Consistency of near-infrared spectroscopy chromophores during a 10-minute vascular occlusion procedure of the thigh over 5 consecutive days

* * ! ! ! ! by!! Cameron!Eve!Mackintosh! B.A.,!University!of!Victoria,!2013! ! ! ! ! ! ! ! ! ! ! ! ! A!Thesis!Submitted!in!Partial!Fulfillment!of!the!! Requirements!for!the!Degree!of!! MASTER!OF!SCIENCE! In!the!Department!of!Exercise!Science,!Physical,!and!Health!Education! ! ! ! ! ! ! ! ! ! ! ! ©!Cameron!Eve!Mackintosh,!2016! University!of!Victoria! ! All!rights!reserved.!This!thesis!may!not!be!reproduced!in!whole!or!in!part,!by! photocopy!or!other!means,!without!the!permission!of!the!author.!!! !! *

Supervisory*Committee* ! ! ! ! ! ! Consistency!of!NearRInfrared!Spectroscopy!chromophores!during!a!10Rminute! vascular!occlusion!procedure!of!the!thigh!over!5!consecutive!days!! ! ! ! ! ! ! ! By! Cameron!Eve!Mackintosh! B.A.,!University!of!Victoria,!2013! ! ! ! ! ! ! ! ! ! ! * * * * * * * * * * Supervisory*Committee* ! Dr.!Kathy!Gaul!(School!of!Exercise!Science,!Physical!and!Health!Education)! Supervisor* * Dr.!Lynneth!StuartRHill!(School!of!Exercise!Science,!Physical!and!Health!Education)! Departmental*Member**

ABSTRACT* * Supervisory*Committee* ! Dr.!Kathy!Gaul!(School!of!Exercise!Science,!Physical!and!Health!Education)! Supervisor* * Dr.!Lynneth!StuartRHill!(School!of!Exercise!Science,!Physical!and!Health!Education)! Departmental*Member** * * The!purpose!of!this!study!was!to!examine!the!consistency!of!nearRinfrared! spectroscopy!(NIRS)!measures!associated!with!a!10!minute!vascular!occlusion!of!the! thigh!during!5!consecutive!days.!A!secondary!purpose!was!to!examine!if!the! repeated!daily!occlusions!modify!the!vascular!response!to!such!occlusions.!The!NIRS! chromophores!of!oxygenated!hemoglobin!(HbO2),!deoxygenated!hemoglobin!(HHb),! total!hemoglobin!(tHb),!hemoglobin!difference!(HbDiff),!and!total!saturation!index! (TSI)!were!monitored!at!the!vastus!lateralis!muscle!during!each!testing!session.! Participants!were!female!(age!18R30!yrs)!and!were!randomly!assigned!to!one!of!two! groups.!The!Test!Group!(age!23!±!2yrs)!completed!an!occlusion!test!every!day!for!5! consecutive!days!while!the!Control!Group!(age!21!±1.6yrs)!was!tested!on!days!1,!3! and!5.!Each!testing!session!involved!10!minutes!rest!in!a!supine!position,!10!minutes! of!femoral!artery!occlusion!(preset!at!250mmHg),!10!minutes!of!recovery.!At!onset! of!occlusion,!the!rapid!cuff!inflation!system!inflated!in!0.3!seconds!to!the!preset! 250mmHg.!Following!the!10!minutes!of!occlusion,!the!cuff!was!deflated!in!0.3! seconds.!Heart!rate!was!monitored!throughout!all!testing!sessions!and!mean!arterial! pressure!was!calculated!and!compared!between!groups.!! There!were!no!significant!group!or!day!main!effects!found,!nor!any! significant!interactions!for!the!following!hemoglobin!chromophores!measures:!

HbO2,!HHb,!HbDiff,!and!TSI.!In!all!participants,!the!NIRS!response!pattern!to!rest,! occlusion!and!recovery!was!consistent!for!all!testing!days!regardless!of!group.!The! response!pattern!to!the!occlusion!initiated!by!the!rapid!cuff!inflation!was!as!follows:! HbO2,!tHb,!HbDiff,!and!TSI!decreased!then!slowly!plateaued!over!the!10!minutes! while!HHb!increased!initially!and!then!plateaued.!Following!the!rapid!cuff!deflation,! HbO2,!tHb,!HbDiff!and!TSI!increased!to!their!maximum!reperfusion!levels!and!then! plateaued!over!the!remainder!of!the!10!minutes!of!recovery.!During!that!same! period,!HHb!decreased!initially!upon!reperfusion!and!then!plateaued.!Unlike!the! other!measures,!TSI!and!tHb!demonstrated!inconsistencies!in!their!response!pattern! regardless!of!group!or!day.!! The!consistency!of!hemoglobin!chromophores!response!pattern!to!daily!10! minute!vascular!leg!occlusions!over!5!consecutive!days!is!an!important!finding!of! this!study.!This!response!indicates!that!there!is!no!apparent!change!in!vascular! response!to!repeated!occlusions!and!has!implications!for!exercise!studies!that!use! occlusion!in!their!methodology.!The!study!also!emphasizes!the!inconsistency!of!tHb! and!TSI!as!primary!hemoglobin!response!measures!to!occlusion.!Similarly,!the! results!demonstrate!the!potential!misinterpretation!of!results!if!only!relying!on!one! hemoglobin!measure!during!occlusion.! ! ! * * * * * *

Table*of*Contents* Supervisory*Committee………..ii* ABSTRACT………...iii** Table*of*Contents……….v* List*of*Figures……….viii* List*of*Tables………..ix** List*of*Abbreviations……….x* Acknowledgements………..xi* * CHAPTER*1*INTRODUCTION………1* 1.1 Overview………..1!! 1.2 Purpose……….2! 1.3 Research!questions………2! 1.4 Hypotheses………..3! 1.5 Assumptions,!limitations,!delimitations………3! 1.5.1 Assumptions………..3! 1.5.2 Limitations………..4! 1.5.3 Delimitations……….5! 1.6 Operational!definitions………5!! * CHAPTER*2*LITERATURE*REVIEW………..7* 2.1! Physiology!of!ischemia………7! 2.2! Reperfusion!physiology………...10! 2.3! Cardiovascular!response!to!arterial!ischemia………13! 2.4! Occlusion………...15! !!!!!!!!!!2.4.1! Arterial!vs.!venous!occlusion!protocols……….15! !!!!!!!!!!2.4.2! Occlusion!normalization!procedures………...………...……….16! !!!!!!!!!!2.4.3! Reliability!of!NIRS!measures!during!occlusion!………....17! 2.5! Tissue!oxygenation!measurement………....18! !!!!!!!!!!2.5.1! NearRinfrared!spectroscopy………..19! !!!!!!!!!!2.5.2! Limitation:!adipose!tissue!thickness………....20!

!!!!!!!!!!2.5.3! Measurement!during!ischemia………...……….20! !!!!!!!!!!2.5.4! Gender!differences!in!occlusion!desaturation………23! 2.6! Conclusions………..25!! ! !! CHAPTER*3*METHODS………...…26* 3.1! Study!design……….………..……….26! 3.2! Participants……….26! 3.3! Experimental!protocol………..27! !!!!!!!!!3.3.1! Preparation!and!questionnaire………...27! !!!!!!!!!3.3.2! Anthropometric!measures……….28! !!!!!!!!!3.3.4! Tissue!oxygenation!measurement……….28! !!!!!!!!!3.3.5! Arterial!occlusion!test………...29! 3.4! Data!processing!and!analysis………30! !!!!!!!!!3.4.1! Data!processing……….30! !!!!!!!!!3.4.2! Statistical!analysis………...32!! * CHAPTER*4*RESULTS………..33* 4.1!Participant!physical!characteristics……….33! 4.2!NIRS!results……….34! !!!!!!!!!4.2.1! Hemoglobin!variables!main!effects………...34! !!!!!!!!!4.2.2! Response!pattern……….34! !!!!!!!!!4.2.3! Daily!variation:!HbO2!and!HHb………..…….35! !!!!!!!!!4.2.4! Exception!to!response!pattern:!TSI………..37! * CHAPTER*5*DISCUSSION………..39* 5.1! NIRS!Response!pattern……….39! !!!!!!!!!5.1.1! Tissue!desaturation………41! !!!!!!!!!5.1.2! Hemoglobin!resaturation………42! !!!!!!!!!5.1.3! Limitation:!gender………...43! 5.2! Mean!arterial!pressure………...……..44! 5.3! Conclusion………..……..44!

References……….………...46* * APPENDICES……….50* Appendix!1:!Screening!Questionnaire………..…..50! Appendix!2:!PARRQ!Questionnaire………52! Appendix!3:!Participant!Consent!Form………..…53! Appendix!4:!Participant!Data!Collection!Sheet………..57! Appendix!5:!Pictures!of!Participant!Data!Collection!Setup………58! Appendix!6:!tHb!Response!Pattern!at!Onset!of!Occlusion………..………59! Appendix!7:*TSI!Reperfusion!Result……….…60** Appendix!8:!Consistency!of!HbO2!Tau!(τ)……….61! * * * * * * * * * * * * * * * * * * * * * * * * * *

List*of*Figures** * Figure!1.!Injury!during!ischemia.!(From,!Casale!&!Pipinos,!2014)! * Figure!2.!Injury!during!reperfusion.!(From,!Casale!&!Pipinos,!2014)! * Figure!3.!!Muscle!oxygenation!trends!at!the!tibialis!anterior!muscle!during! pneumatic!cuff!ischemia!of!the!thigh!recorded!throughout!lower! leg!surgery!(From,!Shadgan!et!al.,!2012).!! * Figure!4.!Typical!representation!of!NIRS!measures!during!5Rminute!arterial! occlusion!of!the!brachial!artery!and!post!occlusive!reactive! hyperemia.!! * Figure!5.!Average!mean!arterial!pressure!for!Test!and!Control!Groups!on! days!1,!3!&!5.!! * Figure!6.!Hemoglobin!chromophores!(TSI,HbO2,!HHb,!tHb,!HbDiff)!response! pattern!of!a!representative!participant!during!rest,!occlusion,!and! recovery!of!a!single!testing!session.!! * Figure!7.!Consistency!of!HbO2!and!HHb!response!pattern!in!test!and!control!groups.! ! Figure!8:*Consistency!of!Test!Group!HbO2!tau!(τ)!(seconds)!during!Occlusion,! Reperfusion!and!Recovery!over!days!1,!3,!&!5.!! ! Figure!9:*Consistency!of!Control!Group!HbO2!tau!(τ)!(seconds)!during!Occlusion,! Reperfusion!and!Recovery!over!days!1,!3,!&!5.! ! ! ! ! ! ! ! ! ! ! * * * * * * * *

List*of*Tables** * Table!1.!Experimental!Protocol!Events!with!Time,!Event!Label,!NIRS!Marker! and!HR!Marker! ! Table!2.!Physical!Characteristics!of!Test!and!Control!Group!Participants! ! Table!3.!tHb!increase!to!rapid!cuff!inflation!and!start!of!occlusion!! Table!4.!TSI!response!to!reperfusion.!! ! ! ! ! ! ! ! ! ! ! ! * * * * * * *

List*of*Abbreviations** ADP! ! Adenosine!diphosphate! ATP! ! Adenosine!triphosphate! AMP! ! Adenosine!monophosphate! Ca2+_{! ! Calcium!ion!} H+_{! ! Hydrogen!ion!} HbO2! ! Oxyhemoglobin! HHb! ! Deoxyhemoglobin! HbDiff!! Hemoglobin!difference,!HbO2!–!HHb! μM! ! Micromolar,!measure!of!concentration!of!hemoglobin!variables! MAP! ! Mean!Arterial!Pressure!! Mb! ! Myoglobin! mmHg!! millimeters!of!mercury,!measurement!of!pressure! MPTP! ! Mitochondrial!membrane!permeability!transition!pore! Na+_{! ! Sodium!} NIRS! ! NearRinfrared!Spectroscopy!! pH! ! Potential!hydrogen,!measure!of!acidity! Pi! ! Inorganic!phosphate! ROS! ! Reactive!oxygen!species!! τ! ! Tau,!the!time!constant!of!recovery!!(response)! tHb! ! Total!hemoglobin,!HbO2!+!HHb! TSI! ! Tissue!Saturation!Index! !

