a b s t r a c t

Purpose: To assess whether use of Impedance Threshold Device (ITD) during cardiopulmonary resuscitation (CPR) reduces the degree of post-cardiac arrest Acute kidney injury , as a result of improved hemodynam- ics, in a porcine model of ventricular fibrillation (VF) cardiac arrest.

Methods: After 8 min of untreated cardiac arrest, the animals were resuscitated either with active compression- decompression (ACD) CPR plus a sham ITD (control group, n = 8) or with ACD-CPR plus an active ITD (ITD group, n = 8). Adrenaline was administered every 4 min and electrical defibrillation was attempted every 2 min until return of spontaneous circulation (ROSC) or asystole. After ROSC the animals were monitored for 6 h under gen- eral anesthesia and then returned to their cages for a 48 h observation, before euthanasia. Two novel biomarkers, Neutrophil gelatinase-associated lipocalin in plasma and Interleukin-18 (IL-18) in urine, were mea- sured at 2 h, 4 h, 6 h, 24 h and 48 h post-ROSC, in order to assess the degree of AKI. Results: ROSC was observed in 7 (87.5%) animals treated with the sham valve and 8 (100%) animals treated with the active valve (P = NS). However, more than twice as many animals survived at 48 h in the ITD group (n = 8, 100%) compared to the control group (n = 3, 37.5%). Urine IL-18 and plasma NGAL levels were augmented post- ROSC in both groups, but they were significantly higher in the control group compared with the ITD group, at all measured time points.

Conclusion: Use of ITD during ACD-CPR improved hemodynamic parameters, increased 48 h survival and de- creased the degree of post-cardiac arrest AKI in the Resuscitated animals.

(C) 2017

Introduction

cardiac arrest constitutes a major medical problem with sub- stantial socio-economic implications. It is estimated that every year about 350,000-700,000 people suffer CA in Europe, both in hospital and out of hospital [1,2], and the prognosis of the victims remains

* Corresponding author at: 3A Parou st, Melissia, Athens 15127, Greece.

E-mail addresses: [email protected] (P. Niforopoulou), [email protected]

(N. Iacovidou), [email protected] (P. Lelovas), [email protected] (G. Karlis), [email protected] (? Papalois), [email protected] (S. Siakavellas), [email protected] (V. Spapis), [email protected] (G. Kaparos), [email protected] (I. Siafaka), [email protected] (T. Xanthos).

ominous despite the hard efforts of international organizations, such as the European Resuscitation Council (ERC) and the International Liaison Committee on Resuscitation , over the last three decades [3]. Even when cardiopulmonary resuscitation (CPR) is initially success- ful and the victim achieves Return of Spontaneous Circulation (ROSC), long term survival and quality of life are not guaranteed. Survival to hos- pital discharge ranges from 9.5% for out-of-hospital cardiac arrest (OHCA) victims to 24.2% for in-hospital cardiac arrest victims. Of the survivors, 40-50% remains with Cognitive impairments, such as memory and intellectual performance deficits [4]. In severe cases, the patients present a persistent comatose state, becoming completely dependent; others minimally regain consciousness or remain in a vege- tative state; and few come out of coma Neurologically intact [5]. The

http://dx.doi.org/10.1016/j.ajem.2017.01.040

0735-6757/(C) 2017

term “post-cardiac arrest syndrome” describes the myocardial, neuro- logic and other vital organs‘ (lungs, kidneys, liver) dysfunction, causing significant morbidity and mortality after ROSC. The mechanisms underlying this syndrome probably involve a whole-body ischemia and reperfusion injury that triggers a systemic inflammatory response [6-10].

These disappointing statistics can be attributed to several factors, such as the long time interval between the occurrence of CA and the initiation of CPR, the low quality CPR often performed by rescuers [11-13] and the inherent inability of classical CPR to supply vital organs with adequate blood flow during CA. During standard closed chest Manual CPR the venous return from the periphery to the chest during decompression is inadequate. This is because a significant percentage of the negative intrathoracic pressure produced during decompression of the thorax is lost by the influx of inspiratory gases. Without adequate venous return to the heart, the pump will be empty during the subse- quent compression phase and, therefore, Blood supply to the vital organs will be minimized. To be more specific, standard CPR only pro- vides 10-20% of normal blood flow to the heart and 20-30% of normal blood flow to the brain [14-16].

