Advisor: Yonggeun Hong (DVM, PhD) The novel exercise-induced hormone irisin protects against neuronal injury via activation of the Akt and ERK1/2 signaling pathways and contributes to the neuroprotection of physical exercise in cerebral ischemia Prepared by:  Dewan Md. Sumsuzzman  Department of Rehabilitation Science,  Inje University, South Korea. Advisor: Yonggeun Hong (DVM, PhD)

Introduction Stroke  -Stroke is define as a sudden loss of brain function due to disturbance in the cerebral blood supply with symptoms lasting at least 24 hours or leading to death (American Heart Association).  -Stroke is the second leading cause of death worldwide and is the major cause of morbidity, particularly in the middle aged and elderly population (Lee y et al., BioMed Research International, 2014). Fig 1: The 10 leading causes of death in the world  (State of the Nation Stroke statistics - January 2017) 

INTRODUCTION Table 1: Stroke mortality in the UK 2015 (State of the Nation Stroke statistics - January 2017)  Fig 2: Stroke  survivors in the UK 2015  (State of the Nation Stroke statistics - January 2017) 

Fig 3: Hemorrhagic stroke introduction Hemorrhagic strokes are less common, in fact only 15 percent of all strokes are hemorrhagic, but they are responsible for about 40 percent of all stroke deaths.  Hemorrhagic stroke occurs when a blood vessel in your brain leaks or ruptures. Brain hemorrhages can result from many conditions that affect your blood vessels, including uncontrolled high blood pressure (hypertension), overtreatment with anticoagulants and weak spots in your blood vessel walls (aneurysms).  Fig 3: Hemorrhagic stroke  (http://www.stroke.org) 

INTRODUCTION  Ischemic strokes occur when the arteries to brain become narrowed or blocked, causing severely reduced blood flow (ischemia).   About 85 percent of strokes are ischemic strokes.  Fig 4: Ischemic stroke  (http://www.strokecenter.org) 

INTRODUCTION  A transient ischemic attack (TIA) also known as a mini-stroke is a brief period of symptoms similar to those you'd have in a stroke. A temporary decrease in blood supply to part of brain causes TIAs, which often last less than five minutes.   Like an ischemic stroke, a TIA occurs when a clot or debris blocks blood flow to part of your brain. A TIA doesn't leave lasting symptoms because the blockage is temporary.  Fig 5: transient ischemic attack  (American heart association) 

INTRODUCTION Fig 6: Post ischemic inflammatory response. (Magdalena Hofer et al, Journal of Translational Medicine, 2009)  Fig 7: Immune signaling of microglia and astrocyte after ischemic stroke.    (Jong Youl Kim et al, Experimental neurobiology, 2016) 

INTRODUCTION Irisin is a recently identified proliferator-activated receptor- gamma coactivator-1α (PGCγ-1α)-dependent myokine, secreted by skeletal muscle and myocardium into circulation during exercise as a cleavage product of the extracellular portion of type I membrane protein fibronectin type III domain containing 5 (FNDC5) (Bostrom et al., 2012).  It was initially discovered as a kind of hormone responsible for the beneficial effects of exercise on the browning of white adipose tissues and increases in energy expenditure (Bostrom et al., 2012).  It has also been demonstrated to reduce oxidative stresses and apoptosis in different models (Park et al.,2015; Zhu et al., 2015). 

iNTRODUCTION Fig 8: Candidate signaling pathways of irisin in myocytes.  (Nikolaos Perakakis et al., nature, 2017) 

Exercise without intervention Exercise+Irisin NA Inj. MATERIALS & METHODS  Control Group Sham control Sedentary Exercise Group Exercise without intervention Exercise+IgG Inj. Exercise+Irisin NA Inj.

MATERIALS & METHODS (A) Extra Fine Graefe Forceps - 0.5 mm Tips Slight Curve.   (B) Ceramic Coated Dumont #5 Forceps.   (C) Extra Fine Bonn Scissors, straight.   (D) Round 3/8 (16 mm) Suture Needles.   (E) NOTE: Suture needles may be shortened via wire cutters according to user preference. After shortening with wire cutters, suture needles should be sterilized.   (F) 6-0 Braided Silk Suture.   (G) 30 gauge needle, ½ in length.  Fig 9: Surgical Tools Required for MCAO  (Melissa F. Davis et al., J. Vis. Exp. 2013) 

MATERIALS & METHODS Anesthetize the rat Surgical nylon monofilament with rounded tip.  Occlusion for 1h.  Fig 10: Surgical Tools Required for MCAO  (Melissa F. Davis et al., J. Vis. Exp. 2013) 

DOSE & ADMINISTRATION Recombinant irisin administration Recombinant irisin to be injected directly into the tail vein 30 min after MCAO operation.    Dose: 0.2 microgram/g body weight. Irisin neutralizing antibody administration  Irisin neutralizing antibody to be injected via tail vein 1h before the MACO operation.  Dose: 20 microgram/rat 

RESULT & DISCUSSION Fig 11: Changes of plasma irisin level and intramuscular irisin precursor FNDC5 expression after cerebral ischemic stroke.

RESULT & DISCUSSION  Fig 12: Correlations between plasma irisin levels and brain infarction volume, neurological deficit score and plasma pro-inflammatory factors.

Fig 13: Effects of irisin treatment on neuronal injury in MCAO mice. RESULT & DISCUSSION Fig 13: Effects of irisin treatment on neuronal injury in MCAO mice.

RESULT & DISCUSSION  Fig 14: Effects of irisin treatment on post-ischemic neuroinflammation in MCAO mice.

Fig 15: Effects of irisin treatment on oxidative stress in MCAO mice. RESULT & DISCUSSION  Fig 15: Effects of irisin treatment on oxidative stress in MCAO mice. 

RESULT & DISCUSSION  Fig 16: Involvement of ERK1/2 and Akt signaling pathways in the neuroprotection of irisin.

RESULT & DISCUSSION   Fig 17: Irisin contributes to the neuroprotection of physical exercise against ischemic stroke.

SUMMARY Irisin inhibited brain infarct volume, reduced neuroinflammation and oxidative stress on post cerebral ischemia. Irisin activated the Akt and ERK ½ signaling pathways in brain tissues. Irisin contributes neuroprotection of physical exercise.

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