1. Supervisor: Yonggeun Hong (DVM, PhD)
Skeletal muscle derived myokine irisin protects neuronal injury via activation of the PI3K-Akt Signaling pathways and contributes to the neuroprotection at evening exercise in cerebral ischemia.
PREPARED BY: DEWAN MD. SUMSUZZMAN
DEPARTMENT OF REHABILITATION SCIENCE,
INJE UNIVERSITY, SOUTH KOREA.
Supervisor: Yonggeun Hong (DVM, PhD)

2. 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)

3. 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)

4. 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 (

5. 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 (

6. Fig 5: transient ischemic attack (American heart association)
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)

7. INTRODUCTION
Fig 6: Mechanism of cerebral ischemia. Jagid, Jonathan, National Neurotrauma Symposium, 2011.

8. INTRODUCTION
Fig 7: Important pathways in ischemic stroke. Seyed Esmaeil Khoshnam et al., Neurol Sci, 2017

9. Introduction Fig 8: Post ischemic inflammatory response.
(Magdalena Hofer et al, Journal of Translational Medicine, 2009)
Fig 9: Immune signaling of microglia and astrocyte after ischemic stroke.  
(Jong Youl Kim et al, Experimental neurobiology, 2016)

10. (Quanguang Zhang et al., JOURNAL OF NEUROLOGY AND NEUROSCIENCE, 2017)
Introduction
Fig 10: the effects of cerebral ischemia injuries on mitochondrial dysfunction, oxidative stress, release of pro-apoptotic factor and proinflammatory cytokines, and the eventually neuronal death
(Quanguang Zhang et al., JOURNAL OF NEUROLOGY AND NEUROSCIENCE, 2017)

11. introduction A B C D E
Fig 11: A. Putative mechanism of IL‑6 in cerebral ischemia, (B+C) Neuroprotective effect of IL-6 against cerebral ischemia, (D+E) Effect of IL‑6 on the inflammatory IL‑1β and TNF-α levels following cerebral ischemia. (QILIN FENG et al., EXPERIMENTAL AND THERAPEUTIC MEDICINE, 2015)

12. Ulrich Dirnagl et al., Trends Neurosci, 1999.
introduction
Fig 12: Putative cascade of damaging events in focal cerebral ischemia. 
Ulrich Dirnagl et al., Trends Neurosci, 1999.

13. introduction
Fig 13 :  PI3K/AKT mediated signaling promotes cell survival.
Ashley L. Hein et al., International Journal of Oncology, 2014.
Fig 14: Alteration of the PI3K pathway after ischemia and reperfusion. Cas-3: caspase-3, Cyto C: cytochrome C, JNK: c-Jun N-terminal kinases.

14. introduction
Fig 15: Central role of PGC-1α in the regulation of skeletal muscle cell plasticity. (Biozentrum et al., /j.bone, 2015.)

15. Fig 16: Signaling pathways regulated by exercise and/or IGF-I.
introduction
Fig 16: Signaling pathways regulated by exercise and/or IGF-I.
(Tiago Fernandes et al., INTECH, 2012)

16. Introduction
Fig 17: Aerobic exercise training alters key intracellular signaling pathways. (Adam R. Konopk et al., American College of Sports Medicine, 2014.)

17. Introduction
Fig 18: Interactions between the circadian clock and exercise.
( Wakamatsu-cho et al., J Phys Fitness Sports Med, 2014)
Fig 19: The circadian clock controls exercise performance..
(  Shinjuku-ku et al., J Phys Fitness Sports Med, 2014)

18. INTRODUCTION
Fig 20: Positive and negative regulators of skeletal muscle mass. Rexford S. Ahima et al., Endocrinology & Metabolism, 2015.

19. (Tong-Yan Liu et al., clinsci, 2015)
Introduction
Fig 21: Irisin activated PI3K/AKT pathway & inhibited hepatic gluconeogenesis .
(Tong-Yan Liu et al., clinsci, 2015)

20. Introduction
Fig 22: Anti-inflammatory treatments in stroke pathophysiology.
(Fabrizio Montecucco et al, IJMS, 2016)

21. Previous study
Fig 23: Changes of plasma irisin level & intramuscular irisin precursor FNDC5 expression after cerebral ischemic stroke.
(Dong-Jie Li et al, Metabolism, 2016)
Fig 24: Effects of irisin on neuronal injury in MCAO mice.
(Dong-Jie Li et al, Metabolism, 2016)

22. (Dong-Jie Li et al, Metabolism, 2016)
Previous study 
Fig 25: Irisin contributes to the neuroprotection of physical exercise against ischemic stroke.
(Dong-Jie Li et al, Metabolism, 2016)

23. Previous Study
Fig 26: Cortisol output over 24 hrs period.
(M. Debono et al., J Clin Endocrinol Metab, 2009)
Fig 27: Diurnal variations of the salivary testosterone: cortisol ratio. 
(Lawrence D Hayes., Sport SPA Vol.9, 2017)

24. Aim of study
To determine & compare fndc5 protein expression on soles & gastrocnemius muscle on post ischemic stroke.
To evaluate the therapeutic effect of irisin treatment on brain infarct volume, neurological deficit, neuro inflammation, apoptosis and intracellular signaling pathway activation (PI3K-AKT). 
To assess the potential involvement of irisin in the neuroprotection of physical exercise and differentiate the effectiveness between morning session & evening session. exercise

