Which remote ischemic preconditioning protocol is favorable in renal ischemia-reperfusion injury in the rat?

Abstract

BACKGROUND:

The optimal timing of remote ischemic preconditioning (RIPC) in renal ischemia-reperfusion (I/R) injury is still unclear. We aimed to compare early- and delayed-effect RIPC with hematological, microcirculatory and histomorphological parameters.

METHODS:

In anesthetized male CrI:WI Control rats (n = 7) laparotomy and femoral artery cannulation were performed. In I/R group (n = 7) additionally a 45-minute unilateral renal ischemia with 120-minute reperfusion was induced. The right hind-limb was strangulated for 3×10 minutes (10-minute intermittent reperfusion) 1 hour (RIPC-1 group, n = 7) or 24 hour (RIPC-24 group, n = 6) prior to the I/R. Hemodynamic, hematological parameters and organs’ surface microcirculation were measured.

RESULTS:

Control and I/R group had the highest heart rate (p < 0.05 vs base), while the lowest mean arterial pressure (p < 0.05 vs RIPC-1) were found in the RIPC-24 group. The highest microcirculation values were measured in the I/R group (liver: p < 0.05 vs Control). The leukocyte count increased in I/R group (base: p < 0.05 vs Control), also this group’s histological score was the highest (p < 0.05 vs Control). The RIPC-24 group had a significantly lower score than the RIPC-1 (p = 0.0025 vs RIPC-1).

CONCLUSION:

Renal I/R caused significant functional and morphological, also in the RIPC groups. According to the histological examination the delayed-effect RIPC method was more effective.

Keywords

Acute kidney injury ischemia-reperfusion remote ischemic preconditioning microcirculation histopathology

1 Introduction

During kidney transplantation, major cardiovascular intervention or arterial occlusion the kidneys can be affected by ischemia reperfusion injury (IRI). Acute kidney injury (AKI) may develop, which is associated with an increased long-term risk of mortality [1, 2]. In most of the cases, the only definitive treatment, where IRI may occur, is the transplantation [2 –6]. Several drugs and methods are available to protect organs from IRI, however, the optimal process has not been clarified yet [7 –9].

Numerous surgical methods can be used for decreasing IRI. A technique called ischemic conditioning was first described by Murry at al. in 1986 on animal myocardium [10]. Since then, we have known that the process can be performed on other organs such as the liver or kidneys, and different techniques have been also evolved [11, 12]. Remote ischemic precondition (RIPC) is a process that includes intermittent short-term interruptions of the blood flow of an organ or extremity before the ischemia-reperfusion occurs in another organ. Depending on the period between the ischemia and the RIPC early and delayed forms are known. In case of the early-effect protocol, RIPC should be performed a few hours before the ischemia, while in case of the delayed-effect one, the process should be carried out more than 24 hours before the ischemic intervention. The protecting mechanisms differ. In the early RIPC neural and humoral pathways might have a great importance, in the delayed version systematic pathways have enough time to be activated, while some other protecting effects decrease [13 –18]. However, the detailed pathomechanisms are still not clarified.

It is known that RIPC is effective on myocardium, intestines and liver, while only a few studies have focused on the renal IRI [19 –22]. However the optimal RIPC protocol in case of renal IRI is still unknown. Our hypothesis in this study was that renal IRI causes significant changes in hematological, microcirculatory and histomorphological parameters, and the different RIPC protocols might decrease it. To decide which protocol is better, we aimed to compare the early- and delayed-effect RIPC protocols, using microcirculatory, hematological and histological examinations.

2 Materials and methods

2.1 Animals, operative techniques and experimental protocol

The experiment was performed on 27 male Crl:WI rats (body weight: 301.6±38.6 g) (permission registration number: 25/2016/UDCAW, University of Debrecen Committee of Animal Welfare), in accordance with national (The Hungarian Animal Protection Act, Law XVIII/1998) and European Union regulations (EU Directive 63/2010). The rats were kept in a conventional animal facility and received rodent chow and water ad libitum. All the surgical interventions were made under general anesthesia. For the atraumatic techniques an operating microscope (Leica Wild M650) and microsurgical approach was used. During the entire experiment an electrical heating helped to maintain the body temperature.

Anesthesia was performed in all groups with thiopental: a short-term anesthesia 24 hours (40 mg/bwkg, i.p.), and a deeper one 1.5 hours prior to the operation (60 mg/bwkg, i.p.). Rats were divided into four experimental groups as sham-operated (Control, n = 7), ischemia-reperfusion (I/R, n = 7), early-effect remote organ ischemic precondition (RIPC-1, n = 7) and delayed-effect remote organ ischemic precondition (RIPC-24, n = 6) groups.

