its opioid receptor antagonizing effect. More recently, (-)-naloxone and its opioid receptor inactive (+) enantiomer have been shown to possess antiinflammatory effects and to reduce microglial activation. (+)-Naloxone therapy, started one day post-stroke and continued for 7 days, decreased the infarct size and microglia/macrophage activation, and reduced behavioral deficits. This work broadens knowledge of the post-stroke neuroinflammation and secondary pathology of the thalamus in the cortical infarct model and shows for the first time that endogenous MANF protein is expressed in the activated, phagocytic immune cells in the infarcted human brain. This work also provides evidence on the recovery-promoting effects of post-stroke MANF and (+)naloxone therapy and links both therapies with immunomodulatory functions.