Remote Ischemic Preconditioning Reduces Cerebral Oxidative Stress Following Hypothermic Circulatory Arrest in a Porcine Model

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Remote Ischemic Preconditioning Reduces Cerebral Oxidative Stress Following Hypothermic Circulatory Arrest in A Porcine Model Oiva Arvola MD, Henri Haapanen MD, Johanna Herajärvi MB, Tuomas Anttila MB, Ulla Puistola MD PhD, Peeter Karihtala MD PhD, Hannu Tuominen MD PhD, Vesa Anttila MD PhD, Tatu Juvonen MD PhD www.elsevier.com/locate/buildenv

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S1043-0679(16)00019-8 http://dx.doi.org/10.1053/j.semtcvs.2016.01.005 YSTCS818

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Semin Thoracic Surg

Cite this article as: Oiva Arvola MD, Henri Haapanen MD, Johanna Herajärvi MB, Tuomas Anttila MB, Ulla Puistola MD PhD, Peeter Karihtala MD PhD, Hannu Tuominen MD PhD, Vesa Anttila MD PhD, Tatu Juvonen MD PhD, Remote Ischemic Preconditioning Reduces Cerebral Oxidative Stress Following Hypothermic Circulatory Arrest in A Porcine Model, Semin Thoracic Surg, http://dx.doi.org/10.1053/j.semtcvs.2016.01.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.


 

Remote ischemic preconditioning reduces cerebral oxidative stress following hypothermic circulatory arrest in a porcine model Oiva Arvola MD1, Henri Haapanen MD1, Johanna Herajärvi MB1, Tuomas Anttila MB1, Ulla Puistola MD PhD2, Peeter Karihtala MD PhD3, Hannu Tuominen MD PhD4 Vesa Anttila MD PhD1, Tatu Juvonen MD PhD5

1 Oulu University Hospital, Department of Surgery, Oulu, Finland 2 Oulu University Hospital, Department of Obstetrics and Gynecology, Oulu, Finland 3 Oulu University Hospital, Department of Oncology and Radiotherapy, Medical Research Center Oulu, Finland 4 Oulu University Hospital, Department of Pathology, Oulu, Finland 5 1) Department of Surgery University of Oulu 2) Department of Cardiac Surgery, Heart and Lung Center HUCH, Helsinki

Keywords: remote ischemic preconditioning, cardiovascular procedures, neuroprotection, central nervous system

This study was supported by grant money from the Finnish Foundation for Cardiovascular Research and Sigrid Juselius Foundation.

The authors report no conflicts.

Corresponding author: Prof. Tatu Juvonen, Department of Cardiac Surgery, Heart and Lung Center HUCH, Haarmanninkatu 4, 90029 Helsinki, Finland, [email protected]

 

ABBREVIATIONS

•OH

Hydroxyl radical

8-OHdG

8-hydroxydeoxyguanosine

ARE

Antioxidant response element

CI

Cardiac index

CNS

Central nervous system

CPB

Cardiopulmonary bypass

DJ-1/PARK7

Protein deglycase

ELISA

Enzyme-Linked Immunosorbent Assay

EtCO2

End-tidal carbon dioxide in expirated air

EtO2

End-tidal oxygen

HCA

Hypothermic circulatory arrest

HE

Hematoxylin-eosin

HIF-1-Į

Hypoxia-inducible factor-1-Į

KATP

ATP-sensitive potassium

LMM

Linear mixed model

MAP

Mean arterial pressure

mPTP

Mitochondrial permeability transition pore

Nrf2

Nuclear factor erythroid 2-related factor 2

OGG1

8-oxoguanine glycosylase

PCWP

Pulmonary capillary wedge pressure

PKC

Protein kinase C mediated pathway

RIPC

Remote ischemic preconditioning

ROS

Reactive oxygen species

SD

Standard deviation

 

ABSTRACT

Objective Remote ischemic precondition has become prominent as one of the most promising methods to mitigate neurological damage following ischemic insult. The purpose of this study was to investigate whether the effects of remote ischemic preconditioning can be seen in the markers of oxidative stress or in redox-regulating enzymes in a chronic porcine model.