In order to enhance CPR efficiency numerous mechanical devices

have been developed [17]. The Impedance Threshold Device (ITD) is a small valve with a silicon diaphragm that can be attached to an endotra- cheal tube, a supraglottic device or a face mask. It is designed to selectively impede inspiratory gas flow into the patient during the de- compression phase, whereas it allows both active patient ventilation and passive exhalation. This way it enhances negative intrathoracic pressure and increases venous return to the heart and, therefore, cardiac output [18-22]. Numerous experimental and clinical studies have proved its value; ITD improves hemodynamics, perfusion of vital or- gans, ROSC, short-term survival, ICU admissions, survival to hospital discharge and long term survival with favorable neurologic function [14,20,23-51]. However, none of the above studies has investigated the effect of ITD use during CPR on the degree of post-CA Acute Ischemic injury of other vital organs, except the myocardium and the brain. The aim of the present study is to assess whether use of ITD during CPR re- duces the degree of post-CA Acute kidney injury in a porcine model of VF cardiac arrest. Our hypothesis was that if ITD use improves hemodynamics during CPR, blood supply to the kidneys would be im- proved and, therefore, the degree of post-CA AKI would be lessened. In order to assess the degree of AKI two novel biomarkers were mea- sured; Neutrophil Gelatinase-Associated Lipocalin (NGAL) in plasma and Interleukin-18 (IL-18) in urine.

Materials and methods

After approval of the General Directorate of Veterinary Services of Prefecture of Athens, Attica, Greece (permit no. 2980/9-5-2012), 16 fe- male Landrace/Large-White piglets, aged 10-15 weeks, with an average weight of 19 +- 2 kg, all from the same breeder were studied. Before any procedure, animals were randomized into two groups with the use of sealed envelopes indicating the animals’ assignment to either the ac- tive/functional ITD group (ITD group, n = 8) or the sham/nonfunctional ITD group (control group, n = 8). Sham ITDs were externally identical to the active ITDs, but internally were modified; the silicon diaphragms were removed so as the devices functioned as hollow conduits. This way, the investigators were blinded to the device function (active or sham).

Preparatory phase

The animals were prepared in a standardized fashion previously de- scribed [52], at ELPEN pharmaceuticals Experimental-Research Centre (Pikermi, Attica, Greece). Premedication was achieved by IM injection of ketamine hydrochloride (10 mg/kg), midazolam (0.5 mg/kg) and at- ropine (0.05 mg/kg). Anesthesia was induced with propofol (2 mg/kg)

via the marginal auricular vein. Then, intubation was performed with a 4.0 or 4.5 mm cuffed endotracheal tube (Portex, ID Smiths Medical, Keene, NH). Subsequently, the animals were connected to a volume- controlled ventilator (Soxil, Soxitronic, Felino, Italy), with room air (FiO₂ 21%) and a tidal volume of 15 ml/kg. End-tidal CO₂ (ETCO₂) was monitored with a side-stream infrared CO₂ analyzer and the respiratory frequency was adjusted to maintain ETCO₂ between 35 and 40 mm Hg. Cisatracurium (0.15 mg/kg) was administered to ascertain synchrony with the ventilator. Continuous infusion of propofol 150mcg/kg/min or more, if needed, was used to maintain anesthesia and fentanyl 4 mcg/kg was administered to ensure satisfactory analgesia. Cardiac rhythm and heart rate were monitored by Electrocardiography , using leads I, II, III, aVR, aVL and aVF. Pulse oximetry was monitored con- tinuously on the animals’ tongue. Right carotid artery and Right internal jugular vein were surgically prepared and cannulated under aseptic conditions. aortic pressures were measured using a normal saline filled catheter (model 6523, USCI CR, Bart, Papapostolou, Athens, Greece) ad- vanced via the right carotid artery into the thoracic aorta. Mean arterial pressure (MAP) was determined by electronic integration of the aortic blood pressure waveform. Right internal jugular vein was catheterized with a 6F sheath and a central vein catheter was inserted through the sheath into the right atrium for continuous measurement of right atrial pressures. All catheters were calibrated before use and their correct position was verified by the presence of the typical pressure waveforms. coronary perfusion pressure was calculated as the difference between decompression diastolic aortic pressure and time- coincident right atrial pressure, measured at the end of each minute of compressions.

Experimental protocol

Baseline data were obtained after a stabilization period of 30 min. VF was then induced with a 5F pacing wire (Pacel(TM), 100 cm, St Jude Medical, Ladakis, Athens, Greece) advanced into the right ventricle through the exposed right internal jugular vein, using a 9 V cadmium battery, as previously described [52]. VF was confirmed by ECG tracing and a sudden drop in MAP. Mechanical ventilation and administration of anaesthetics were discontinued simultaneously with the onset of VF and the animals were left untreated for 8 min (representing the average time of EMS arrival in OHCA in Europe [53]).