Acknowledgements** * Thank!you!to!my!incredible!parents,!Trip!and!Whitney,!whose!love!and! unwavering!support!throughout!this!degree!and!my!life!is!invaluable.!Your! enthusiasm!for!life!and!learning!has!shown!me!that!with!dedication!and!passion! anything!is!possible.!! ! ! Thank!you!to!my!siblings:!Terra,!Peter!and!Nenani!for!being!my!corner!upon! which!I!know!I!can!always!fall!back.!You!are!my!best!friends!and!I!am!blessed!to! have!you!as!family.!! ! ! To!the!love!of!my!life!and!future!husband,!Bryson,!thank!you!for!all!of!the! love!and!support!throughout!this!journey.!You!were!there!for!me!every!step!of!the! way!and!I!am!so!grateful!to!have!you!by!my!side.!!! ! ! Thank!you!to!my!fellow!graduate!students,!Emily,!Holly,!and!Stephanie!for! sharing!this!experience!with!me.!You!kept!me!grounded!and!reminded!me!that! when!all!else!fails,!laughter!is!the!best!distraction.!! ! ! Thank!you!to!all!of!the!women!who!volunteered!their!time!to!participate!in! this!study!and!to!Josh!for!all!of!his!help!during!data!collection.!Thank!you!Rebecca! and!Christine!for!all!of!your!assistance.!Thank!you!Greg!and!Marc!for!all!of!your! support,!patience,!and!guidance!throughout!this!whole!process.!Thank!you!Lynneth! for!your!contributions!to!this!thesis!and!willingness!to!brainstorm!ideas.!Kathy,! thank!you!for!believing!in!me!and!giving!me!the!opportunity!to!accomplish!this!goal.! Your!guidance!and!patience!as!I!found!my!footing!in!this!process!is!greatly! appreciated.!Thank!you!for!supporting!my!professional!development!and!allowing! me!to!explore!my!love!of!exercise!science.!! !! * * * * * * *

* NearRinfrared!spectroscopy!(NIRS)!has!been!used!since!the!1980’s!to! measure!cerebral!and!muscle!oxidative!metabolism!(Ferrari!et!al.,!2011).!NIRS!takes! advantage!of!the!differential!absorption!properties!of!oxygenated!and!deoxygenated! hemoglobin/myoglobin!(Neary,!et!al.,!2002).!It!is!a!common!nonRinvasive! measurement!methodology!used!in!a!variety!of!fields!ranging!from!exercise! research!to!monitoring!tissue!oxygenation!during!peripheral!surgery!(Neary,! McKenzie,!&!Bhambhani,!2002;!Shadgan!et!al.,!2012).!NIRS!measures!the!following! hemoglobin!chromophores:!oxygenated!hemoglobin!(HbO2),!deoxygenated! hemoglobin!(HHb),!total!hemoglobin!(tHb),!hemoglobin!difference!(HbDiff),!and! total!saturation!index!(TSI).! ! Exercise!tests!using!NIRS!measures!of!skeletal!muscle!oxygen!kinetics!are! commonly!used!in!conjunction!with!vascular!arterial!occlusion.!The!vascular! occlusion!test!can!enhance!the!data!gathered!by!the!NIRS!by!indirectly!examining! microcirculatory!function!and!oxygen!utilization!(Gerovasili!et!al.,!2010).!Some! researchers!scale!the!exercise!data!using!the!occlusion!and!reperfusion!results!to! compute!an!individual’s!physiological!minimum!and!maximum!(Ryan,!Erickson,! Brizendine,!Young,!&!Mccully,!2012;!Shibuya!&!Tanaka,!2004;!Southern,!Ryan,! Reynolds,!&!Mccully,!2014).!! ! The!NIRS!chromophore!response!pattern!to!vascular!arterial!occlusion!has! been!established!by!previous!studies!(Gomez,!Torres,!Kim,!Zenker,!&!Pinsky,!2008;! Lacroix!&!Gremeaux,!2012;!Shadgan!et!al.,!2012).!As!arterial!occlusion!restricts!

blood!flow!to!and!from!the!limb,!oxygenated!hemoglobin!in!the!affected!tissue! decreases!while!deoxygenated!hemoglobin!increases!at!onset!of!occlusion.!Both! measures!eventually!plateau!(in!approximately!6R10!minutes).!While!this!response! pattern!has!been!established!in!many!singleRday!studies,!there!are!conflicting!results! regarding!inconsistencies!observed!in!studies!that!test!the!response!pattern!on! separate!occasions!over!a!given!number!of!days/months!(Gomez!et!al.,!2008;! Lacroix!&!Gremeaux,!2012).!It!is!also!unknown!what!the!residual!effects!of!occlusion! are!on!skeletal!muscle!tissue!and!microvasculature,!an!important!consideration! when!using!this!methodology!during!exercise!studies.!! 1.2*Purpose** The!purpose!of!this!research!was!to!examine!the!consistency!of!nearRinfrared! spectroscopy!(NIRS)!measures!during!and!following!vascular!occlusion!of!the!thigh! over!5!consecutive!days.!A!secondary!purpose!was!to!examine!if!repeated!daily! occlusions!modify!the!vascular!response!to!such!occlusions.!! 1.3*Research*questions** The!following!research!questions!were!addressed!in!the!study:!! 1) What!is!the!impact!of!daily!10Rminute!bouts!of!vascular!leg!occlusion!on!the! following!hemoglobin!variables!over!5!consecutive!days?! a. Deoxygenated!hemoglobin!! b. Oxygenated!hemoglobin!! c. Total!saturation!index!! d. Hemoglobin!difference!! e. Total!hemoglobin!!

2) Does!5!consecutive!days!of!10Rminute!occlusion!impact!the!rate!of!response! of!hemoglobin!measures!following!the!onset!of!rapid!occlusion?!! 3) Does!5!consecutive!days!of!10Rminute!occlusion!impact!the!rate!of!response! of!hemoglobin!measures!following!reperfusion!caused!by!the!rapid!release!of! occlusion?!! 4) Is!there!a!difference!between!the!rate!and!pattern!of!response!of!hemoglobin! measures!between!the!5Rday!test!group!and!a!control!group!that!experiences! fewer!occlusion!protocols!within!the!same!5Rday!period?!! 1.4*Hypotheses** 1) H0:!There!is!no!difference!in!hemoglobin!variable!response!to!10Rminute! bouts!of!vascular!thigh!occlusion!over!5!consecutive!days.!! 2) H0:!There!is!no!group!difference!in!hemoglobin!variable!response!to!10R minute!bouts!of!vascular!thigh!occlusion!over!5!consecutive!days.!! 3) H0:!The!rate!of!response!of!hemoglobin!measures!during!the!onset!of!rapid! occlusion!does!not!change!from!day!1,!3!to!5.!! 4) H0:!The!rate!of!response!of!hemoglobin!measures!during!reperfusion!from! the!rapid!release!of!occlusion!does!not!change!from!day!1,!3!to!5.! 5) H0:!There!is!no!difference!in!the!hemoglobin!variable!rate!of!response!to! occlusion!and!reperfusion!between!the!control!and!test!groups!when! compared!on!days!1,!3!&!5!of!vascular!thigh!occlusion.!! * * *

1.5*Assumptions,*limitations,*delimitations* * 1.5.1$Assumptions$ 1) Participants!followed!all!criteria!required!to!participate!in!the!study!related! to!alcohol,!drugs!and!physical!activity.!! 2) No!participant!was!pregnant! 3) Participants!maintained!regular:! a. !Aerobic!physical!activity! b. !Caffeine!consumption!! c. !Meals!&!hydration! d. !Sleep!pattern!!! Assumptions!associated!with!NIRS!data!! The!following!assumptions!are!stated!in!the!Artinis!Portalite!Manual!(Artinis! Medical!Systems!BV,!2011,!p.21):! 1) Slope!estimations!are!based!on!the!assumption!that!the!sourceRdetector! separation!is!much!larger!than!the!source!size!and!the!scattering!mean!free! pathlength! 2) The!algorithm!assumes!homogeneous!and!infinite!tissue! 3) The!light!enters!the!tissue!perpendicular!and!without!any!airRtissue! transition! 4) A!constant!scattering!coefficient!is!assumed,!and!it!is!estimated!to!follow!a! linear!relation!to!the!wavelength!! $ $

1.5.2$Limitations$$ 1) Each!participant!arrived!at!the!lab!within!a!2!hour!window!each!day!to! minimize!time!of!day!effects!over!the!5!days!of!testing! 2) Hormonal!birth!control!was!not!controlled!and!varied!among!participants.!! 3) Phase!of!menstrual!cycle!was!recorded!but!not!controlled!for!! 4) Results!applicable!to!dominant!leg!only!! 5) Exercise!and!caffeine!consumption!was!recorded!but!not!controlled!!! 6) Results!are!only!applicable!to!resting!(nonRexercise)!NIRS!measures!! 1.5.3$Delimitations$$ 1) Participants!were!female!aged!18R30!yrs,!healthy,!with!no!injury!or!chronic! diseases!! 2) All!participants!met!the!Canadian!Society!for!Exercise!Physiology!(CSEP)! physical!activity!guidelines!of!150!minutes!of!moderate!to!vigorous!physical! activity!per!week!(Tremblay!et!al.,!2011)! 3) Participants!were!not!currently!involved!in!strength!focused!resistance! training!! 1.6*Operational*definitions** 1) Physically!active:!150!minutes!of!moderate!to!vigorous!physical!activity!per! week!! 2) Healthy:!No!chronic!diseases!including!any!vascular!disorders!! 3) Rest:!10!minutes!lying!relaxed!in!supine!position!preceding!the!arterial! occlusion! 4) Occlusion:!Complete!arterial!occlusion!at!250mmHg!for!10!minutes!!!

5) Reperfusion:!Time!period!from!cuff!deflation!to!maximum!NIRS!hemoglobin! value!(calculated!per!hemoglobin!variable)!! 6) Recovery:!Time!from!maximum!to!end!test!(calculated!per!value)!! 7) Range:!The!change!in!a!variable!from!a!given!start!value!to!end!value! 8) Tissue!or!muscle!oxygenation:!changes!in!oxygenated!hemoglobin,! deoxygenated!hemoglobin,!hemoglobin!difference,!total!hemoglobin!and! tissue!saturation!index!measured!at!the!vastus!lateralis!muscle!of!the!tested! leg!using!a!NIRS!! 9) Test!Group:!participants!who!completed!a!10Rminute!vascular!occlusion!test! intervention!for!5!consecutive!days!(days!1R5)! 10) !Control!Group:!participants!who!completed!a!10Rminute!vascular!occlusion! test!intervention!on!days!1,!3!&!5! ! * * * * * * * * * *

CHAPTER*2*LITERATURE*REVIEW* * Peripheral!vascular!occlusion!has!been!used!in!combination!with!NIRS!to! examine!tissue!response!to!ischemic!conditions.!The!application!of!NIRS!with! occlusion!ranges!from!medical!research!to!exercise!physiology.!Previous!studies! have!examined!skeletal!muscle!tissue!response!to!occlusion!on!singular!occasions!as! well!as!responses!tested!on!separate!days.!It!is!important!to!understand!the! physiological!response!to!peripheral!ischemia!in!order!to!interpret!the!results!of! consecutive!vascular!limb!occlusions.!Similarly,!there!are!potential!complications!or! contraindications!related!to!the!physiological!response!to!exercise!following!limb! occlusion.* * 2.1*Physiology*of*ischemia* ! The!lack!of!sufficient!blood!flow!to!an!area!or!limb,!ischemia,!results!in!local! as!well!as!“full!body”!physiological!responses.!Some!of!these!responses!are! immediate!while!others!are!slower!to!respond.!The!severity!of!these!changes!and! potential!physiological!damage!is!related!to!the!duration!of!ischemia!as!well!as!the! pressure!of!the!tourniquet!(if!used)!causing!ischemia!(Appell,!Glöser,!Duarte,! Zellner,!&!Soares,!1993;!Casale!&!Pipinos,!2014).! At!the!onset!of!occlusion,!there!is!a!progressive!decline!in!oxygen!and!a! subsequent!increase!in!CO2!within!the!muscle!(Deloughry!&!Griffiths,!2009).!During! ischemia,!mitochondria!can!no!longer!produce!adenosine!triphosphate!(ATP)!by! oxidative!phosphorylation!due!to!a!lack!of!oxygenated!blood!reaching!the!tissue! (Casale!&!Pipinos,!2014).!Along!with!the!decrease!in!ATP!stores,!creatine!phosphate! levels!eventually!become!exhausted!as!well,!however!total!depletion!of!both!ATP!

and!creatine!phosphate!are!exhausted!after!roughly!2R3!hours!(Deloughry!&! Griffiths,!2009).!As!cellular!ATP!declines!quickly!there!is!a!concomitant!increase!in! inorganic!phosphate!(Pi),!adenosine!monophosphate!(AMP),!and!adenosine! diphosphate!(ADP).!In!an!attempt!to!maintain!ATP,!glycolysis!is!stimulated.! However,!it!is!unable!to!produce!sufficient!amounts!of!ATP!that!are!needed,! particularly!during!exercise!or!nonRrest!conditions(Casale!&!Pipinos,!2014).!The! accumulation!of!lactic!acid!decreases!the!intracellular!pH,!increasing!CO2!levels!and! leading!to!intracellular!acidosis!(Deloughry!&!Griffiths,!2009).!The!lactic!acid! buildup!further!inhibits!glycolysis!and!activates!Na+_/H+_{!antiporter!that!pumps!H}+_! out!of!the!cell.!Due!to!the!shortage!of!ATP,!the!Na+_/Ca2+_{!ATPase!does!not!pump!the!} Na+_{!ions!that!enter!the!cell!out.!The!accumulation!of!Na}+_{!causes!intracellular!edema!} via!osmotic!effect!(Appell,!Glöser,!et!al.,!1993).!The!high!levels!of!intracellular!Na+_! inhibits!the!Na+_/Ca2+_{!antiporter,!which!leads!to!high!levels!of!Ca}2+_{.!The!increased!} intracellular!Ca2+_{!activates!degradative!enzymes,!including!phospholipase!A2!and!} proteases!such!as!calpains!(see!Figure!1)!(Appell,!Glöser,!et!al.,!1993;!Casale!&! Pipinos,!2014).!Additionally,!the!ATP!shortage!impairs!ATPRdependent!repair! processes!(Halestrap!&!Pasdois,!2009).!

! Figure!1.!“Injury!during!ischemia.!Decreased!oxygen!supply!activates!a!complex! cascade!of!metabolic,!inflammatory,!and!prothrombotic!pathways!and!sets!the! conditions!for!MPTP!opening!during!reperfusion.”!(Casale!&!Pipinos,!2014)! ! After!15!minutes!of!arterial!occlusion,!muscle!biopsies!of!the!vastus!lateralis! have!shown!observable!pathological!reactions!that!included!intrafibre!edema!in!the! subsarcolemmic!space,!separating!myofibrils,!and!in!the!interstitial!space!(Appell,! Glöser,!et!al.,!1993).!The!edema!may!be!caused!by!the!cellular!changes!to!the! microvascular!permeability!as!a!result!of!the!disruption!of!ion!pumps!(Figure!1).! Similarly!after!15!minutes,!affected!capillaries!display!a!thickening!of!their! basement!membrane!as!well!as!total!occlusion.!The!capillary!occlusion!could!be! caused!by!red!blood!cells!obstructing!the!capillaries!or!their!walls!having!collapsed.! It!is!proposed!that!the!endothelial!cell!basement!membrane!thickening!is!not! protection!against!the!ischemia,!but!perhaps!protection!for!the!reperfusion!pressure! increase!(Appell,!Gloser,!Duarte,!Zellner,!&!Soares,!1993;!Hammersen!et!al.,!1989).!!! The!progression!of!ischemia!eventually!leads!to!the!production!and! accumulation!of!reactive!oxygen!species!(ROS)!within!the!mitochondria!(Clanton,!

2007;!Halestrap!&!Pasdois,!2009).!A!combination!of!factors!including!increased! cytosolic!Ca2+_{,!elevated!Pi,!increased!mitochondrial!matrix!ROS,!and!reduction!of!} adenine!nucleotides!creates!a!condition!that!eventually!open!the!mitochondrial! membrane!permeability!transition!pores!(MPTP).!This!will!ultimately!cause!necrotic! cell!death!(Halestrap,!2010;!Loor!et!al.,!2011).!The!extent!of!tissue!damage!is! determined!by!the!duration!of!the!MPTP!opening,!which!is!caused!by!the!magnitude! of!ROS!damage!to!the!mitochondria!(Halestrap!&!Pasdois,!2009).!During!ischemia,! the!decreased!intracellular!pH!prohibits!the!pore!opening.!However,!the!pH!changes! during!reperfusion,!allowing!the!pores!to!open.!! The!lack!of!oxygenated!blood!to!the!tissue!during!ischemia!causes!tissue! hypoxia!and!an!accumulation!of!vasoactive!metabolites!such!as!nitric!oxide! (Klabunde,!2005).!This!causes!dilation!of!arterioles!and!a!decrease!in!vascular! resistance.!When!blood!flow!is!restored!to!the!tissue,!a!hyperemic!reperfusion! response!occurs!due!to!increased!flow!caused!by!the!decreased!vascular!resistance! (Klabunde,!2005).!!! 2.2*Reperfusion*physiology*! ! Reperfusion!occurs!when!blood!flow!to!a!previously!occluded!tissue!is! restored!following!ischemia!(Casale!&!Pipinos,!2014).!While!reperfusion!restores! blood!flow!to!the!tissue,!there!is!a!risk!of!reperfusion!injury!causing!tissue!damage.! When!reperfusion!is!initiated!there!is!an!increase!in!blood!volume!to!the!affected! region!as!flow!becomes!elevated!above!normal!due!to!the!reduced!vascular! resistance!initiated!during!ischemia!(Klabunde,!2005).!The!increased!flow!brings! oxygenated!hemoglobin!into!the!area!and!flushes!deoxygenated!hemoglobin!away!

(Gerovasili,!Dimopoulos,!Tzanis,!AnastasiouRnana,!&!Nanas,!2010).!The!first!few! minutes!of!reperfusion!with!its’!concomitant!hyperemia!allow!rapid!resaturation!of! hemoglobin!and!flushing!of!vasodilator!metabolites!such!as!nitric!oxide!(Klabunde,! 2005).!Eventually,!the!vascular!tone!is!restored!and!the!blood!flow!to!the!region!is! returned!to!normal!levels.!This!reperfusion!rate!is!thought!to!be!dependent!on!the! function!of!vascular!endothelium!vasodilation!(Gerovasili!et!al.,!2010).!The!longer! the!occlusion,!the!greater!the!demand!on!vasodilation!and!therefore!the!increase!in! peak!hyperemic!flow!(Klabunde,!2005).!! ! Three!processes!characterize!reperfusion:!1.!A!burst!of!ROS!production! occurring!primarily!by!the!electron!transport!chain!in!the!mitochondria;!2.!An! increase!of!Ca2+_{!uptake!into!the!mitochondrial!matrix;!3.!An!intracellular!pH!shift!} back!to!neutral!from!acidic!during!ischemia!(Halestrap,!2010).!The!increase!of!ROS! production!during!both!ischemia!and!reperfusion!can!cause!necrosis!and!apoptosis.! Both!of!these!cellular!death!programs!are!mediated!through!the!mitochondria.!The! degree!of!potential!reperfusion!injury!is!primarily!dependent!on!the!total!time!of! ischemia.!!! ! Cellular!death!can!occur!during!reperfusion,!however!this!is!typically!only!a! concern!for!severe!(amount!of!pressure!causing!ischemia)!and!prolonged!ischemia! (over!2!hours)!(Casale!&!Pipinos,!2014;!Deloughry!&!Griffiths,!2009).!The!length!of! time!and!the!pressure!causing!ischemia!are!both!factors!in!the!speed!of!tissue! damage.!In!the!case!of!cellular!death,!proapoptotic!factors,!BAX!and!BAK,!work!to! promote!the!death!of!the!cell.!BAK!initiates!apoptosis!by!releasing!proteins!such!as! cytochrome!c.!This!occurs!as!a!result!of!increased!outer!membrane!permeability!of!