25. Inhibition apoptotic cell death Suppress pro-inflammatory mediators
hYPOTHESIS
Cerebral Ischemia
Inflammation
Apoptosis
IRISIN
Inhibition apoptotic cell death
Suppress pro-inflammatory mediators
Angiogenesis
Neuroprotection

26. Materials & Methods

27. Experimental design Experimental groups Experimental groups Purposes
Sham control group
To compare among groups.
MCAO(Vehicle treated) 
To determine  the neuroprotective effect without any intervention.
MCAO+Irisin neutralizing antibody+exercise 
To block the effectiveness of irisin.
MCAO+Recombinant irisin+Morning shift exercise
To evaluate the neuroprotective effect of irisin with morning shift exercise.
MCAO+Recombinant irisin+evening shift exercise
To evaluate the neuroprotective effect of irisin with evening shift exercise. 

28. EXPERIMENTAL DESIGN
Experimental plan

29. Middle cerebral artery occlusion
(A) Extra Fine Graefe Forceps 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 28: Surgical Tools Required for MCAO
(Melissa F. Davis et al., J. Vis. Exp. 2013)

30. Middle cerebral artery occlusion
Anesthetize the rat
Surgical nylon monofilament with rounded tip.
Occlusion for 1h.
Fig 29: Surgical set up of the permanent distal middle cerebral artery occlusion model
(Christina Wayman, Journal of Visualized Experiments, 2016)

31. Plasma Irisin and pro-inflammatory cytokines measurement
Blood to be obtained from an intravenous cannula at 4, 6, 8, 10, 12 h and 24h following onset. Blood to be allowed to clot at room temperature for 1 h, and after centrifugation the serum to be stored at -80°C until used. 
Plasma levels of irisin to be measured using a commercial ELISA kit (Phoenix Pharmaceutical, Burlingame, CA) with a 1:2 dilution of each plasma sample (50 μL).
Plasma concentrations of pro-inflammatory cytokines, including TNF-α, IL-6 and IL-1β, to be measured using ELISA kits from Boster Co. Ltd. (Wuhan, China). 

32. NEUROLOGICAL FUNCTIONING SCORING
Grip test:
The grip test is used to measure the maximal muscle strength of forelimbs and combined forelimbs and hind limbs as a primary phenotype screen. 
Three trials are carried out consecutively measuring forelimb- strength only, followed by three trials testing consecutively the combined forelimb/hindlimb grip strength. 
Fig 30: Grip test

33. Neurological functioning scoring
Beam Walking test:
The animals are first pre-trained across a plank. This helps to make sure that the behavior during testing is more stable and more accurately reflects motor coordination.  
After the pre-training, the animals can be tested on the balance beam for the latency to cross the beam and the number of slips. A B
Fig 31: An overview of balance beam test.
(Dr. Maria Gulinello, Albert Einstein College of Medicine)

34. Brain infarct volume measurement
Nissl staining
-Brain tissue. 
-Cryostat microtome(MICROM International GmbH, Germany). 
-Nissl staining to be done of brain tissues at 3 days after MCAO.
TTC  staining
-Infarction volume to be determined by 2,3,5-triphenyltetrazolium chloride (TTC) staining.
- Brain tissue slices to be stained with TTC & measured with the set scale function of the ImageJ program.

35. INHIBITION OF pi3k/akt PATHWAYS & INHIBITORY EFFECT OF IRISIN
To test whether the activation of PI3K and AKT is essential for the neuroprotection of irisin, I will use LY and MK2206 to inhibit the PI3K and AKT signaling pathways, respectively.
Thereafter, I will determine the neurological outcome of irisin by performing beam walking test.
Furthermore, the inhibitory effect of irisin on Bax, Foxo-1, TNF-alpha, IL-1 beta and IL-6 to be measured.

36. 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

37. Target genes and proteins
FNDC5
Apoptosis (Bax, FOXO-1)
Inflammation (TNF-α, IL-6 and IL-1β)
House-keeping (Tubulin)

38. EXERCISE & Irisin blocking treatment
-The irisin neutralizing antibody to be used to specifically block the irisin activity.
-After 2 weeks of physical exercise (Morning & evening shift), the sedentary & exercise rat to be subjected to the MCAO operation. 
-Irisin neutralizing antibody to be injected 1h before MACO operation.
Finally, three days after MCAO, the rat to be evaluated for neurological function, brain infarct volume and other biochemical parameters.  

39. PREDICTIVE RESULT
1. Both the plasma and skeletal muscle levels of irisin decrease following cerebral ischemia. And, the irisin precursor FNDC5 protein expression to be found lowest at gastrocnemius muscle as compared to the soleus muscle. 2. The regression analysis among 4, 6, 8, 10, 12, 24 hrs intervals will show that, higher blood irisin levels are associated with smaller brain infarct volumes, better neurological function and lower level of plasma pro-inflammatory cytokines in rat with cerebral ischemia. 3. Administration of recombinant irisin confers potent neuroprotection in MCAO model, indicating by the reduce brain infarction, improve neurological function, significantly attenuate apoptosis and neuroinflammation. 4. Activation of the PI3K-AKT signaling pathways by irisin is involved in this neuroprotection. 5. Evening season exercise irisin contributes to the neuroprotective effect on ischemic stroke model.

40. Thank you 
"Research is what i am doing when i do not know what i am doing"  Wernher von Braun..