After 8 min of untreated cardiac arrest, CPR begun; a bolus dose of adrenaline (0.02 mg/kg) was administered, mechanical ventilation was resumed with 100% oxygen and automatic continuous precordial compressions were initiated. The sham or active ITD (ResQPOD, Ad- vanced Circulatory Systems, Eden Prairie, MN) was placed directly on the proximal end of the ETT and the ventilator’s breathing system was then directly connected to it. LUCAS CPR device (LUCAS, Jolife, Lund, Sweden) is a pneumatically driven, automated compression-decom- pression device, which provided high-quality chest compressions at a rate of 100/min and a compression depth equivalent to 1/3 of the anteroposterior diameter of the animals’ thorax, with active recoil of the chest to the full resting position after each compression.

After 2 min of CPR, a 4 J/kg monophasic waveform shock was delivered. CPR was resumed for another 2 min after each defibrillation attempt. Further bolus doses of adrenaline (0.02 mg/kg) were adminis- tered every four minutes during CPR. This sequence was continued until ROSC or asystole. ROSC was defined as the presence of a perfusing cardiac rhythm with a MAP of at least 60 mm Hg for a minimum of 5 min. After ROSC, the ITD was removed and the animals were mechanical- ly ventilated and closely monitored for 6 h, under general anesthesia, at the pre-arrest settings. No other interventions (drugs, cardioversion or defibrillation attempts) were made after ROSC. After 6 h all catheters were removed. The animals were allowed to recover from anesthesia and, then, were extubated and transferred to their observation cages. They remained under observation for 48 h after ROSC and then

euthanized with an IV bolus dose of propofol 40 mg, followed by 2 g of thiopental IV.

Measurements

Hemodynamic data (heart rate, aortic and right atrial pressures), ECG and ETCO₂ were continuously recorded before and during the ar- rest and 6 h post-ROSC. Coronary perfusion pressure was calculat- ed as described above. The number of defibrillations needed to achieve ROSC and time to ROSC were noted on each experiment. arterial blood gases (ABGs) were measured at baseline and after ROSC. A neurological alertness score was performed at 48 h by a veterinarian blinded to sham/active valve use, as previously described [54]. Alertness was scored from 0 (coma) to 100 (fully alert). Blood and Urine samples were collected at baseline and 2 h, 4 h, 6 h, 24 h and 48 h post-ROSC. Each blood sample was centrifuged at 4000 rpm for 10mins, while urine samples were centrifuged at 2000 rpm for 10mins. The superna- tants were collected and stored in liquid nitrogen at -70 ?C until re- quired. The plasma levels of NGAL were determined at baseline, 2 h, 4 h, 6 h, 24 h and 48 h post-ROSC using a commercially available pig ELISA kit (KIT 044, Bio-Porto Diagnostics, Gentofte, DK), while the serum levels of creatinine were determined at baseline and 48 h after ROSC. The urine levels of IL-18 were determined at baseline, 2 h, 4 h, 6 h, 24 h and 48 h after ROSC using a commercially available pig ELISA kit (KIT BMS672/BMS672TEN, eBioscience, US).

Statistical analysis

Data are expressed as mean +- standard deviation (SD) for quantita- tive variables and as frequency (%) for categorical variables. The normal- ity of the distributions was assessed using the Kolmogorov-Smirnov test and graphical methods. Comparisons of continuous data were per- formed using independent samples t-test, or Mann-Whitney test in case of violation of normality. Categorical variables were compared with the chi-square test, or Fisher’s exact test, as required. Spearman’s p was calculated to examine linear relationships between variables. Rel- ative risk of the two groups was derived from cross tabulations. Be- tween-group differences of all variables at each time point were analyzed with ANCOVA covariance model controlling for baseline dif- ference using the value at each time point as dependent variable and baseline measurements as covariates. All tests were two sided. Statisti- cal significance was set at P b 0.05. All analyses were carried out using the statistical package SPSS 17.0 (Statistical Package for the Social Sci- ences, SPSS Inc., Chicago, Ill., USA)

Results

Hemodynamic outcomes

Before cardiac arrest, there were no statistically significant differ- ences regarding Hemodynamic variables, ETCO2, and pH, pO2, pCO2

Table 1

Baseline measurements in the two groups (presented as means +- SD); ITD, Impedance Threshold Device.

Measurement	Control group	ITD group	p
Systolic pressure, thoracic aorta (mm Hg)	120 +- 9.0	120.3 +- 8.1	NS
Diastolic pressure, thoracic aorta (mm Hg)	100.1 +- 10.7	97.8 +- 5.7	NS
Systolic pressure, right atrium (mm Hg)	15.1 +- 4.0	12.1 +- 1.5	NS
Diastolic pressure, right atrium (mm Hg)	10 +- 3.5	7.6 +- 1.6	NS
Coronary perfusion pressure (mm Hg)	90.1 +- 10.3	90.1 +- 5.8	NS
End-tidal CO2 (mm Hg)	36.5 +- 2.0	36.4 +- 1.8	NS
Heart Rate (beats/min)	111.1 +- 10.8	107 +- 5.4	NS
pH	7.41 +- 0.07	7.41 +- 0.05	NS
PO2	78.6 +- 4.8	75.9 +- 5.1	NS
PCO2	39.5 +- 4.2	41.1 +- 7.0	NS
HCO3	24.9 +- 3.0	25.7 +- 2.3	NS

(Table 1). By the end of the eighth minute of cardiac arrest (non-

flow), mean arterial pressure decreased significantly from 106.8 +-

9.7 mm Hg to 22.3 +- 4.9 mm Hg in the control group, and from

105.2 +- 6.2 mm Hg to 19.6 +- 2.5 mm Hg in the ITD group (P = not sig- nificant between the two groups).

CPP at the first minute of CPR was 1.44-times higher in the ITD group (P = 0.015) and remained significantly elevated throughout the experiment, apart from the second minute of CPR, when there was an elevation in the ITD group that did not reach statistical significance (P = 0.093) (Fig. 1). A significant difference in diastolic aortic pressure was also noted between groups at the first minute of CPR, and while the difference did not reach statistical significance at the second minute, it achieved significance for the rest of the experiment. The same was true for systolic aortic pressure as well, as it was significantly elevated in the ITD group in the first minute of CPR but not in the second, and the significant difference remained until the end of CPR.

ETCO₂ rose more rapidly and to higher peak values in the ITD group compared with the control group. From the first minute of CPR ETCO2 was significantly higher in the ITD group and remained significantly higher throughout the experiment.

All animals that were successfully resuscitated were monitored for 6 h. During the post-resuscitation period ETCO₂ was significantly higher in the ITD group (P = 0.032). One minute post-resuscitation, systolic and diastolic aortic pressures were elevated in the ITD group but not sig- nificantly, while 15 min post-resuscitation this elevation was significant again (P = 0.01).

Survival and biomarkers

ROSC was observed in seven (87.5%) animals treated with the sham valve and eight (100%) animals treated with the active valve (P = NS). However, more than twice as many animals survived at 48 h in the ITD group (n = 8, 100%) compared to the control group (n = 3, 37.5%).

More specifically, five animals in the control group and eight animals in the ITD group achieved ROSC after the first defibrillation attempt, while two animals in the control group achieved ROSC after the second defibrillation. The mean number of Electric shocks was, thus, marginally higher for successfully resuscitated animals in the control group than in the ITD group (P = NS). Mean time to ROSC was higher, but not signif- icantly, in the control group compared to the ITD group.

Neurological alertness score was higher, but not statistically signifi- cant, in the 48-h survivors that received treatment with the ITD com- pared with the control group (93.8 +- 17.7 vs. 83.3 +- 28.9, P = NS). Seven out of eight survivors of the ITD group had a completely normal

Fig. 1. Coronary Perfusion Pressure fluctuation during the experiment; asterisk represents

P b 0.05. ITD, Impedance Threshold Device.

neurologic score. On the other hand, two out of three survivors treated with the sham valve had intact neurologic function (P = NS).

Urine IL-18 and plasma NGAL levels were augmented in the ensuing hours post-resuscitation in both groups (Table 2, Table 3), but they were significantly elevated in animals treated with the sham valve at all measured time points, when compared to the ITD group (Table 4, Fig. 2, Fig. 3). On the other hand, Creatinine levels did not increase signif- icantly 48 h post-ROSC when compared to baseline values and, also, there was no significant difference observed between the two groups.

Discussion

The ITD has been the subject of numerous studies in the past, both experimental and clinical. Some of these studies used the ITD alone, whereas others combined it with active compression-decompression devices (ACD-CPR).

When the ITD was used for resuscitation of animal models of cardiac arrest, it significantly decreased intrathoracic pressure and defibrillation energy requirements. Moreover, it improved hemodynamics, such as Coronary Perfusion Pressure (CPP), cerebral perfusion pressure , vital organ blood flow, ETCO₂, systolic and diastolic blood pressures. Also, it increased ROSC, 24 h and 48 h survival and neurologic outcome (evaluated with both clinical and Biochemical parameters) [14,20,23-32].

These spectacular results were further confirmed in several clinical studies. In two trials [33,34] the ITD was used in victims of OHCA and the resuscitation results were compared with those of historical control groups which were resuscitated with standard CPR. The comparison re- vealed a statistically significant difference in ROSC and short term sur- vival. Numerous observational studies compared the resuscitation results before and after the implementation of the 2005 AHA guidelines, including the use of ITD, and showed a statistically significant increase in hospital discharge rates [35-39]. Except from these observational studies, several prospective, randomized, blinded studies confirmed the importance of ITD use [40-44]. In the groups resuscitated with ITD there was a statistically significant rise in ETCO₂, CPP, DAP and SAP [40,43] and, also, in ROSC, 1 h and 24 h survival [41,42]. Moreover, ac- cording to Wolcke et al. [41], the overall neurological function in the ITD group trended higher, though the difference was not statistically significant. In 2011, the ResQTrial was published in Lancet. It was a multicentre, randomized trial which included 1653 patients with

Table 2

Control group: urine IL-18 and serum NGAL measurements.

Animal	Measurement	Urine IL-18 (pg/ml)	Serum ?GAL (pg/ml)
Control 1	Baseline	0	157542
	2 h	86	4014780
	4 h	542	3623466
	6 h	3518	2241162
	24 h	412	1905750
	48 h	284	2083620
Control 2	Baseline	0	167706
Control 3	Baseline	0	127050
	2 h	0	3999534
	4 h	312	3557400
	6 h	388	2429196
	24 h	284	1417878
	48 h	224	2159850
Control 4	Baseline	0	137214
	2 h	0	2164932
	4 h	216	1285746
	6 h	284	1240008
	24 h	196	1087548
	48 h	156	1128204
Control 5	Baseline	0	157542
Control 6	Baseline	0	188034
Control 7	Baseline	0	152460
Control 8	Baseline	0	172788

Table 3

ITD group: urine IL-18 and serum NGAL measurements; ITD, Impedance Threshold Device.

Animal	Measurement	Urine IL-18 (pg/ml)	Serum ?GAL(pg/ml)
ITD 1	Baseline	0	167706
	2 h	0	640332
	4 h	100	609840
	6 h	156	559020
	24 h	112	487872
	48 h	100	457380
ITD 2	Baseline	0	113456
	2 h	0	426888
	4 h	86	370986
	6 h	100	249018
	24 h	86	238854
	48 h	0	198198
ITD 3	Baseline	0	177870
	2 h	0	609840
	4 h	112	594594
	6 h	130	548856
	24 h	100	381150
	48 h	86	396396
ITD 4	Baseline	0	116886
	2 h	0	950334
	4 h	168	924924
	6 h	196	863940
	24 h	130	797874
	48 h	112	747054
ITD 5	Baseline	0	126478
	2 h	0	472626
	4 h	86	391314
	6 h	112	340494
	24 h	86	289674
	48 h	0	279510
ITD 6	Baseline	0	157542
	2 h	0	472626
	4 h	86	406560
	6 h	112	396396
	24 h	130	350658
	48 h	86	365904
ITD 7	Baseline	0	172788
	2 h	0	421806
	4 h	100	396396
	6 h	100	289674
	24 h	86	223608
	48 h	0	193116
ITD 8	Baseline	0	137214
	2 h	0	792792
	4 h	112	650496
	6 h	168	594594
	24 h	130	457380
	48 h	100	431970

OHCA of presumed cardiac cause, resuscitated with either standard CPR or ACD-ITD CPR. This study demonstrated a 53% increase in survival to hospital discharge with favorable neurologic function and a 50% in- crease in 1-Year survival in the ACD-ITD group [45]. A secondary analy- sis of the ResQTrial by Frascone et al. [46] included 2738 patients with

Table 4

P values for urine IL-18 and serum NGAL comparison between the two groups, at all mea- sured time points.

Biomarker	Measurement	P values
IL-18	Baseline	1.000
	2 h	0.102
	4 h	0.013
	6 h	0.014
	24 h	0.013
	48 h	0.013
NGAL	Baseline	0.429
	2 h	0.014
	4 h	0.014
	6 h	0.014
	24 h	0.014
	48 h	0.014

Fig. 2. Neutrophil Gelatinase-Associated Lipocalin (NGAL) fluctuation; ITD, Impedance Threshold Device.

non-traumatic OHCA, regardless of etiology. Even with the inclusion of a large segment of non-Utstein cardiac arrest patients, this post-hoc study showed a 39% increase in survival to hospital discharge with favorable neurologic function and a 39% increase in 1-year survival. Another im- portant study was the NIH PRIMED in 2011, which included 8718 vic- tims of OHCA resuscitated with either standard CPR or ITD CPR. This trial showed no difference in ROSC, Survival to hospital admission, sur- vival to hospital discharge or survival to hospital discharge with favor- able neurologic function between the two groups [47]. However, a further analysis of the NIH PRIMED study revealed that when acceptable Quality of CPR was performed, the use of ITD increased survival to hos- pital discharge with favorable neurologic function. On the contrary, when the CPR quality was poor, the use of ITD led to a worse outcome [48].

Three meta-analyses evaluated the ITD. The first one by Cabrini et al. in 2008 included 833 patients with non-traumatic OHCA, from 5 high quality randomized studies. It showed a statistically significant increase in ROSC, early survival and Favorable neurologic outcome [49]. A second meta-analysis by Biondi-Zoccai et al. included 11 254 victims of OHCA from 7 trials. It did not demonstrate an overall increase in ROSC, favorable neurologic outcome or long term survival. However, explor- atory analysis showed that the combined use of ITD with ACD-CPR

Fig. 3. Interleukin-18 (IL-18) fluctuation; ITD, Impedance Threshold Device.

significantly increased ROSC, favorable neurologic outcome and long term survival [50]. In 2015 a third meta-analysis including 16 088 pa- tients from 15 trials, initially failed to reveal any differences in ROSC, survival or Neurologic outcome at hospital discharge. However, after ad- justment of two important prognostic factors (witnessed status and re- sponse time), the use of ITD appeared to improve ROSC [51].

Results from our study support previous research findings regarding improvement of hemodynamic parameters and 48 h survival with the use of ITD. CPP is considered to be the only predictive factor for success- ful resuscitation and ROSC [55] and has been associated with more pos- itive long-term outcomes, including 1 to 24-h survival and even 7-day survival [56]. In our study, CPP was significantly elevated in the ITD group throughout the experiment, apart from the second minute of CPR when there was an elevation in the ITD group that did not reach sta- tistical significance. This was translated into better 48 h survival rates.

To our knowledge, this is the first study evaluating the effect of ITD use during CPR on the degree of post-CA AKI. In general, over the past decades, the majority of resuscitation studies focused on post-CA brain injury and myocardial dysfunction, while AKI and other vital organs’ dysfunction have not been well described. However, renal dysfunction is common following resuscitation from CA (12-28%) and it is strongly associated with increased early and long-term morbidity and mortality, as well as development of chronic kidney disease (CKD) [57-59]. The most common mechanism of AKI after CA is renal ischemia, which causes functional impairment by multiple factors, such as renal vaso- constriction, tubular obstruction, tubular back-leakage of glomerular fil- trate, reduced glomerular permeability and apoptosis [60-62]. In our study, the kidneys’ perfusion ceased completely for 8 min, during the untreated phase of CA, while a degree of hypoperfusion was present during the resuscitation phase, depending on the efficiency and dura- tion of CPR until ROSC.

The term AKI implies a potentially reversible kidney injury or dam-

age occurring in a time frame of hours or days. Early detection is imper- ative, in order to initiate treatment that may mitigate renal injury. Although the word “injury” would not necessarily encompass kidney dysfunction, in current clinical practice serum creatinine (sCr) and urine output are used for identification and classification of AKI, accord- ing to the RIFLE (Risk, Injury, Failure, Loss, End-stage) criteria [63], the AKIN (Acute Kidney Injury Network) criteria [64] and the KDIGO (Kid- ney Disease: Improving Global Outcomes) clinical practice guidelines for AKI [65]. Both these indexes are functional, as they are affected from an altered glomerular filtration rate (GFR), therefore identifying more a dysfunction than damage. Moreover, they have serious limita- tions; Oliguria is a non-specific marker of AKI. sCr, on the other hand, is influenced by several non-renal factors such as sex, age, muscle mass, muscle metabolism, medications, nutrition status and hydration status of the patient. Also, sCr concentrations do not reflect the true de- crease in GFR during acute changes, as several hours or days must elapse before a new equilibrium between the steady-state production and the decreased excretion of creatinine is established. But the most serious problem is that sCr and urine output are not ideal for screening the early stages of AKI, because they may be normal until 48-72 h post-in- jury, when 25-50% of kidney function has already been lost [66]. Asa re- sult, the clinical diagnosis of AKI is delayed, long past the therapeutic window of opportunity [67]. All the above suggest that sCr and urine output are delayed and unreliable indicators of AKI.

Thus, early diagnosis of AKI by using reliable biomarkers, preceding filtration function loss was imperative. The quest for such biomarkers has often been referred to as the “search for the renal troponin I”. Over the last decade, such molecules have been identified by genomic, proteomic and metabolomic methods. These biomarkers are specific for kidney injury, sensitive enough to detect even less severe insults, easy and rapid to measure and inexpensive enough to make their use sustainable. The novel renal biomarkers have the potential to detect minor renal injury early, even when creatinine levels are not yet elevat- ed [68]. The term “subclinical AKI” refers to patients who are biomarker-

positive and creatinine-negative. In contrast with the traditional view that a kidney problem is clinically relevant only when a loss of filtration function becomes apparent, recent studies have demonstrated that even tubular damage without glomerular function loss is associated with worse renal and overall outcomes [69-72]. Moreover, the ability to diagnose AKI earlier in the disease course would allow a more Timely intervention and prevention of progression. Consequently, the new bio- markers have been intensively investigated over the last years and have been shown to be potentially suitable not only for early diagnosis of AKI, but also for risk stratification, prediction of AKI progression, severity and outcomes or need for Renal replacement therapy [73-75]. Two of the most promising renal biomarkers are NGAL and IL-18.

NGAL is a 25 kDa glycoprotein of the lipocalin family and it is com- posed of eight beta strands, forming a ?-barrel defining a calyx, which binds and transports low-molecular-weight substances [76]. NGAL was originally identified in neutrophils, but it is also expressed at low levels in a variety of human tissues, including lung, liver, kidney, stom- ach and colon and its expression increases greatly in the presence of in- flammation and injured epithelia [77]. While the majority of NGAL is in a monomeric form, it also occurs as dimers or trimers, as well as in a complex with neutrophil gelatinase [78,79]. The monomeric form is se- creted by injured kidney tubule epithelial cells, whereas the dimeric form is the predominant form secreted by neutrophils [80,81]. NGAL has been implicated in several biological processes, including attenua- tion of apoptosis and differentiation of renal tubule epithelial cells and nephrons [82,83].

In healthy kidneys, NGAL is barely detectable in either plasma or urine. However, in the setting of acute tubular injury it undergoes rapid and profound upregulation, with large increases in both plasma and urine. In animal studies of renal Ischemia-reperfusion injury NGAL was one of the most upregulated genes and proteins in the kidney. NGAL protein was detected in the plasma and urine of animal models of AKI within 2 h of injury, preceding the increase of sCr concentrations [84-86]. NGAL protein expression was detected predominantly in tu- bule epithelial cells that were undergoing proliferation and regenera- tion, suggesting a role in the repair process [85]. NGAL has also been studied in numerous clinical studies, in several situations such as critically ill patients in the ICU, acute heart failure, sepsis and septic shock, radio-contrast procedures, cardiac surgery, toxins and trauma [87-113]. It proved to be a sensitive, specific and highly predictive bio- marker of AKI, increasing as early as 2 h after renal injury.

NGAL fulfils many of the characteristics important for a useful AKI biomarker; its concentration in plasma and urine rises proportionally to the degree of Renal damage. Also, it is expressed early after kidney in- jury, when such damage is potentially limitable or reversible. NGAL fur- ther allows risk stratification, therapy monitoring and prognostication with respect to the need for acute dialysis, duration of hospital stay and mortality. Last, it is easily and reliably measurable on available stan- dardized clinical platforms [114].

NGAL can be measured both in plasma and urine. Each sample has its advantages and disadvantages. Blood NGAL measurements are invasive and may potentially reflect the effect of extrarenal disease. However, samples are taken easily and the measurement can be performed rapid- ly on a point-of-care device. Urine samples are non-invasive and, also, there are less potentially interfering proteins present in urine than in blood. However, many critically ill patients may be oliguric or anuric. In addition to that, urinary NGAL concentrations may be affected by hy- dration status and diuretic treatment [115].

Although NGAL is considered a reliable biomarker of AKI, its use has some limitations. First, plasma NGAL levels may be influenced by a number of coexisting diseases, including CKD, chronic hypertension, Systemic Infections, Inflammatory conditions, anemia, hypoxia and ma- lignancies [116]. In sepsis, NGAL originates not only from the injured kidney but also from leucocytes and liver. Therefore, NGAL levels in pa- tients with septic AKI are substantially higher compared to non-septic AKI, and this may limit the diagnostic utility of NGAL in sepsis [117].

In CKD NGAL levels correlate with the severity of Renal impairment. However, in all the above settings, the increased plasma NGAL is much less than those typically encountered in AKI [118].

IL-18 is an 18 kDa pro-inflammatory cytokine that is induced in proximal tubular epithelial cells in response to injury. Following renal injury, IL-18 is secreted in the urine before a significant decrease in renal function occurs. Several animal studies have demonstrated that IL-18 is not only a biomarker of AKI, but also plays a pathogenic role in ischemic renal injury [119-123]. These findings in animal studies have lead to extensive research of IL-18 as a biomarker of AKI in humans. In a cross-sectional study, urine IL-18 levels were significantly higher and had high sensitivity and specificity for the diagnosis of acute tubular necrosis compared both with healthy subjects and other kidney diseases, such as urinary tract infections, prerenal azotemia, Nephrotic syndrome and chronic kidney disease [124]. IL-18 has proved to be an early and reliable AKI biomarker in several clinical settings, such as ICU patients, post-cardiac surgery, Contrast-induced nephropathy and renal transplant patients [125-131]. Moreover, elevated urine IL-18 con- centrations are correlated with the severity of AKI, as well as longer hos- pitalization, longer ICU stay, higher risk of dialysis and mortality [126, 130-132]. Urine levels of IL-18 begin rising as early as 4 to 6 h following an ischemic renal insult, peak at 12 h and remain increased for up to 48 h [125]. IL-18 levels in urine are elevated up to 48 h before the creat- inine-defined occurrence of AKI [126]. However, this AKI biomarker has some limitations. Considering that it is a pro-inflammatory cytokine, it plays an important role in sepsis and its concentrations are also influ- enced in several other pathophysiological states, such as endotoxemia, inflammatory and autoimmune diseases [132,133].

In our study, we measured plasma NGAL and urine IL-18 levels

before cardiac arrest and 2 h, 4 h, 6 h, 24 h and 48 h post-ROSC, in order to assess the degree of AKI. We found that NGAL levels peaked 2 h post-resuscitation and then began falling gradually, but still remained increased 48 h post-ROSC, in both groups. IL-18 levels, on the other hand, peaked at 4 h, decreased gradually over the next hours and persisted 48 h post-ROSC, in both groups. Both NGAL and IL-18 levels were significantly elevated in animals treated with the sham valve when compared to the active-valve group, at all mea- sured time points (Table 4, Fig. 2, Fig. 3), validating our hypothesis, that ITD reduces the degree of post-CA AKI, by improving hemodynam- ics during CPR.

Moreover, in our study we measured serum creatinine levels before cardiac arrest and 48 h post-ROSC. We found that there was no differ- ence between the two groups and also there was no difference between the baseline and the post-ROSC sCr levels, despite the statistically signif- icant differences in both NGAL and IL-18 levels. This may be given two explanations. The first explanation is that sCr may remain normal until 48-72 h post-injury, as mentioned above. In other words, if we had not euthanized our animals at 48 h we may have seen sCr levels ris- ing the ensuing days. The second explanation is that the degree of AKI was not significant enough to cause an increase in sCr; kidneys have an important glomerular function reserve, and dysfunction becomes ev- ident only when N 50% of the renal mass is compromised. However, as mentioned above, this “subclinical AKI” is associated with worse renal and overall outcomes [69-72]. Another interesting theory is based on an observation of a previous study, regarding resuscitated victims; in this study, in the majority of CA victims the initial post-arrest creatinine concentration dropped dramatically instead of rising, and much more than it could be explained by dilution, due to administration of fluid bo- luses. In patients with no change in creatinine the outcomes were worse and elevated levels of kidney injury biomarkers were observed in the urine. These data imply that cardiac arrest may temporarily cease the production of creatinine [134]. We should always keep in mind that novel AKI biomarkers reflect structural injury and are not elevated be- cause of changes in GFR. Any association with creatinine is, therefore, because the injury reflected by the elevated biomarkers causes a loss of GFR, later reflected by increased creatinine.

There are several limitations to our study. First, the number of ani- mals is relatively small with high risk of type II statistical error. Second, the experiment was conducted on healthy animals and its direct appli- cation to human victims of CA has yet to be addressed. Another problem is that the use of LUCAS device may not accurately reflect the clinical setting, where chest compressions are usually delivered manually. Fi- nally, this was an experimental study and, thus, we should be careful in extrapolating our findings to humans.

Conclusions

Our study demonstrated that use of the ITD decreased the degree of AKI in resuscitated CA animals, by improving hemodynamics and reduc- ing the magnitude of renal ischemia during CPR. This study adds some evidence to the existing literature of ITD-CPR benefits. Further evalua- tion of these results should be undertaken in the future.

Institution

ELPEN pharmaceutical Experimental-Research Centre, Pikermi, Attika, Greece.

Source of funding

This research did not receive any specific grant from funding agen- cies in the public, commercial, or not-for-profit sectors.

Acknowledgments

The authors would like to thank ELPEN pharmaceutical Experimen- tal-Research Centre for providing research facilities and for the constant technical assistance during the experiments.

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