the!mitochondria!(Kubli!&!Gustafsson,!2012).!The!mitochondrial!MPTP!opening!is!a! main!factor!causing!ischemia/reperfusion!injury!(see!Figure!2).!When!this!is!opened! it!allows!free!passage!of!water!and!solutes!less!than!1.5!kDa.!With!the!free!passage! of!water,!osmotic!damage!to!the!mitochondria!occurs!as!well!as!uncoupling!of! mitochondrial!oxidative!phosphorylation.!This!occurs!due!to!the!dissipation!of!pH! differences!and!of!the!electrical!potential!across!the!mitochondrial!membrane! (Halestrap!&!Pasdois,!2009;!Halestrap,!2010).!The!disruption!of!metabolism!ionic! homeostasis!is!promoted!by!mitochondrial!damage!and!cessation!of!oxidative! phosphorylation.!! ! The!vast!changes!in!metabolism!within!the!skeletal!muscle!during! reperfusion!could!impact!performance!during!subsequent!exercise!tests.!Although! performance!changes!from!postRocclusion!exercise!tests!have!not!been!determined,! it!is!recommended!to!wait!10R15!minutes!after!occlusion!release!to!restore!muscle! ATP!levels!(Deloughry!&!Griffiths,!2009).!! ! The!surge!of!mitochondrial!ROS!production!during!reperfusion!as!well!as!the! increased!production!during!ischemia!increases!oxidative!stress!(Figure!2).!The! oxidative!stress!created!by!the!ROS!increase!helps!to!increase!the!sensitivity!of!the! MPTP!pore!opening!to!mitochondrial!Ca!2+_{!concentrations.!Increased!Ca}2+_! sensitivity,!along!with!the!intracellular!pH!increase!during!reperfusion,!work!to! open!the!mitochondrial!MPTP!(Halestrap,!2010).!! !

! Figure!2.!“Injury!during!reperfusion.!The!return!of!oxygen,!influx!of!inflammatory! cells,!and!washout!of!metabolites!contribute!to!cell!death!and!inflammatory,! prothrombotic!milieu!that!exacerbates!tissue!injury.”!(Casale!&!Pipinos,!2014)! Abs=!antibodies;!ARDS=!acute!respiratory!distress!syndrome;!DAMPS=!dangerR associated!molecular!patterns;!METC=!mitochondrial!electron!transport!chain;! NADH=!reduced!nicotinamide!adenine!dinucleotide;!NFRkB=!nuclear!factor!kappa!B;! TLRs=!tollRlike!receptors!! ! ! The!physiological!response!to!single!bouts!as!well!as!repeated!ischemiaR reperfusion!tests!is!important!to!understand,!as!peripheral!vascular!occlusion!is!a! common!test!in!conjunction!to!exercise!studies!using!NIRS.!Changes!within!the! tissue!during!ischemia!and!reperfusion,!as!well!as!residual!effects!during!recovery,! can!affect!skeletal!muscle!function!and!metabolism.!These!physiological!changes!can! impact!performance!in!subsequent!exercise!tests.!! 2.3*Cardiovascular*response*to*arterial*ischemia** ! The!cardiovascular!response!to!limb!occlusion!has!been!reported!in!previous! studies!(Fellahi!et!al.,!2014;!Iida!et!al.,!2007).!Throughout!a!10Rminute!femoral!

artery!occlusion,!blood!pressure!and!heart!rate!remained!consistent!(Fellahi!et!al.,! 2014).!This!absence!of!an!increase!in!heart!rate!or!blood!pressure!during!peripheral! thigh!occlusion!lead!Fellahi!et!al.!(2014)!to!conclude!that!sympathetic!activity!did! not!increase!during!arterial!occlusion.!! However,!an!earlier!study!that!examined!heart!rate!variability!throughout! bilateral!leg!arterial!occlusions!found!an!increase!in!sympathetic!activity!during! occlusion!(Iida!et!al.,!2007).!The!increased!sympathetic!activity!Iida!et!al.!(2007)! reported!was!during!15!minute!bilateral!leg!occlusions!(200mmHg),!as!opposed!to! unilateral!leg!occlusion!in!the!study!by!Fellahi!et!al.!(2014);!therefore!the! cardiovascular!results!may!not!be!directly!comparable.!During!the!bilateral!leg! occlusions,!heart!rate!increased,!stroke!volume!steadily!decreased,!and!sympathetic! activity!increased!while!parasympathetic!activity!decreased,!as!measured!using!HRV! (Iida!et!al.,!2007).!Another!vascular!measure!of!sympathetic!activity,!plasma! concentrations!of!noradrenaline,!also!increased!significantly!during!the!bilateral! occlusion!procedure.!! Sympathetic!control!in!response!to!occlusion!may!not!be!apparent!when!only! examining!heart!rate!and!blood!pressure,!thus!creating!a!need!for!more! comprehensive!testing!measures,!such!as!heart!rate!variability,!of!cardiovascular! response!to!vascular!limb!occlusion.!Monitoring!sympathetic!and!parasympathetic! control!during!and!after!occlusion!tests!could!be!a!valuable!tool!when!conducting! tests!in!conjunction!with!exercise!tests!as!this!can!affect!performance!results.!! * *

2.4*Occlusion*Protocols* 2.4.1$Arterial$vs.$venous$occlusion$protocols$$ ! Occlusion!tests!have!been!conducted!using!both!arterial!and!venous! protocols.!Arterial!occlusion!limits!all!blood!flow!to!and!from!the!tissues!while! venous!occlusion!only!limits!blood!flow!out!of!the!tissue.!Venous!occlusion!is! performed!with!a!pressure!of!~60mmHg.!It!has!been!used!to!examine!resting!blood! volume!change,!which!is!represented!by!the!total!hemoglobin!(tHb)!increase!during! venous!occlusion!(Southern!et!al.,!2014;!Van!Beekvelt,!Colier,!Wevers,!&!Van! Engelen,!2001).!! ! Both!arterial!and!venous!occlusion!methods!are!used!in!research,!however! the!focus!in!this!review!will!be!on!arterial!occlusions!as!they!are!commonly!used!in! conjunction!to!NIRS!exercise!research.!Arterial!occlusion!restricts!all!blood!flow!to! and!from!the!tissue.!It!is!performed!using!a!supraRsystolic!pressure!usually!set! between!250R300mmHg!(Fellahi!et!al.,!2014;!Shibuya!&!Tanaka,!2004).!During! arterial!occlusion!there!is!a!lack!of!oxygenated!blood!delivered!to!the!occluded! tissue.!The!initial!drop!of!HbO2!and!subsequently!hemoglobin!difference!(HbDiff)! following!the!initiation!of!occlusion!can!be!used!to!demonstrate!the!rate!of!oxygen! consumption!in!the!tissue!(Van!Beekvelt!et!al.,!2001).!! The!current!literature!does!not!state!a!standard!protocol!for!the!duration!of! arterial!occlusion.!It!has!been!suggested!that!using!a!fixed!duration!method!is! preferable!as!it!ensures!the!same!time!of!occlusion!is!applied!to!every!participant! (Gerovasili!et!al.,!2010).!This!fixed!duration!method!has!been!used!in!earlier!studies! (Gomez!et!al.,!2008;!Lacroix!&!Gremeaux,!2012).!However,!not!all!studies!utilize!this!

method!and!some!determine!a!set!maximum!desaturation!value!(40%!decrease!in! TSI!from!rest)!at!which!occlusion!will!cease!(Fellahi!et!al.,!2014;!Gomez!et!al.,!2008).! Other!researchers!have!set!a!minimum!duration!of!5!minutes,!but!stop!any! time!after!that!when!the!participant!expresses!discomfort!(Shibuya!&!Tanaka,! 2004).!This!lack!of!protocol!standardization!leads!to!confusion!as!well!as! misunderstanding!when!generalizing!results!across!studies.!Similarly,!it!likely!leads! to!inaccurate!determination!of!an!absolute!physiological!minimum!and!maximum.!! 2.4.2$Occlusion$normalization$procedures$$ ! Arterial!occlusion!tests!have!been!used!in!conjunction!with!skeletal!muscle! NIRS!measurements!in!a!broad!scope!of!research!settings.!These!studies!range!from! exercise!physiology!studies,!peripheral!limb!surgeries!and!tissue!monitoring!in! trauma!in!order!to!monitor!tissue!oxygenation!in!various!skeletal!muscles!(Gomez! et!al.,!2008;!Ryan!et!al.,!2012;!Shadgan!et!al.,!2012;!Shibuya!&!Tanaka,!2004;! Southern!et!al.,!2014).!With!such!a!wide!range!of!uses!for!NIRS!measures!during! occlusion,!there!are!an!equal!number!of!protocols!reported!in!the!literature.!It!is! unknown!if!the!occlusion!data!on!one!day!is!truly!representative!for!that!individual! across!time!(or!if!it!could!change!daily).!As!well,!there!are!multiple!proposed! methodologies!of!using!the!occlusion!data!to!scale!exercise!NIRS!data.!! There!are!a!few!proposed!methodologies!that!use!NIRS!occlusion!data!in! conjunction!with!exercise!test!data.!One!method!involves!occlusion!used!with!NIRS! research!to!normalize!the!participant’s!NIRS!data!from!the!exercise!portion!of! testing!to!their!physiological!minimum!and!maximum!found!using!the!occlusion!test.! The!minimum!(used!as!the!participant’s!physiological!0%)!is!achieved!at!end!of!full!

