Does Early Inhibition of Interleukin‐1 and Interleukin‐18, Adds to The Cardioprotection as Seen with Delayed Interleukin Inhibition Given at Reperfusion in Donation after Circulatory Death Murine Heart Model?
Authors: Quader Mohammed , Mezzaroma Eleonora , Kenning Kristine , Toldo Stefano 
Published: April 2020

BackgroundDonation after circulatory death (DCD) donors can expand the heart donor pool for transplantation, which primarily is dependent on donation after brain death (DBD) donors. Inherent to the DCD process is ischemia and reperfusion injury, predominantly mediated by interleukin‐1 (IL‐1) and IL‐18, the downstream mediators of inflammasome.HypothesisPharmacologic blockage of IL‐1 or IL‐18 with recombinant IL‐1 receptor antagonist (IL‐Ra) or IL‐18 binding protein (IL‐18 BP) prior to initiation of DCD process is superior to the myocardial protection with IL‐1 or IL‐18 blockade given at reperfusion alone.MethodsFollowing clinical protocol, DCD mice model was developed with in‐situ warm ischemia time maintained at 40 minutes. Mice (strain C57bl6/j) were randomly assigned to DCD control, DCD IL‐1Ra early and late or DCD IL‐18 BP early and late groups (n = 8–10 each). Pharmacologic inhibition of IL‐1 and IL‐18 was obtained with recombinant IL‐1Ra or IL‐18BP, respectively. Anesthetized mice were ventilated while monitoring heart with EKG and echocardiography. After muscle paralysis with vecuronium, ventilatory support was terminated and cardiac asystole observed. Hearts were procured and reanimated for 90 minutes on a Langendorff system with Krebs Henseleit (KH) buffer at 37°C. Physiologic parameters including heart rate, perfusion rate, developed pressure (DP), +/− dP/dt were obtained at 15‐minute intervals. After reanimation, hearts were collected for molecular and histological analysis. In early inhibition groups IL‐1 receptor antagonist (10 mg/kg) or IL‐18 binding protein (1 mg/kg) were given intraperitoneally, 30 minutes prior to DCD process (termination of ventilation). In the late inhibition groups IL‐1Ra and IL‐BP, 1μg/ml and 1μg/ml respectively were added to KH buffer. Coronary sinus samples were analyzed for cardiac troponin‐I (cTnI) levels.ResultsHeart rate and perfusion flow rates were comparable in all groups. In IL‐1 inhibition group, myocardial contractility was better (improved developed pressure, dP/dt compared to control group, however the differences attained statistical significance for DP and −ve dP/dt only). No additional incremental benefit was noted with early inhibition of IL‐1. In IL‐18 group, there was better physiologic function of heart compared to control (DP, +/−dP/dt and rate pressure product, all P = <0.05). Compared to late IL‐18 inhibitor group the early IL‐18 inhibitor group did not have significant improvements in myocardial function parameters. Markers of cell injury were significantly better with IL‐18 inhibition compared to control and IL‐1 inhibition groups.ConclusionPharmacologic blockade of IL‐1 or IL‐18 is protective to the DCD mice hearts, with better protection with IL‐18 inhibition compared to IL‐1 inhibition. Early treatment with IL‐1Ra or IL‐18BP did not afford any additional benefits compared to what is observed with late inhibition given at reanimation. Our study findings are relevant to the clinical practice as only late interventions are allowed in clinic DCD process.Support or Funding InformationWork supported by Merit Review Award and American Association grant to Dr. Quader

DCD
Control

DCD
IL‐1 Inhibition

DCD
IL‐18 Inhibition

N=10

Early
IL‐1Ra
N = 13

Late
IL‐1Ra
N = 8

Early
IL‐18BP
N = 8

Late
IL‐18BP
N=9

Functional Data

Heart Rate b/min mean±SEM

396±14

418±13

401±15

445±20

440±10

Perfusion rate ml/min

1.8±0.1

2.2±0.1

2.2±0.3

2.8±0.2

2.4±0.1

Developed pressure mmHg mean±SEM

78.7±5.8

99.3±6.5
#

94.1±9.3

99.5±8.1
#

108.3±6.5
#

Rate Pressure Product

29,233±3,293

37,344±3,005

34,198±3,634

41781±4327
#

43082±3191
#

Rate of positive developed pressure dt/dp mmHg/ms mean±SEM

3256±266

3595±481

3272±695

4923±44
#

4744±204
#

Rate of negative developed pressure −dt/dp mmHg/ms mean±SEM

−2278±18

−2991±19
#

−2992±28
#

−3256±29
#

−3632±29
#

Makers of myocyte damage

Eluate troponin level ng/ml

7.79±0.98

6.16±1.12

7.68±0.85

4.21±0.67
#

3.82±0.65
#

TUNEL assay

2.09±0.53

2.13±0.54

2.02±0.76

0.66±0.18
#

0.99±0.23

DCD = Donation after Circulatory Death, IL‐IRa = IL‐1 receptor antagonist, IL‐18 BP IL‐18 receptor binding protein, SEM = Standard Error of Mean,
#P<0.05 vs DCD control