Methods Twelve female piglets were randomly assigned to 2 groups. The study group underwent an intervention of 4 cycles of 5-minute ischemic preconditioning on the right hind leg. All piglets underwent

60-minute

hypothermic

circulatory

arrest.

Oxidative

stress

marker

8-

hydroxydeoxyguanosine (8-OHdG) was measured from blood samples with ELISA. After 7 days of follow-up, samples from the brain, heart, kidney, and ovary were harvested for histopathologic examination. The immunohistochemical stainings of hypoxia marker HIF-1-Į, oxidative stress marker 8-OHdG, DNA repair enzyme 8-oxoguanine glycosylase (OGG1), and antioxidant response regulators nuclear factor erythroid 2-related factor 2 (Nrf2) and DJ-1/PARK7 were analysed.

Results The level of 8-OHdG referred to baseline was decreased in the sagittal sinus’s blood samples in the study group after a prolonged deep hypothermic circulatory arrest at 360 minutes after reperfusion. Total histopathological score in the study group was 3.8 (1.8-6.0) and was 4.4 (2.5-6.5) in the control group (p=0.72),demonstrating no statistically significant difference in cerebral injury .

Conclusion Our findings demonstrate that the positive effects of remote ischemic preconditioning can be seen in cellular oxidative balance regulators in a chronic animal model after 7 days of preconditioned ischemic insult.

 

PERSPECTIVE STATEMENT

Procedures for repairing complex heart defects and Stanford type A aortic dissection have high rate of neurological complications. Remote ischemic preconditioning is one of the most promising methods to mitigate neurological damage. In this chronic porcine model, longer lasting alteration of cellular oxidative balance was revealed using this method.

CENTRAL PICTURE Remote ischemic preconditioning alters cellular oxidative balance after following ischemia

CENTRAL MESSAGE Remote ischemic preconditioning also alters cellular oxidative balance after subsequent ischemic insult.

INTRODUCTION Stanford type A aortic dissection is extremely life-threatening, with a mortality rate close to 90% without surgical intervention. Even though the operative management of lesions of the transverse aortic arch, techniques used, perioperative care, and intense care have improved during the past few decades, replacement of the aortic arch continues to have an in-hospital mortality rate of 8% to 15% and following neurological complications from 3 to 20%. (1-6). Deep hypothermic circulatory arrest mitigates ischemia-reperfusion damage by decreasing the metabolic rate of the central nervous system (CNS). Various perfusion strategies are also used to maintain the supply of metabolic demand of the CNS. Other approaches to suppress the neurological damage consist of drug therapies and remote ischemic preconditioning(7). Remote ischemic preconditioning (RIPC) has risen as one of the most promising methods to reduce the neurological damage in ischemia reperfusion injury. The mechanisms behind the beneficial effects of RIPC are considered in terms of triggers, mediators, and effectors. Protein kinase C mediated pathway (PKC) is considered to lower the production of reactive oxygen species

 