In the Control group, there were no interventions until the operation. Before the operation the abdomen and the left hind limb were shaved. After disinfection with Betadine solution and isolation, the left femoral artery was cannulated (BD Neoflon, 26G) through a 1 cm skin incision. The left kidney and its artery were gently exposed with atraumatic preparation via median laparotomy. After this only a 165-minute monitoring period and the measurements were performed.

In the I/R group, the protocol was the same as in the Control group until the exposure of the kidney that was followed by placing an atraumatic microsurgical clip on the left renal artery to provide a 45-minute warm renal ischemia. After removing the clip we observed a 120-minute reperfusion.

In RIPC groups a tourniquet was applied on the right hind limb beneath the level of the inguinal ligament for 3x10 minutes with 10-minute intermittent reperfusion periods. In the RIPC-1 group this process was performed one hour prior to the operation, while in the RIPC-24 group one day before the I/R. In both groups after the RIPC, the operating protocol was the same as in the I/R group. At the end of the experiment the rats were euthanized by exsanguinations.

2.2 Samplings and measurements

Blood samplings were taken and parameters were measured after the median laparotomy (base), at the end of the renal ischemic period (I45), and at the 30th, 60th and 120th minute of the reperfusion (R30, R60, R120). Between measurements, the abdominal cavity was covered with warm, wet gauze.

Heart rate and mean arterial pressure (MAP) were measured invasively (HaemoSys, Experimetria Ltd., Hungary), while the respiratory rate was measured directly by counting for 60 seconds. Rectal temperature was assessed with a digital thermometer.

An LD-01 Laser Doppler Flowmeter (Experimetria Ltd., Hungary) was used to monitorize the microcirculation on the surface of the liver and the kidneys. Illuminating single-frequency light beams penetrate the tissue or are reflected from it. If the laser beam that hits moving erythrocytes, its frequency changes. The velocity and the count of erythrocytes correlate well with the change of frequency, accordingly the device expresses blood flux unit (BFU). The signal was recorded by S.P.E.L. Advanced Kymograph software (Experimetria Ltd., Hungary) at 1 kHz sampling rate for 20–40 seconds. Off-line analysis of the BFU was made by curve parameterization of a noise-free 10-second representative section [23, 24]. In parallel, an infrared thermometer (Rudolf Riester GmbH) was used on the surface of the median lobe of the liver and on the front surface of both kidneys.

Blood samples (400μl/each) were taken from the cannulated right femoral artery into K3-EDTA anticoagulated tubes, and an equal volume of physiological saline solution was given intravascularly. Hematological parameters (red blood cell count, hematocrit, white blood cell count, platelet count) were tested with a Sysmex K-4500 automated hematology analyzer (TOA Medical Electronics Corp. Ltd., Japan).

Just after the animals were euthanized, tissue samples were taken from the liver and both kidneys, and periodic acid-Schiff (PAS) staining was performed. The histological scoring evaluated tubular epithelial cell nuclear staining, tubular necrosis, hyaline globules in the tubular epithelial cells, hydropic degeneration of the tubular epithelial cells, glomerular congestion, peritubular capillary congestion, congestion of the vasa recta, and brush border (0 to 3 points per each). The liver samples were scored according to the level of stagnation in hepatic sinusoids, cell necrosis, and vacuolization (0 to 3 points per each).

2.3 Statistical analysis

All data were expressed as the mean±standard deviation (SD). For single pair-wise inter-group comparison Student t-test or Mann–Whitney rank-sum tests, for multiple inter-group and intra-group comparisons one-way and repeated measures ANOVA tests (Dunn’s, Bonferroni’s or Student–Newman–Keuls method) were applied, depending on the normality of data distribution. P values less than 0.05 were considered as statistically significant.

3 Results

3.1 Vital parameters

Heart rate. The base values were lower in the I/R group and increased significantly by the end of the 45-minute ischemia (p = 0.0112 vs Base), and kept increasing during the reperfusion (p < 0.0001 vs R30, R60, R120). A mild elevation was also recorded in the Control group, reaching a significant level from the 60th minute of reperfusion (p = 0.096 vs R60, p = 0.0085 vs R120). The values of the RIPC groups were stable throughout the experiment (Fig. 1, A).

Fig. 1

Changes of (A) heart rate, (B) mean arterial pressure, (C) respiratory rate, and (D) rectal temperature. Base: after laparotomy, I45: the end of 45-minute ischemia, R30: the 30th minute of reperfusion, R60: the 60th minute of reperfusion, R120: the 120th minute of reperfusion. I/R: ischemia-reperfusion group; RIPC-1: early-effect remote ischemic preconditioning group; RIPC-24: delayed-effect remote ischemic preconditioning group. means±(SD); *p < 0.05 vs Base, ⁺p < 0.05 vs I45, ^xp < 0.05 vs Control, ^#p < 0.05 vs I/R, °p < 0.05 vs RIPC-1, ^∨p < 0.05 vs All.

Mean arterial pressure. The MAP values were maintained in all groups, however, it significantly increased in RIPC-1 (at I45: p = 0.0154 vs I/R; R30: p = 0.0074 vs Control, p = 0.0013 vs I/R, p = 0.0228 vs RIPC-24, p = 0.0262 vs base; R60: p = 0.036 vs RIPC-24; R120: p = 0.0115 vs I/R, p = 0.0153 vs RIPC-24, p = 0.0139 vs base) (Fig. 1, B).

Respiratory rate. The RIPC-24 group had the lowest values (base: p < 0.0001 vs Control and RIPC-1, p = 0.0003 vs I/R; at I45: p = 0.0012 vs Control, p = 0.0359 vs I/R, p = 0.0048 vs RIPC-1; at R30: p = 0.0006 vs Control, p = 0.0487 vs RIPC-1; at R60 p = 0.0006 vs Control, p = 0.0279 vs I/R; at R120: p = 0.0046 vs Control, p = 0.0149 vs I/R). A mild elevation could be observed (Fig. 1, C).

Rectal temperature. Rectal temperature was stable in the Control and I/R groups, while rats in the RIPC-24 group had significantly lower body temperature till the 30th minute of the reperfusion (base: p = 0.0073 vs Control, p = 0.0057 vs I/R, p = 0.0053 vs RIPC-1; at I45: p = 0.0388 vs I/R; R30: p = 0.016 vs Control, p = 0.0033 vs I/R, p = 0.0141 vs RIPC-1). By the end of the reperfusion, the animals of the RIPC-1 group had the lowest values (p = 0.0071 vs base, p = 0.0002 vs I45, p < 0.0001 vs R30 and Control, p = 0.0179 vs R60, p = 0.0008 vs I/R, p = 0.0139 vs RIPC-24) (Fig. 1, D).

3.2 Microcirculation

There were remarkable differences in the relative values of BFU (versus base) of the liver in the Control and I/R groups during the reperfusion, being significant at R30 (p = 0.0205). Values of both RIPC groups were stable. In case of the right, non-ischemic kidney there were not any notable changes, but in the left, ischemic kidney a moderate increase could be seen in the I/R and RIPC-24 groups during the reperfusion. The BFU values reached the highest values in the RIPC-24 group at R60 (p = 0.0085 vs I45), and later it normalized, while the data of the I/R group were still increasing till the end of the experiment (p = 0.0011 vs I45, p = 0.0322 vs RIPC-1) (Fig. 2).

Fig. 2

Changes in microcirculation by the 120th minutes of the reperfusion expressed as relative values versus base (R-120/base). Base: after laparotomy, I45: the end of 45-minute ischemia, R30: the 30th minute of reperfusion, R60: the 60th minute of reperfusion, R120: the 120th minute of reperfusion. I/R: ischemia-reperfusion group; RIPC-1: early-effect remote ischemic preconditioning group; RIPC-24: delayed-effect remote ischemic preconditioning group. means±(SD); ⁺p < 0.05 versus I45, ^xp < 0.05 versus Control, ^#p < 0.05 vs I/R.

3.3 Organ surface temperatures

Regarding the relative values of the organ surface temperatures, there were no significant changes in either group or investigated organ. However, a tendentious increase could be observed in the RIPC-24 group, while the most stable data were shown in the I/R and RIPC-1 groups (Fig. 3).

Fig. 3

Changes in surface temperature by the 120th minutes of the reperfusion expressed as relative values versus base (R-120/base). Base: after laparotomy, I45: the end of 45-minute ischemia, R30: the 30th minute of reperfusion, R60: the 60th minute of reperfusion, R120: the 120th minute of reperfusion. I/R: ischemia-reperfusion group; RIPC-1: early-effect remote ischemic preconditioning group; RIPC-24: delayed-effect remote ischemic preconditioning group. means±(SD).

3.4 Hematological parameters

Red blood cell count (RBC). The highest RBC count was measured in the RIPC-24 group (base, R30, R60, R120: p < 0.0001 vs Control, I/R; at R30 and R60: p < 0.0001 vs RIPC-1), while the Control group had the lowest number (base, R30, R60, R120: p < 0.0001 vs RIPC-24; at R30, R60: p < 0.0001 vs RIPC-1; at R30: p = 0.0003 vs I/R; at R60: p = 0.0002 vs I/R) (Fig. 4, A). In parallel, the highest hematocrit values were found in the RIPC-24 group (base: p = 0.0053 vs I/R; R30: p < 0.0001 vs I/R, p = 0.0011 vs RIPC-1, R60: p < 0.0001 vs I/R; R120: p = 0.0010 vs I/R, p = 0.0339 vs RIPC-1) (Fig. 4, B).

Fig. 4

Hematological parameters. Changes of (A) red blood cell count, (B) hematocrit, (C) leukocyte count, and (D) platelet count. Base: after laparotomy, I45: the end of 45-minute ischemia, R30: the 30th minute of reperfusion, R60: the 60th minute of reperfusion, R120: the 120th minute of reperfusion. I/R: ischemia-reperfusion group; RIPC-1: early-effect remote ischemic preconditioning group; RIPC-24: delayed-effect remote ischemic preconditioning group. Means±(SD); *p < 0.05 vs Base, ^p < 0.05 vs R30, ^-p < 0.05 vs R60, ^xp < 0.05 vs Control, ^#p < 0.05 vs I/R, °p < 0.05 vs RIPC-1.

White blood cell count. The most stable leukocyte count was measured in the RIPC groups, while there was a peak in both the Control (R60: p = 0.0307 vs base, p = 0.039 vs RIPC-1) and I/R groups (R30: p = 0.0102 vs base, p = 0.0006 vs R120, p = 0.0004 vs Control, p < 0.0001 vs RIPC-1, p = 0.0005 vs RIPC-24) (Fig. 4, C).

Platelet count. RIPC-24 group showed the lowest platelet count during the experiment (base: p < 0.0001 vs I/R, p = 0.0416 vs RIPC-1; R30: p = 0.0043 vs I/R, p = 0.0279 vs RIPC-1; R60: p = 0.0161 vs I/R). In the I/R group a decrease could be seen, which became significant by the end of the reperfusion (p = 0.0465 vs base) (Fig. 4, D).

3.5 Histological scores

Kidney. The ischemic left kidney had significantly higher scores than the non-ischemic right kidney (I/R: p < 0.0001), except for the Control and RIPC-24 groups. The lowest score in the left kidney was measured in the Control group (p < 0.0001 vs I/R and RIPC1, p = 0.0025 vs RIPC-24), while the I/R group had the highest values (p = 0.0017 vs RIPC-24). Among the ischemic groups, the RIPC-24 showed the lowest scores in the left kidney (p = 0.0025 vs RIPC-1) (Figs. 5, A and 6).

Fig. 5

Histopathological scores for (A) the kidneys and (B) the liver. I/R: ischemia-reperfusion group; RIPC-1: early-effect remote ischemic preconditioning group; RIPC-24: delayed-effect remote ischemic preconditioning group. Means±(SD); *p < 0.05 vs Right kidney, ^xp < 0.05 vs Control, ^#p < 0.05 vs I/R, °p < 0.05 vs RIPC-1.

The highest level of hyaline globules and hyaline degradation in the tubular epithelial cells were found in the I/R group, and also the most damaged glomeruli were found in this group. The congestion of the vasa recta and glomeruli were more pronounced in the RIPC-1 group. Pathological brush border was observed in all the ischemic groups. The tubular epithelial cell nuclear staining decreased and the tubular necrosis increased due to the ischemia, which was moderated by the RIPC protocols. In the ischemic kidneys, the hyaline degeneration increased, and foamy lesions appeared in the cytoplasm of the tubular epithelial cells therefore, the hyaline globules level decreased. Both preconditioning protocols could decrease the severity of these changes. The operation itself did not cause notable congestion in the glomeruli, in the peritubular capillaries, or the vasa recta. The I/R resulted in a light or moderate congestion in the affected (left) kidney, while there was no elevation in the right kidneys. In parallel, the RIPC-1 protocol caused significant congestion in both kidneys, while the RIPC-24 group has lower scores of peritubular capillary and vasa recta congestion. The brush border of the proximal epithelial cells was normal in both side kidneys in case of the control group, while in the I/R group there were parts where the brush border subtotal disappeared or intermittently missed. In the case of the RIPC-24 group, only small gaps could be seen, while in the RIPC-1 group there were gaps and intermittently missing parts too. (Table 1, Fig. 6)

Table 1

Histopathological scores in intact (right) and ischemically affected (left) kidneys

	Control		I/R		RIPC-1		RIPC-24
Scored parameter	Right kidney	Left kidney	Right kidney	Left kidney	Right kidney	Left kidney	Right kidney	Left kidney
Tubular epithelial cell nuclear staining	0	0	0	1.67±0.58	0	1.43±0.53	0	0.83±0.41
Tubular necrosis	0	0	0	1.67±0.58	0	1.43±0.53	0	0.83±0.41
Hyaline globules in the tubular epithelial cells	1.33±0.58	1.33±0.58	1.67±1.15	0.33±0.58	1.29±0.49	0.29±0.49	1.5±0.55	0.83±0.76
Hydropic degeneration of the tubular epithelial cells	0.33±0.58	0.33±0.58	0.67±0.58	3±0	0.29±0.49	2±0	0	0.83±0.41
Glomerular congestion	0	0	0	0.67±0.58	1.14±1.21	0.86±0.9	1.3±0.52	1.17±0.76
Peritubular capillary congestion	0	0	0	0.33±0.58	0.57±0.79	0.71±0.76	0.17±0.41	0
Congestion of the vasa recta	0.33±0.58	0.33±0.58	0	0.67±0.58	0.86±1.07	1.29±0.76	0.17±0.41	0.17±0.41
Brush border	0	0	0.33±0.58	2.67±0.58	0.14±0.38	1.86±0.38	0.17±0.41	1.5±0.84
Cumulate	2±1.73	2±1.73	2.67±1.53	11±1.73 ^,x*	4.29±3.4	9.86±1.86^,x*	3.33±1.21	6.17±1.47^{x, # , ∘}

I/R: ischemia-reperfusion group; RIPC-1: early-effect remote ischemic preconditioning group; RIPC-24: delayed-effect remote ischemic preconditioning group. Means± (SD); *p < 0.05 vs right kidney, ^xp < 0.05 vs Control, ^#p < 0.05 vs I/R, °p < 0.05 vs RIPC-1.

Fig. 6

Representative Periodic Acid Schiff stained histological samples. I/R: ischemia-reperfusion group; RIPC-1: early-effect remote ischemic preconditioning group; RIPC-24: delayed-effect remote ischemic preconditioning group. Black arrow: tubular necrosis, white arrow: hyaline globules, bordered area: loss of integrity, *: damaged glomeruli.

Liver. The highest scores were found in the RIPC-1 group, where the high level of stagnation in hepatic sinusoids was the most marked pathological difference. The lowest scores were in the RIPC-24 (Fig. 5, B and 6).

4 Discussion

In numerous studies, it has been demonstrated that RIPC can moderate the ischemic-reperfusion injury in various organs. However, clinical results are controversial [25 –29]. The comparison of early- and delayed-effect remote ischemic preconditioning might be useful in experimental surgery and clinical practice. In this study, our aim was to compare these RIPC protocols. Renal IRI caused significant worsening in the examined hematological and microcirculatory parameters and also resulted in higher histological scores. We found, that both RIPC protocols were able to alleviate the level of worsening in most measured parameters. However, the influence on the IRI was different in the early- and the delayed-effect RIPC.

The microcirculation of the liver increased most markedly in the I/R group during the reperfusion, while in the right kidney, we found the most increased BFU values in the I/R and RIPC at the last hour of reperfusion. During Laser Doppler flowmetry, an often-used measuring method of the microcirculation, several factors may influence the results including temperature, tremor, movements of the target organ, drying of the surface, evaporation of the tissue, and oxygenation, among others [30]. It has been proved that reperfusion causes local hyperperfusion and also increases the perfusion of the liver, and RIPC protocols could decrease the hyperperfusion [31]. Our results verified these findings. However, as a limitation, the effect of the thiopental on the measured parameters cannot be excluded. It is known that thiopental decreases systemic blood pressure and respiratory rate, and Ahiskaligou et al. showed that thiopental causes oxidative stress [32 –34]. In 1982, Franke et al. demonstrated that phenobarbital caused alterations in capillary density and perfusion, via opening arterio-venous shunts [35]. It is also proven that barbiturates, such as the thiopental, can alter microcirculation through autonomic sympathetic and parasympathetic nerves of the vascular smooth muscles [36]. This study focused on the short-term changes of the I/R injury. The follow-up period (120 min reperfusion) could not be elongated more because of the limited amount of blood taking, and the invasively measured cardiac parameters.

We found an elevated leukocyte count. The effect of immobilization, the anesthesia (as happened twice), the surgical intervention (incision, preparation, cannulation, laparotomy, etc) all may cause acute phase reaction. The ischemia-reperfusion injury leads to an inflammatory response, therefore the preconditioning procedure may have increased the white blood cell count as well. Although the preconditioning can decrease the systemic inflammatory response, the effect of strangulation cannot be excluded [37].

The postischemic inflammation caused by the kidney IRI was initiated by the dysfunction of endothelial and tubular cells which release inflammatory cytokines, such as IL-1 β, IL-6, and IL-8, TNF-α, TGF-β, and MCP-1 [38, 39]. Studies are controversial about the effectiveness of RIPC protocols in decreasing inflammatory modulators. MacAllister et al. found no significant changes in serum levels of IL-1β, IL-6, TNF-α, and INF-γ between a control group and a RIPC group [40], while a recent study of Zapata-Chavira et al. measured significant increase in some of these inflammatory modulators in a RIPC group [41]. Pan et al. demonstrated that delayed RIPC had a renoprotective effect in case of septic AKI via miR-21 due to modulation of inflammatory response [42].

The histopathological examination of the liver showed no significant changes between the groups. The values of the control and the I/R groups were almost the same, while in the RIPC-1 groups the score was higher, and lower in the RIPC-24 group. In case of the ischemic and non-ischemic kidneys the histopathological scores were significantly different. The ischemic left kidneys had significantly higher scores, as expected. Although scores of all preconditioned groups were significantly higher compared to the base, the RIPC-24 group had lower values compared to the other two ischemic groups.

Oral et al. studied renal I/R injury and an early remote ischemic preconditioning protocol that was followed by a 4-hour reperfusion. They found significantly lower histological scores in the early RIPC group compared to the I/R. In comparison with our findings, there were no significant changes between the RIPC-1 and the I/R groups. However, the values of the RIPC-24 group were significantly lower than that of the I/R. The reason for the difference might be the duration of the observed reperfusion period [27, 43]. Motta et al. observing only a 15-minute reperfusion, did not find significant changes in histopathological evaluation between early RIPC and warm I/R injury groups [44].

The effectiveness of RIPC may depend on the target organ. In a study of Magyar et al. similar protocols were used in a rat model of partial liver ischemia-reperfusion, but the histopathological evaluation showed different results [45]. The whole pathway of these alterations is unknown and forms a very complicated network. In the first line, the initiating effect is the mechanical trauma to blood, the hypoxia in parallel with the release of free radicals and tissue damage. Due to metabolic alterations, the rheological parameters worsen in the level of macro- and micro-rheology, similarly to different hematological parameters which leads to a decrease in blood flow. In this point, a vicious circle is formed because the decrease in blood flow worsens the local metabolic status of the organ [46 –48]. Further well-planned research is necessary to find the ideal preconditioning methods which examine co-morbidities and inter-species differences [47, 49].

5 Conclusion

Renal I/R injury causes significant alterations in hematological and microcirculatory parameters in the rat. Both delayed and early RIPC protocols can decrease the injury. Based on the vital, hematological, and microcirculatory parameters, it cannot decide which RIPC protocol is more effective. However, the histological scoring showed that the delayed-effect RIPC protocol is better in this model.

Footnotes

Acknowledgments

The authors are grateful for the technical staff of the Department of Operative Techniques and Surgical Research, the Faculty of Medicine, the University of Debrecen.

The authors comply with the Ethical Guidelines for Publication in Clinical Hemorheology and Microcirculation as published on the IOS Press website and in Volume 63, 2016, pp. 1-2. of this journal.

The experiment was supported by the ÚNKP-17-3, ÚNKP-18-2 New National Excellence Program of The Ministry of Human Capacities, EFOP-3.6.3-VEKOP-16-2017-00009 (co-financed by EU and the European Social Fund), and the Bridging Fund of the Faculty of Medicine of The University of Debrecen.

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