arterial!occlusion!and!their!maximum!(used!as!their!physiological!100%)!is! achieved!at!maximum!of!their!reperfusion.!This!has!been!performed!using!a!rapid! inflation!pneumatic!cuff!occlusion!(Ryan!et!al.,!2012;!Shibuya!&!Tanaka,!2004;! Southern!et!al.,!2014).!However,!this!is!not!a!standardized!protocol!in!that!not!all! NIRS!research!uses!an!occlusion!test!to!normalize!participant!data.!! Other!research!findings!do!not!depend!on!calculating!the!absolute!values! based!on!NIRS!occlusion!physiological!minimum!and!maximum.!Instead,!the!kinetics! of!the!response!signal!is!determined!using!the!time!constant!of!recovery!(τ)! (McCully!&!Hamaoka,!2000).!The!time!constant!of!recovery!assumes!that!the! changes!in!oxygen!kinetics!are!not!linear!but!rather!exponential.!This!method!does! not!give!absolute!measurements,!but!rather!calculates!the!change!of!a!variable!over! a!specific!timeframe.!! 2.4.3$Reliability$of$NIRS$measures$during$occlusion$ ! The!reliability!of!NIRS!measures!collected!during!a!vascular!occlusion!test! has!been!examined!previously!(Bhambhani,!Maikala,!Jeon,!&!Bell,!1998).!Same!day! repeated!leg!occlusions!(8!minutes)!with!a!period!of!rest!(6!minutes)!between!tests! were!determined!reliable!in!both!upper!and!lower!limb!skeletal!muscle!(Bhambhani! et!al.,!1998).!Similarly,!intraRday!reliability!was!demonstrated!using!4!repeated! vascular!occlusion!tests!on!the!thenar!eminence!separated!by!>5!minutes!of!rest! (Gomez!et!al.,!2008).!While!intraRday!reliability!of!NIRS!measures!during!vascular! occlusion!has!been!reported,!interRday!reliability!is!less!consistent.!! ! Variability!of!interRday!reliability!of!NIRS!variables!has!been!noted!in!upper! limb!occlusion!studies.!In!the!study!by!Gomez!et!al.!(2008)!changes!in!tissue!oxygen!

saturation!in!the!thenar!eminence!during!vascular!occlusion!tests!were!monitored! to!measure!oxygen!consumption!and!cardiovascular!reserve!in!a!healthy!and!trauma! population.!The!healthy!participants!completed!the!same!vascular!occlusion!test!6! months!apart.!The!second!occlusion!test!showed!that!there!was!significant!variation! in!the!response!pattern!compared!to!their!initial!vascular!occlusion!test.!In!addition,! the!same!study!repeated!the!vascular!occlusion!test!with!trauma!patients!every!day! for!3!days.!There!were!significant!differences!in!the!desaturation!rates!across!the!3! days!of!vascular!occlusion!tests.!Conversely,!Lacroix!et!al.!(2012)!found!the! hemoglobin!measures!did!not!respond!differently!to!a!pre/post!vascular!arm! occlusion!test!separated!by!7R30!days.!These!contradicting!findings!indicate!a! potential!variability!of!response!to!occlusion!when!separated!over!time,!and!should! be!explored!further.! These!studies!indicate!the!potential!variability!of!NIRS!measures!to!occlusion! on!separate!testing!sessions.!Both!of!these!studies!were!conducted!on!the!upper! limb!therefore!creating!a!gap!to!examine!the!consistency!of!lower!limb!skeletal! muscles!to!repeated!occlusion!tests.!!! 2.5*Tissue*oxygenation*measurement* * The!monitoring!of!tissue!oxygenation!is!important!when!performing!limb! occlusion!for!both!research!and!safety!purposes.!With!the!development!of!nonR invasive!imaging!technology,!such!as!nearRinfrared!spectroscopy!(NIRS),!it!is! possible!to!examine!tissue!responses!throughout!vascular!occlusions.!!!! $ $

2.5.1$NearDinfrared$spectroscopy$$$ ! NearRinfrared!spectroscopy!(NIRS)!has!been!used!since!the!1980’s!to!nonR invasively!measure!cerebral!and!muscle!oxidative!metabolism!(Ferrari,!Muthalib,!&! Quaresima,!2011).!It!is!based!on!the!differential!absorption!properties!of! chromophores!hemoglobin!and!myoglobin!in!the!nearRinfrared!range!of!700R1000! nm!(Mancini!et!al.,!1994;!Neary!et!al.,!2002).!At!the!nearRinfrared!wavelength!of! 760nm!deoxygenated!hemoglobin!(HHb)!has!a!higher!absorbency!than!oxygenated! hemoglobin!(HbO2).!At!850nm!oxygenated!hemoglobin!has!higher!absorbency!than! deoxygenated!hemoglobin.!! The!effect!of!myoglobin!(Mb)!concentration!on!NIRSRmeasured!variables!is! unknown!due!to!the!absorbency!signals!of!hemoglobin!and!myoglobin!overlapping! in!the!nearRinfrared!range!(Ferrari,!Mottola,!&!Quaresima,!2004).!Therefore,!it!is! common!practice!for!NIRS!data!to!be!reported!as!a!combined!value!including!both!! Mb!!and!hemoglobin!measures!(Ferrari!et!al.,!2011;!Lacroix!&!Gremeaux,!2012;! Neary!et!al.,!2002).!From!the!differential!absorbency!patterns!of!HHb!and!HbO2!it!is! possible!to!infer!the!oxidative!status!of!the!skeletal!muscle!tissue!of!interest.!! ! NIRS!measures!concentration!changes!of!the!following!chromophores:!HbO2,! HHb,!total!hemoglobin!(tHb),!and!hemoglobin!differences!(HbDiff).!Total! hemoglobin,!tHb,!is!the!summation!of!HbO2!and!HHb.!Hemoglobin!difference,!HbDiff,! is!the!difference!between!HbO2!and!HHb.!Most!commonly,!the!ratio!of!HbO2!and!tHb! is!also!monitored!and!reported!as!tissue!saturation!index!(TSI).!!

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2.5.2$Limitation$of$NIRS$accuracy:$adipose$tissue$thickness$$! ! NIRS!measures!in!skeletal!muscle!assess!any!changes!in!tissue!oxygenation! through!photon!pathways!of!minimal!absorbers!such!as!arterioles,!capillaries,!and! venules!(Mancini!et!al.,!1994).!However,!if!surface!adipose!tissue!thickness!is!too! great,!it!can!influence!the!light!propagation!into!the!underlying!skeletal!muscle! tissue!and!create!inaccurate!measurements!(Niwayama,!Suzuki,!Yamashita,!&! Yasuda,!2012).!Therefore,!while!NIRS!can!be!used!on!a!broad!age!range!from! children!to!adults,!adiposity!is!a!limiting!factor!for!using!NIRS!and!limits!the! population!to!those!with!smaller!adipose!tissue!thickness!(Ferrari!et!al.,!2011).!This! limitation!of!the!NIRS!does!not!allow!for!studies!on!some!clinical!populations!as!well! as!athletes!with!larger!layers!of!adipose!tissue.!! 2.5.3$NIRS$Measurement$during$ischemia$$ ! NIRS!has!been!used!to!evaluate!and!monitor!tissue!oxygenation!response!to! rapid!onset!occlusion!and!reactive!hyperemia!in!the!lower!and!upper!limbs!(Fellahi! et!al.,!2014;!Lacroix!&!Gremeaux,!2012;!Shadgan!et!al.,!2012).!The!NIRS!variables! respond!in!a!distinct!pattern!to!arterial!vascular!occlusion.!This!pattern!(see!Figure! 3)!has!been!well!established!with!many!lower!and!upper!limb!occlusion!tests! (Gomez!et!al.,!2008;!Lacroix!&!Gremeaux,!2012;!Shadgan!et!al.,!2012;!Shibuya!&! Tanaka,!2004).!At!onset!of!rapid!arterial!occlusion!HHb!rapidly!increases!while! HbO2!rapidly!decreases,!initially!in!a!linear!manner!then!leveling!off.!The!decrease!of! HbO2!reflects!the!rate!of!tissue!oxygen!consumption.!Total!hemoglobin!(tHb)!stays! between!HbO2!and!HHb,!sometimes!slightly!increasing!or!decreasing!initially,!but! ultimately!plateauing!during!occlusion!(Shadgan!et!al.,!2012).!The!plateau!typically!

occurs!after!approximately!5R7!minutes.!tHb!reflects!whether!blood!is!entering!or! exiting!the!occluded!area,!so!with!a!complete!vascular!occlusion!tHb!should!be!in!the! middle!of!HHb!and!HbO2.!HbDiff!decreases!below!that!of!HbO2!as!it!is!the!difference! of!HbO2!and!HHb.!At!rapid!release!of!occlusion!HbO2,!HbDiff!and!tHb!all!rapidly! increase!in!a!hyperemic!response.!HHb!rapidly!decreases!as!the!blood!is!flushed!out! of!the!limb.!All!variables!eventually!return!to!baseline!measures!after!a!few!(5R10)! minutes!of!recovery!post!reperfusion.!! ! These!patterns!of!rest,!occlusion,!reperfusion!and!recovery!are!clearly! demonstrated!in!a!variety!of!skeletal!muscles!in!multiple!anatomical!locations! (upper!and!lower!limbs)!and!durations!(ranging!from!5R30!minutes)!of!occlusion.! For!example,!Figure!3!demonstrates!a!representative!tissue!oxygenation!response!of! the!tibialis!anterior!muscle!during!lower!leg!surgery,!monitored!by!NIRS!(Shadgan! et!al.,!2012).! ! ! !

! ! Figure!3.!Muscle!oxygenation!trends!at!the!tibialis!anterior!muscle!during!pneumatic! cuff!ischemia!of!the!thigh!recorded!throughout!lower!leg!surgery!(Shadgan!et!al.,! 2012).*!! A=!tourniquet!inflation!time,!B=!cuff!release!time,!C=!point!of!maximum!hyperemia,! D=!point!of!maximum!HbO2!recovery.!!! *tHb!decrease!during!occlusion!was!suspected!to!be!due!to!blood!loss!from!the! surgical!field.! ! The!same!response!pattern!has!been!found!in!the!upper!limb!during!vascular! occlusion!tests!(Lacroix!&!Gremeaux,!2012)!(Figure!4)!with!similar!response!of! hemoglobin!chromophores!as!seen!in!Figure!3.!!!

!!!!!!!!!!!! ! Figure!4.!Typical!representation!of!NIRS!measures!during!5Rminute!arterial! occlusion!of!the!brachial!artery!and!“postRocclusive”!reactive!hyperemia!(Lacroix!&! Gremeaux,!2012).!! ! ! The!consistency!in!response!pattern!of!NIRS!measures!throughout!and!after! arterial!vascular!occlusion!has!been!demonstrated!in!singleRday!sessions!in!the! upper!and!lower!limbs!(Figure!3!&!4).!It!is!clear!that!post!occlusion!there!is!a! reperfusion!and!recovery!timeframe!of!approximately!5R10!minutes!that!is!needed! to!return!NIRS!measures!to!preRocclusion!baseline.!This!recovery!timeframe!is! especially!important!when!subsequent!exercise!tests!are!performed.!To!date,!there! has!been!no!report!indicating!if!there!is!a!consistency!in!NIRS!response!pattern!over! consecutive!days.!! 2.5.4$Gender$differences$in$occlusion$desaturation$$ ! Males!and!females!have!similar!baseline!NIRS!measures!however!they!have! been!found!to!respond!differently!to!arterial!occlusion!(Fellahi!et!al.,!2014;!Shadgan! et!al.,!2012).!While!males!and!females!follow!the!same!pattern!of!NIRSRmeasured!

hemoglobin!chromophores!response!throughout!rest,!occlusion,!and!recovery,!the! rates!of!desaturation!and!resaturation!vary!between!genders!(Fellahi!et!al.,!2014).!! Males!have!been!shown!to!have!faster!rates!of!TSI!desaturation!than!females.! In!the!study!by!Fellahi!et!al.!(2014)!females!demonstrated!higher!minimal!oxygen! saturation!during!occlusion!compared!to!males.!The!higher!minimal!saturation! during!occlusion!was!thought!to!impact!the!speed!of!resaturation!rate!post! occlusion.!Males!in!the!same!study!demonstrated!lower!minimal!oxygen!saturation! and!faster!resaturation!rates!post!occlusion!than!females.!Therefore,!a!relationship! between!the!lower!minimum!oxygenation!saturation!during!occlusion!and!the!speed! of!resaturation!rate!in!males!could!help!explain!the!resaturation!rate!differences! found!between!males!and!females!(Fellahi!et!al.,!2014).!! Previous!studies!support!the!results!that!males!have!faster!desaturation! rates!compared!to!females!(Wüst,!Morse,!de!Haan,!Jones,!&!Degens,!2008).!Wüst!et! al.!(2008)!suggest!that!there!are!intrinsic!sexRrelated!differences!in!the!rate!of! skeletal!muscle!energy!utilization!as!well!as!contractile!speed.!These!physiological! differences!in!male!and!female!skeletal!muscles!are!suggested!to!help!explain!the! difference!in!desaturation!rate.!While!the!response!pattern!differences!between! genders!are!understood,!it!is!necessary!to!further!explore!potential!changes!to! occlusion!patterns.!This!is!especially!important!when!performing!occlusion!studies! immediately!preceding!exercise!tests,!as!there!may!be!physiological!changes!to! occlusion!that!can!affect!exercise!performance!results.!! ! * *

2.6*Conclusions** ! Vascular!limb!occlusion!creates!an!ischemic!response!in!the!tissue.!The! physiological!response!to!ischemia!and!reperfusion!are!well!established.!NonR invasive!imaging!technology!such!as!NIRS!allows!for!further!exploration!of!the! hemoglobin!chromophores!response!to!occlusion!and!subsequent!reperfusion!tests.! The!consistency!of!hemoglobin!response!to!repeated!vascular!occlusions,!especially! in!the!lower!limb,!is!not!established!and!requires!further!exploration!in!order!for!it! to!be!used!in!exercise!physiology!test!protocols.!As!well,!the!impact!of!repeated!daily! occlusion!procedures!on!the!tissue!(muscle)!function!has!not!yet!been!examined.! This!could!have!implications!for!exercise!research!that!uses!occlusion,!as!there!may! be!residual!effects!from!the!occlusion!procedures,!which!could!negatively!impact! performance.!! * * * * * * * * * * *

CHAPTER*3*METHODS* 3.1*Study*Design** The!design!of!this!study!was!experimental!with!two!equivalent!groups! recruited!through!purposeful!sampling.!TwentyRfour!female!volunteers!(age!range! 18R30!yrs)!participated!in!this!research!study!and!were!randomly!assigned!to!either! a!5Rday!intervention!(“Test!Group”)!or!3Rday!intervention!(“Control!Group”).!All! participants!in!both!groups!underwent!a!10Rminute!arterial!occlusion!procedure!on! the!same!thigh!during!each!testing!session.!The!Test!Group!completed!the! intervention!procedure!every!day!for!five!consecutive!days!(days!1R5).!!The!control! group!completed!the!intervention!procedure!3!times,!once!every!other!day!for!five! consecutive!days!(days!1,!3,!5).!Each!participant!was!required!to!come!into!the!lab! for!approximately!40!minutes!for!each!testing!session.!The!Human!Research!Ethics! board!at!the!University!of!Victoria!approved!this!research!study!and!each! participant!provided!written!informed!consent.!! 3.2*Participants** Volunteer!participants!were!recruited!through!word!of!mouth,! announcements!to!undergraduate!courses,!and!posters!at!the!University!of!Victoria.! All!participants!were!healthy!with!no!chronic!health!conditions!including!vascular! disease.!Participants!were!not!pregnant,!nonRsmokers!and!did!not!use!drugs! (prescription!or!non,!with!the!exception!of!birth!control).!All!participants!met!the! CSEP!physical!activity!guidelines!of!150!minutes!of!moderate!to!vigorous!physical! activity!per!week!(Tremblay!et!al.,!2011)!and!none!were!currently!involved!in! structured!strengthRfocused!resistance!training!programs.!Participants!were!asked!

to!refrain!from!vigorous!physical!activity!and!alcohol!consumption!for!the!duration! of!the!testing.!If!they!regularly!drank!caffeine!they!were!asked!to!continue!normal! consumption.!They!were!asked!to!continue!regular!aerobic!physical!activity,!sleep,! hydration,!and!meals!during!the!testing!days.!! Of!the!twentyRfour!volunteers!who!participated,!data!from!3!participants!in! the!5Rday!Test!group!were!excluded!from!analysis:!One!participant!chose!to!leave! the!study!after!completing!one!day!due!to!anxiety!of!testing.!Data!from!two!other! participants!were!excluded!due!to!unexplained!error!in!the!NIRS!data!collected.!This! left!a!total!of!12!participant!data!sets!in!the!5Rday!Test!group!and!twelve!in!the!(3! day)!Control!group.!!! The!number!of!participants!needed!was!determined!using!G*Power!(Faul,! Erdfelder,!Lang,!&!Buchner,!2007).!ANOVA!with!repeated!measures!was!used!with!a! α!error!probability!of!0.05!and!an!effect!size!f!of!0.25.! 3.3*Experimental*protocol** * 3.3.1$Preparation$and$questionnaire$$ Following!recruitment!and!confirmation!of!having!met!the!inclusion!criteria,! participants!were!randomly!assigned!to!the!control!or!testing!group!before!their! initial!testing!session.!All!testing!took!place!at!the!University!of!Victoria.!Upon!initial! testing!session,!the!experimental!procedures!were!explained!to!each!participant! verbally!by!the!researcher!(Appendix!1).!Following!the!explanation,!participants! completed!a!ParRQ!questionnaire!(Appendix!2)!and!signed!the!written!consent! (Appendix!3).!Age,!birth!control!type!and!phase!of!menstrual!cycle!were!also! recorded!on!the!data!collection!form!(Appendix!4).!!

The!control!group!participated!in!testing!every!other!day!for!5!consecutive! days!(days!1,!3,!5)!while!the!testing!group!completed!the!occlusion!procedure!every! day!for!5!consecutive!days.!For!each!individual!participant,!all!of!their!testing! sessions!were!conducted!at!the!same!time!every!day!to!maintain!consistency!and!to! limit!potential!diurnal!effects.!Participants!were!asked!to!refrain!from!physical! activity!or!eating!at!least!an!hour!before!each!testing!session.! 3.3.2$Anthropometric$measures$$ Height!and!weight!were!measured!with!participants!wearing!shorts,!tRshirts! and!no!shoes!using!a!stadiometer!(Tanita,!USA)!and!a!scale!(HealthRoRmeter,! Continental!Scale!Corporation,!USA),!respectively.!!One!skinfold!measure!was!taken! with!calipers!(John!Bull!Indicators,!England)!at!the!site!of!NIRS!sensor!placement!on! the!thigh!of!the!participant!(over!vastus!lateralis!muscle,!see!section!3.3.3)!to! measure!adiposity!at!the!site!of!measurement.!The!tested!leg!was!determined!as!the! lead!leg!used!to!step!up!onto!a!platform.!The!same!leg!was!tested!each!day!for!each! participant.!All!data!were!recorded!on!the!collection!form!(Appendix!4).! Heart!rate!was!measured!throughout!all!testing!sessions!for!safety!purposes! using!a!heart!rate!monitor!(Polar!model!H2)!secured!around!the!chest!at!the!level!of! the!xiphoid!process.!Heart!rate!data!throughout!the!entire!data!collection!procedure! was!recorded.!! 3.3.3$Tissue$oxygenation$measurement$$ Throughout!the!experimental!procedure!for!the!arterial!occlusion!test!(see! section!3.3.4)!a!continuous!wave!NearRInfrared!Spectroscopy!(NIRS)!monitor! (Portalite,!Artinis!Medical!Systems!BV,!The!Netherlands;!Oxysoft!version!2.1.6)!

measured!changes!in!concentrations!of:!oxyhemoglobin!(HbO2),!deoxyhemoglobin! (HHb),!total!hemoglobin!(tHb),!and!hemoglobin!difference!(HbDiff)!in!µM.!Total! Saturation!Index!(TSI)!was!expressed!as!a!ratio!of!HbO2!to!tHb.!The!data!were! collected!at!10Hz!with!a!differential!pathlength!factor!(DPF)!of!4!and!wavelengths! preset!at!760m!and!850nm.!! The!NIRS!sensor!was!placed!10R14cm!from!the!anterior!boarder!of!the! patella!of!the!test!leg!following!previously!accepted!protocols!(Belardinelli,!Barstow,! Porszasz,!&!Wasserman,!1995;!Bhambhani,!Maikala,!&!Esmail,!2001).!This! placement!was!marked!with!indelible!ink!while!the!participant!was!seated!with! their!knee!fully!extended!and!other!leg!placed!flat!on!the!ground.!The!NIRS!sensor! was!placed!over!the!vastus!lateralis!muscle!parallel!to!the!muscle!fibers!and!taped! down!using!athletic!tape.!It!was!then!covered!with!a!black!cloth!leg!band!and!then! covered!with!a!black!nylon!sleeve!(see!Appendix!5).!The!events!marked!in!the!NIRS! data!are!outlined!in!section!3.3.4.!! 3.3.4$Arterial$occlusion$test$$ Immediately!prior!to!all!arterial!occlusion!tests,!participant!resting!blood! pressure!taken!at!the!brachial!artery!was!recorded!using!a!stethoscope!(Almedic)! and!manual!arm!blood!pressure!cuff!(Almedic)!while!seated!in!a!chair.! !The!participant!then!lay!in!supine!on!a!gymnastics!mat!(1!inch!thick)!on!the! floor.!A!fitted!pressure!cuff!was!placed!around!the!test!thigh!just!proximal!to!the! NIRS!sensor!and!leg!was!supported!under!the!knee!by!a!rolled!towel!(see!Appendix! 5).!A!rapid!inflation!system!(E20/AG101!system,!Hokanson!Inc.,!Bellevue,!WA,!USA)! was!used!for!the!arterial!occlusion!test.!The!participant!was!instructed!to!lie!supine!

and!relaxed!(see!Table!1)!for!10!minutes!of!rest.!After!8!minutes!of!rest,!another! brachial!blood!pressure!measurement!was!recorded.!The!rapid!inflation!cuff! pressure!was!preset!to!250mmHg,!which!was!achieved!in!0.3!seconds!once!the! inflation!system!was!initiated.!The!data!acquisition!system!was!simultaneously! marked!with!Event!B!“Start!Occlusion”!(see!Table!1).!The!pressure!was!maintained! for!10!minutes!at!which!point!the!cuff!was!rapidly!autoRdeflated!in!0.3!seconds!and! the!NIRS!marker!Event!C!“Stop!occlusion”!(see!Table!1)!was!noted.!The!participant! was!instructed!to!lie!still!for!an!additional!10!minutes!of!postRocclusion!rest.! Following!10!minutes!of!rest!the!NIRS!marker!D!“End!test”!was!noted!and!Heart! Rate!recording!stopped.!! ! Table!1.!Experimental!Protocol!Events!with!Time,!Event!Label,!NIRS!Marker!and!HR! Marker!