(ROS) through inhibition of the KATP channel. This inhibits the mitochondrial permeability transition pore (mPTP) during ischemia, ultimately resulting in lower production of ROS. The effectors are considered to consist of a neuronal pathway, a humoral pathway or a systemic response, or the combination of both (8-10). Both hypoxia and the reintroduction of oxygen to hypoxic cells result in the creation of reactive oxygen species (ROS). The hydroxyl radical (•OH) is the most unstable ROS, and its interaction with DNA leaves a specific and stable footprint, 8-hydroxydeoxyguanosine (8-OHdG), the expression of which can be reliably assessed with specific antibodies. OGG1 glycosylase excises and removes 8-OHdG from the damaged DNA, and thereafter it is secreted to the bloodstream and ultimately to the urine (11, 12). Nrf2 is the key sensor of cellular redox status, and under oxidative stress, it translocates from cytoplasm to nucleus and binds to antioxidant response element (ARE) in DNA together with small Maf proteins (13). Nrf2 expression associates with neuronal cell protection during ischemia (14). Hypoxia-inducible factor-1-Į (HIF-1-Į) is a constantly produced subunit that stabilises in hypoxic conditions, binding with other subunits, and that regulates hypoxia-inducible gene expression. Both Nrf2 and HIF-1-Į target to mitigate ROS production (15, 16). DJ-1 is a ubiquitous, multifunctional redox-regulating protein, and it is found in most tissues, including the brain. It is associated with countering oxidative stress (17). In our earlier study, leukocyte filtration during cardiopulmonary bypass reduced the neurological damage and decreased the number of cerebrocortical adherent leukocytes after hypothermic circulatory arrest (HCA). We also demonstrated that remote ischemic preconditioning altered in vivo adherent leukocytes in cerebral vessels after HCA (18, 19). The primary aim of the present study was to investigate whether the effects of remote ischemic preconditioning can be seen in the markers of oxidative stress or in the regulators of cellular oxidative balance in a chronic animal model. Therefore, the other principal aim of this study was to investigate whether the mechanism can be explained by reducing the oxidative damage

 

of the DNA and mitochondria, as a previously described trigger. We also sought to take a closer look at the systemic blood count to determine whether the humoral effector can be seen in leukocytes.

MATERIALS AND METHODS Experimental Setup Twelve female piglets from native stock were randomly assigned to 2 groups. Six of the animals were assigned to the intervention group, and 6 animals were assigned to the control group. Both groups underwent a prolonged 60-minute HCA. The animals of the intervention group were preconditioned with 4 cycles of a 5-minute ischemia, followed by a 5-minute reperfusion phase, in the right hind leg. The control group received a sham treatment, but no preconditioning. The randomisation was performed using sealed envelopes.

Preoperative Care All animals received humane care following the instructions of the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources, National Resource Council (Published by National Academy Press, revised 1996). This study was approved by the Research Animal Care and Use Committee of the University of Oulu.

Anesthesia Protocol and Hemodynamic Monitoring All piglets were sedated with intramuscular injection of ketamine (350 mg), midazolam (35 mg), and medetomidine (1.5 mg). The anesthesia induction consisted of intravenous injection of thiopental (5-7 mg/kg) and fentanyl (50 µg/kg) prior to endotracheal intubation with 6.5 mm cuffed tube. Anaesthesia was maintained with continuous infusion of fentanyl (25 µg/ [kg·h]), midazolam

 

(0.25 µg/ [kg·h]), and rocuronium (1.5 mg/ [kg·h]), with inhalation anesthesia of 1.5% sevoflurane throughout the experiment, excluding HCA. The piglets were ventilated 20 times per minute with positive-pressure of 5 cmH2O. The end-tidal carbon dioxide in expirated air (EtCO2) was kept at 5.0 kPa, and end-tidal oxygen (EtO2) was ensured at 50% in expirated air with End-Tidal Control of the GE Aisys CarestationTM (GE Healthcare, Madison, WI, USA). An arterial line was inserted in the left femoral artery for arterial blood pressure monitoring and arterial blood sampling. Cardiac output, pulmonary capillary wedge pressure (PCWP), central venous pressure, systemic blood temperature, and pulmonary pressure were monitored, and systemic blood samples were collected via pulmonary artery thermodilution catheter (CritiCath 7F; Ohmeda GmbH & Co, Erlangen, Germany), which was introduced through the left femoral vein. Cefuroxime at 750 mg was administered intravenously at anesthesia induction. Electrocardiographic monitoring was carried out throughout the operation.

Cranial Procedures A cranial window (35 mm · 40 mm) was created in the middle of the scull at coronal suture with a 14 mm/11 mm disposable cranial perforator (200-253 DGR-II, