Carbon Monoxide For Therapy?The Protective Characteristics of Carbon Monoxide Used to Treat Ischemia Reperfusion Injury

Joshua A. Mickle

Advisor:Prof. Jennifer Hancock

Tuesday, March 30th, 2010

BackgroundIschemia reperfusion injury (IRI)

◦Damage caused by the occlusion and subsequent return of blood supply to tissue

http://hyperbaricworx.com.au/images/How2.png

Why does reperfusion cause damage?

Combination of factors◦Local and systemic inflammatory

response Leukocyte activation Adherence to vessel walls Transmigration to interstitial space Increased edema, clotting, permeability

Collard & Gelman, 2001, 1134

Why does reperfusion cause damage?

Ischemic conditions - Anaerobic Metabolism

Oxidative enzymes produce Reactive Oxygen

Species (ROS)

ROS damage cell membranes - lipid

peroxidation

Cell Death

ReperfusionLarge influx of O2

What is Carbon Monoxide (CO)?Colorless, odorless, poisonous gas

◦CO greater affinity than O2 for Hemoglobin

◦Hb Unable to deliver O2

◦Disrupts cell respiration and triggers hypoxia

http://www.jems.com/Images/CO_Fig1_tcm16-186547.jpg

Why use CO for treatment?

CO produced naturally in bodyHeme Oxygenase-1 system (HO-

1)◦ HO-1 converts heme molecule into

CO, iron, and biliverdin

http://www.bioscience.org/2007/v12/af/2130/figures.htm

Why use CO for treatment?Oxidative stress shows increase

in HO-1◦Leads to increase in CO

concentration in blood◦Possibly a mechanism of protection

against reactive oxygen speciesAvailabilityRelatively well-understood

toxicologyLOW CONCENTRATIONS

Current Treatments for IRIIschemic preconditioningTherapeutic hypothermiaAnti-leukocyte therapyIntroducing radical scavengers

and antioxidantsTreatment of symptoms following

damage

HypothesisCO therapy is a viable option for

further investigation and eventual use on humans alongside or replacing the current treatment options for IRI.

CO Treatment Following StrokeStroke induced on rats

◦(Zeynalov E, Dore`, 2008)◦90 min. occlusion of Middle Cerebral

Artery

Moore & Dalley, 2006, 929

CO TreatmentDuring reperfusion

◦subjected to various concentrations of CO

◦exposed at 0, 1, or 3 hrs following reperfusion Air

At 0h125ppm

At 0h250ppm

At 0h

250ppmAt 1h

250ppmAt 3h

All for 18h of total exposure

Analysis of RecoveryNeurological Deficit Scores (NDS)

◦Conducted at: Onset of reperfusion 24hrs 48hrs

◦Scores range from 0 – 4Scor

eDescription

0 Normal motor function

1 Flexion of torso and contralateral forelimb when held by tail

2 Circling to the contralateral side, but normal posture at rest

3 Leaning to the contralateral side at rest

4 No spontaneous movement

Good

Bad

Results

Hours after Reperfusion

Neurological Deficit Score

0ppm CO

125ppm CO

250ppm CO

250ppm CO

0h 2 2 2 2

24h 2 1.5 2 1

48h 2 2 1 1

Also AnalyzedBrain tissue

◦Sliced into 2mm sections◦Stained to show metabolic activity

Posterior

Anterior

Brain Edema◦Ipsilateral and contralateral

hemispheres separated◦% Brain Water = [(Wet – Dry)/Wet] x

100

Also Analyzed

Not only in the BrainCO treatment in rat heart

transplant model◦Akamatsu et al., 2004

Comparison of CO therapy during reperfusion to:◦HO-1 activation◦Air inhalation

ProceduresHeart transplant

◦Success/failure – % survivalCO Exposure

◦Varying exposure to donor, graft, and/or recipient

◦400ppm

• Induces HO-1 expressionCobalt

protoporphyrin (CoPPIX)

• Reverses protective effect of HO-1

Zinc protoporphyrin

(ZnPPIX)

Reagents and Results

ResultsHighest survival rate

◦Donor and graft exposure◦Donor, graft, and recipient exposure

Interesting to note◦Donor and Recipient exposure only

Other studiesSimilar results in liver and lung

grafts◦Both in vivo and ex vivo◦(Amersi et al., 2002; Kohmoto et al.,

2006)Upregulation of HO-1 system

◦Increases CO endogenously◦(Amersi et al., 1999)

Current goal◦Investigate mechanisms behind

these results

Limitations to current studiesSmall sample sizesSmall animal models (rats and

mice)Performed only on healthy

specimensAlthough better, still low survival

rate◦Need to improve before human trials

Long term effectsDosage and duration of CO

treatment

Contradiction among studiesMechanisms of action

◦Anti-inflammatory◦Anti-apoptotic◦Other HO-1 byproducts

Conclusions & Implications

Compared to no IRI treatment:◦CO therapy shows significantly

improved tissue functioningPromising for future treatments

of IRIMust further understand

mechanism before clinical implementation

References Akamatsu Y, Haga M, Tyagi S, Yamashita K, Graca-Souza AV,

Ollinger R, Czismadia E, May GA, Ifedigbo E, Otterbein LE, Bach FH, Soares MP. 2004. Heme- oxygenase-1-derived carbon monoxide protects hearts from transplant-associated ischemia reperfusion injury. The FASEB Journal. 18: 771-782.

Amersi F, Shen X, Anselmo D, Melinek J, Iyer S, Southard D J, Katori M, Volk H, Busuttil R W, Buelow R, Kupiec-Weglinski J W. 2002. Ex-vivo exposure to carbon monoxide prevents hepatic ischemia/reperfusion injury through p38MAP Kinase pathway. Hepatology. 35(4): 815-823.

Amersi F, Buelow R, Kato H, Ke B, Coito AJ, Shen X, Zhao D, Zaky J, Melinek J, Lassman CR, Kolls JK, Alam J, Ritter T, Volk HD, Farmer DG, Ghobrial RM, Busuttil RW, Kupiec-Wegelski JW. 1999. Upregulation of heme oxygenase-1 protects genetically fat Zucker rat livers from ischemia/reperfusion injury.

The Journal of Clinical Investigation. 104(11): 1631-1639. Kohmoto J, Nakao A, Kaizu T, Tsung A, Ikeda A, Tomiyama K, Billiar

TR, Choi AMK, Murase N, McCurry KR. 2006. Low-dose carbon monoxide inhalation prevents ischemia/reperfusion injury of transplanted rat lung grafts. Surgery. 140(2): 179-185.

Zynalov E, Doré S. 2009. Low doses of carbon monoxide protect against experimental focal brain ischemia. Neurotoxicity Research. 15(2): 133-137.

AcknowledgementsProf. HancockDr. BrownCapstone ClassmatesTeammates

Questions?