Time*(min)* Event*Label** Details* NIRS*Marker** HR*Marker**

0!R10! Start!Rest!! Supine,!quiet! A! 1! 10R20! Start!Occlusion!! Arterial!occlusion! B! 2! 20R30! Stop!Occlusion!! Reperfusion! C! 3! 30! End!Test!! Sit!upright,!stand! D! 4! ! 3.4* Data*processing*and*analysis* * 3.4.1$ Data$processing$** * The!blood!pressure!measures!taken!after!8!minutes!of!rest!were!calculated! into!mean!arterial!pressure!(MAP)!using!the!formula:!! MAP=diastolic+(0.33*(systolicRdiastolic))! The!NIRS!data!were!averaged!over!each!second!and!separated!into!sections! representing!the!events!of!Rest,!Occlusion,!and!Recovery.!The!time!constant!of! Recovery!tau!(τ)!(seconds)!was!the!time!in!seconds!it!took!for!the!NIRS!variables!

(TSI,!HbO2,!HHb,!tHb)!to!reach!63%!of!each!event!section.!Tau!was!calculated!post! test!for!each!of!three!periods:!occlusion,!reperfusion,!and!recovery.!In!the!context!of! this!study!tau!was!considered!the!time!constant!of!response.!As!indicated!in!Table!1,! occlusion!was!defined!as!the!10!minutes!from!the!beginning!of!cuff!inflation!(Event! B)!to!the!cuff!deflation!(Event!C)!and!reperfusion!was!defined!as!the!time!from!cuff! deflation!(Event!C)!to!reperfusion!maximum!(minimum!for!HHb)!value.!The! reperfusion!maximum!value!was!found!for!each!variable!(TSI,!HbO2,!HHb,!tHb).! Recovery!was!defined!as!the!time!from!reperfusion!maximum!value!to!the!end!of!the! test!(Event!D).!! Examples!of!the!3!equations!to!derive!τ:!! 1.!τ!Occlusion=!rest!avgR((rest!avgRocclusion!2!min!plateau)*0.63)! 2.!τ!Reperfusion!=!((Max!ReperfusionRocclusion!2!min!plateau)*0.63)+occlusion!2! min!plateau! 3.!τ!Recovery!=!Max!ReperfusionR((Max!ReperfusionRRecovery!2!min!plateau)*0.63! The!occlusion!2Rminute!plateau!was!calculated!as!an!average!over!the!last!2! minutes!of!occlusion!(minutes!18R20!respectively).!Recovery!2Rminute!plateau!was! calculated!as!an!average!of!the!last!2!minutes!of!recovery!(minutes!28R30! respectively).! ! Magnitude!of!change,!referred!to!as!“range”!in!TSI,!HbO2,!HHb,!tHb,!HbDiff! throughout!each!of!the!events,!was!determined!as!the!difference!between!a!given! start!and!end!value.!Four!range!values!were!calculated:! 1.!Rest!Average!–!Occlusion!2!min!plateau!! 2.!Occlusion!2!min!plateau!–!Max!Reperfusion!(Minimum!for!HHb)!

3.!Max!Reperfusion!–!Recovery!Average! 4.!Recovery!Average!–!Occlusion!2!min!plateau!!! 3.4.2$Statistical$analyses$ A!multivariate!twoRway!repeated!measures!analysis!of!variance!was! performed!to!test!for!the!main!effect!of!time!(days!1,!3!&!5),!group!(test!vs.!control)! and!the!interaction!between!time!and!group!for!each!of!the!NIRS!variables!(TSI,! HbO2,!HHb,!tHb,!HbDiff):! 1.!Rest!average!(calculated!from!last!2!minutes!of!rest,!8R10!respectively)!! 2.!τ!for!Occlusion! 3.!Occlusion!2Rminute!plateau! 4.!τ!for!Reperfusion! 5.!Reperfusion!maximum!! 6.!τ!for!Recovery! 7.!Recovery!2Rminute!plateau! 8.!Four!ranges! Mean!and!standard!deviation!(SD)!are!reported!for!all!variables.!The! significant!alpha!level!was!set!at!0.05!(95%!confidence!interval).!SPSS!(IBM!SPSS! Statistics!for!Windows,!Version!23.0.!Armonk,!NY:!IBM!Crop)!was!used!for!all! statistical!analyses.!! ! * * *

CHAPTER*4*RESULTS* 4.1*Participant*physical*characteristics** The!physical!characteristics!of!both!groups!are!shown!in!Table!2.! Participants!in!the!test!and!control!groups!had!an!average!age!of!23!(±2)!and!21! (±1.6)!years,!respectively.!There!were!no!significant!differences!between!groups! with!regard!to!height,!weight,!and!test!leg!NIRS!placement!side!(vastus!lateralis)! skinfold.!Resting!mean!arterial!pressure!(MAP)!was!compared!between!groups!and! across!days!1,!3!&!5.!No!significant!differences!existed!between!groups!or!over!days! for!MAP.!The!results!of!each!group’s!average!MAP!for!days!1,!3!&!5!are!shown!in! Figure!5.!* Table!2.!Physical!Characteristics!of!Test!and!Control!Group!Participants! ! Test*Group=12* Control*Group=12* * ! Mean* SD* Mean* SD* p* Height*(cm)* 169.5! 4.8! 168.3! 4.8! .549! Weight*(kg)* 65.8! 14.4! 63.0! 9.9! .583! Test*leg*Vastus*Lateralis* skinfold*(mm)* 27.9! 3.3! 26.7! 3.2! .384! MAP*Day*1* 86.2! 10.6! 90.7! 8.0! .253! MAP*Day*3* 85.5! 8.7! 90.0! 6.4! .180! MAP*Day*5* 86.0! 8.8! 85.5! 4.8! .842! * ! ! ! ! ! ! ! Figure!5.!Average!Mean!Arterial!Pressure!for!Test!and!Control!Groups!on!days!1,!3!&! 5.!! 60! 70! 80! 90! 100! 1! 3! 5! M ea n *A rt er ia l*P re ssu re * Days* Test!Group!! Control!Group!!

4.2*NIRS*Results** 4.2.1$Hemoglobin$variables$main$effects$* There!were!no!significant!main!effects!for!group!or!day,!nor!any!significant! interaction!for!the!following!hemoglobin!chromophore!measures:!! HbO2:!rest!average,!occlusion!tau,!occlusion!2Rminute!plateau,!reperfusion! tau,!reperfusion!max,!recovery!tau,!recovery!2Rminute!plateau,!range!1R4.!! HHb:!rest!average,!occlusion!tau,!occlusion!2Rminute!plateau,!reperfusion! tau,!reperfusion!max,!recovery!tau,!recovery!2Rminute!plateau,!range!1R4.! tHb:!rest!average,!occlusion!2Rminute!plateau,!reperfusion!tau,!reperfusion! max,!recovery!tau,!recovery!2Rminute!plateau,!range!2R4.!! HbDiff:!rest!average,!occlusion!tau,!occlusion!2Rminute!plateau,!reperfusion! tau,!reperfusion!max,!recovery!tau,!recovery!2Rminute!plateau,!range!1R4.! TSI:!rest!average,!occlusion!tau,!occlusion!2Rminute!plateau,!reperfusion!tau,! reperfusion!max,!recovery!tau,!recovery!2Rminute!plateau,!range!1R4.! There!was!a!significant!day!main!effect!of!tHb!for!occlusion!tau,!which! decreased!from!day!3!(Mean:!249.42,!SD:!158.24)!to!5!(Mean:!184.33,!SD:!113.96).!! As!well,!a!significant!day!main!effect!for!tHb!range!1!(rest!averageRocclusion!2! minute!plateau)!was!observed!indicating!an!increase!from!day!3!(Mean:!4.29,!SD:! 2.59)!to!day!5!(Mean:!5.42,!SD:!3.14).!!However!there!were!no!group!differences!nor! were!there!any!significant!group!by!day!interactions.! 4.2.2$Response$pattern$$ For!all!participants,!the!NIRS!response!pattern!to!rest,!occlusion!and! recovery!was!consistent!for!all!testing!days!regardless!of!group!(Figure!6).!The!

response!to!the!10Rminute!rest!period!was!consistent!and!demonstrated!stable! measures!for!all!participants!on!all!days.!The!response!pattern!to!the!occlusion! initiated!by!the!rapid!cuff!inflation!was!as!follows:!HbO2,!tHb,!HbDiff,!and!TSI! decreased!then!slowly!plateaued!over!the!10!minutes!while!HHb!increased!initially! and!then!plateaued.!Following!the!rapid!cuff!deflation,!HbO2,!tHb,!HbDiff!and!TSI! increased!to!their!maximum!reperfusion!levels!and!then!plateaued!over!the! remainder!of!the!10!minutes!of!recovery.!During!that!same!period,!HHb!decreased! initially!upon!reperfusion!and!then!plateaued.!This!response!pattern!was!consistent! for!all!participants!for!all!testing!days!regardless!of!group.! ! ! ! ! Figure!6.!Hemoglobin!chromophore!(TSI,HbO2,!HHb,!tHb,!HbDiff)!response!pattern! of!a!representative!participant!during!rest,!occlusion,!and!recovery!of!a!single! testing!session.!! $ 4.2.3$Daily$variation:$HbO2$and$HHb$ The!HbO2!and!HHb!averages!of!both!Test!and!Control!Groups!during!rest,! occlusion!2Rminute!plateau,!and!recovery!2Rminute!plateau!are!provided!graphically! R30! R25! R20! R15! R10! R5! 0! 5! 10! R25! R5! 15! 35! 55! 75! 95! 0! 200! 400! 600! 800! 1000! 1200! 1400! 1600! 1800! Co n ce n tr at io n *C h an ge *μ *M*** T issu e* Sa tu ra ti on *In d ex *( % )* Time*(s)* * TSI! HbO2! HHb! tHb! HbDiff!!

in!Figures!7ARD.!The!graphs!represent!the!consistency!within!and!between!groups,! as!there!were!no!significant!group!or!day!main!effects!observed.!Similar!graphs!for! the!consistency!of!HbO2!tau!results!are!provided!in!Appendix!7!(Figures!8!and!9).!!! ! A.! ! B.! ! R16! R14! R12! R10! R8! R6! R4! R2! 0! 2! 4!

Rest! Occlusion! Recovery!!

Hb O2 *C on ce n tr at io n *C h an ge ** μM* ** Test*Group*(Average*Over*Last*2*Minutes)* Day!1! Day!3! Day!5! R16!R14! R12! R10!R8! R6!R4! R2!0! 2! 4!

Rest!! Occlusion! Recovery!!

Hb O2 *C on ce n tr at io n *C h an ge * μM* * Conrol*Group*(Average*Over*Last*2*Minutes)* Day!1! Day!3! Day!5!

C.! ! D.! ! ! Figure!7aOd.!Consistency!of!HbO2!and!HHb!response!pattern!in!test!and!control! groups.! ! The!tHb!response!to!the!onset!of!occlusion!was!not!as!consistent!as!the!other! hemoglobin!variables!(Appendix!6).!At!onset!of!occlusion,!4!of!12!Test!group! participants!experienced!an!increase!in!!tHb!!which!then!plateaued!on!one!or!more! days!of!testing.!In!the!control!group,!4!of!12!participants!also!showed!an!increase!of! tHb!then!a!plateau.!The!control!group!increases!were!all!isolated!to!day!1!of!testing.! The!occurrence!of!tHb!increase!at!occlusion!onset!did!not!occur!in!a!particular! R4! R2! 0! 2! 4! 6! 8! 10!

Rest!! Occlusion! Recovery!!

H H b *C on ce n tr at io n *C h an ge *** μ*M* * Test*Group*(Average*Over*Last*2*Minutes)** Day!1! Day!3! Day!5! R3! R2! R1!0! 1! 2! 3! 4! 5! 6! 7! 8!

Rest!! Occlusion! Recovery!!

H H b *C on ce n tr at io n *C h an ge ** μ*M* ** Control*Group*(Average*Over*Last*2*Minutes)* Day!1! Day!3! Day!5!

pattern.!In!the!remainder!of!participants!in!both!groups!tHb!decreased!(following! the!path!of!HbO2)!then!plateaued!during!the!remainder!of!occlusion.!! 4.2.4$Exception$to$response$pattern:$TSI$! In!the!majority!of!participants!across!both!groups!and!throughout!all!testing! days,!TSI!overshot!during!reperfusion!then!plateaued!to!recovery!average! (calculated!as!an!average!of!the!last!2!minutes!of!recovery).!However,!this!overshoot! was!not!present!in!5!participants!in!the!test!group!for!one!or!more!days.!The! overshoot!was!also!not!observed!in!6!control!group!participants!and!of!those!6! participants,!4!showed!the!lack!of!overshoot!on!two!or!more!days.!The!lack!of! reperfusionRinduced!overshoot!did!not!appear!in!any!particular!pattern!(see!Table!4! in!Appendix!8).!! ! * * * * * * * * * * *

CHAPTER*5*DISCUSSION* ! This!study!examined!the!reliability!of!NIRS!chromophores!during!vascular! occlusion!of!the!thigh!repeated!daily!over!5!consecutive!days.!The!findings!influence! the!understanding!of!NIRS!measurement!with!vascular!occlusion!and!have! important!implications!for!exercise!research!that!utilizes!both!occlusions!along!with! exercise!tests.!!! 5.1*NIRS*response*pattern** ! The!response!pattern!to!the!vascular!occlusion!test!was!consistent!across! days!1,!3!and!5!both!within!and!between!test!and!control!groups.!There!were!no! significant!differences!between!days!or!between!test!and!control!groups!with!regard! to!the!response!pattern!of!hemoglobin!variables!to!vascular!occlusion!of!the!thigh.! These!results!support!the!null!hypotheses!1!&!2!(see!section!1.4).!The!response! pattern!demonstrated!in!this!study!is!consistent!with!results!of!previous!research! (Gomez!et!al.,!2008;!Lacroix!&!Gremeaux,!2012;!Shadgan!et!al.,!2012;!Shibuya!&! Tanaka,!2004).!However,!these!studies!examined!the!hemoglobin!variable!response! pattern!to!a!single!testing!session!of!occlusion.!The!present!study!provides!evidence! of!the!consistency!of!this!pattern!over!multiple!days!of!occlusion.!! ! The!lack!of!circadian!variability!variation!(compared!on!day!1,!3!&!5)!in! response!pattern!to!vascular!thigh!occlusion!demonstrated!in!this!study!indicates! that!there!is!no!shortRterm!effect!of!repeated!occlusions.!Although!previous!research! has!shown!that!there!is!a!change!in!response!pattern!of!occlusion!when!examining! the!difference!over!extended!periods!of!time,!the!duration!was!substantially!longer! (6!months)!than!that!of!the!current!study!(Gomez!et!al.,!2008).!The!response!pattern!

change!observed!by!Gomez!et!al.!(2008)!was!for!the!HHb!desaturation!rate.!Perhaps! the!change!in!hemoglobin!variables!response!to!occlusion!does!not!appear!within!a! week,!but!variation!occurs!with!longer!change!of!time.!! ! The!significant!day!main!effect!on!tHb!occlusion!tau!from!day!3!to!5!may!have! been!impacted!by!the!observed!variability!in!tHb!occlusion!response!pattern! (Appendix!6).!This!finding!is!similar!to!that!reported!previously!where!a!lack!of! consistency!in!tHb!at!onset!of!occlusion!has!been!demonstrated!in!other!occlusion! studies!(Shadgan!et!al.,!2012).!The!current!study!supports!the!earlier!findings!that! demonstrate!the!lack!of!consistency!in!response!pattern!of!tHb!to!vascular!leg! occlusion.!While!Shadgan!et!al.!(2012)!reported!similar!variability!within!a!single!!! surgical!occlusion!(average!43!minute);!the!present!study!showed!that!tHb! occlusion!tau!is!inconsistent!across!days!as!well!as!within!and!between!participants.! This!could!be!due!to!minor!changes!in!HbO2!and!HHb!that!are!not!apparent!when! examining!the!individual!variables,!but!become!more!significant!when!examining! them!added!together,!as!is!the!case!of!tHb.!! ! The!main!effect!of!day!on!range!1!(rest!averageRocclusion!2Rminute!plateau)! demonstrated!an!increase!from!day!3!to!5.!However,!this!result!was!most!likely! impacted!by!the!inconsistency!in!the!tHb!occlusion!response.!The!change!of! occlusion!tau!impacts!the!concentration!change!of!occlusion!2Rminute!plateau! therefore!impacting!the!range!calculation.!! ! tHb!is!a!valuable!measure!to!examine!during!occlusion!as!it!can!indicate! blood!flow!to!the!limb.!However,!the!inconsistency!in!response!to!vascular!occlusion!

within!a!participant!and!within/between!days!makes!it!unreliable!as!a!primary! measure!during!occlusion.!! 5.1.1$Tissue$Desaturation$ The!current!study!did!not!find!any!significant!differences!with!regard!to!the! rate!of!desaturation!for!any!of!the!hemoglobin!variables!when!comparing!over!days! 1,!3!and!5!or!between!groups.!!Therefore!the!null!hypothesis!(#3),!which!stated!that! the!rate!of!response!of!hemoglobin!measures!during!onset!of!rapid!occlusion!does! not!change!from!day!1,!3!to!5,!was!accepted.! The!rate!at!which!HbO2!desaturates!following!vascular!occlusion!has!been! commonly!used!as!a!measure!of!oxygen!consumption!within!the!interrogated!tissue! (Van!Beekvelt!et!al.,!2001).!The!consistency!of!the!occlusionRinduced!HbO2! desaturation!over!the!5!consecutive!days!of!testing!implies!that!the!rate!of!oxygen! consumption!within!the!interrogated!vastus!lateralis!muscle!was!unchanged!across! all!5!days!of!testing.!Therefore,!despite!the!repeated,!daily,!vascular!occlusions,!the! stability!of!HbO2!desaturation!rate!indicates!that,!at!least!at!rest,!the!metabolic!rate! of!the!tissue!remains!consistent.!This!result!has!implications!for!exercise!studies! post!occlusion,!as!the!metabolic!rate!of!the!skeletal!muscle!does!not!appear!to! change.!! The!consistency!in!desaturation!rate!and!occlusion!plateau!suggests!that! there!were!no!changes!in!the!arteriole!dilation!(and!vascular!resistance)!or!of! vasodilator!metabolites,!such!as!nitric!oxide,!over!the!5!days!of!occlusion!tests.!This! would!have!been!shown!by!an!increase!in!desaturation!rate!and!a!decrease!in! occlusion!plateau!over!the!week!of!testing.!The!consistency!in!dayRtoRday!

hemoglobin!chromophore!values!during!vascular!occlusion!is!likely!also!responsible! for!the!consistency!observed!in!the!reperfusion!and!hyperemic!responses!post! occlusion.! 5.1.2$Hemoglobin$resaturation$ ! The!hemoglobin!resaturation!rate!immediately!following!the!rapid!cuff! deflation!was!consistent!from!day!1,!3!to!5!within!and!between!groups,!supporting! the!null!hypothesis!#4!(see!section!1.4).!There!were!no!changes!to!the!vascular! recovery!response!over!the!5!consecutive!days!of!occlusion.!Reperfusion!rate!is! representative!of!endothelial!vasodilation!function!(Gerovasili!et!al.,!2010).!Since!no! changes!in!resaturation!rates!or!hyperemic!responses!were!found!over!the!5!days!of! testing,!it!is!assumed!that!endothelial!vasodilation!function!and!vascular!resistance! do!not!change!despite!the!daily!vascular!occlusion!tests.!Appell!et!al.!(1993)! demonstrated!that!endothelial!basement!membranes!thicken!during!ischemia,! perhaps!as!a!protective!measure!for!increasing!pressure!during!reperfusion.! Therefore,!while!the!current!study!found!no!difference!in!hemoglobin!resaturation! rate,!as!measured!by!changes!in!HbO2,!shortRterm!residual!changes!to!the!occluded! tissue’s!microvasculature!may!exist.!An!increase!in!arteriole!dilation,!and!therefore! decrease!of!vascular!resistance,!could!exist!especially!while!vasodilator!metabolites! such!as!nitric!oxide!are!being!flushed!out!of!the!tissue!during!recovery.!These! potential!residual!changes!in!the!microvasculature!could!impact!the!performance!of! exercise!immediately!following!vascular!occlusion!and!should!be!taken!into!account! when!designing!methodologies!that!use!occlusion!before!exercise!tests.!!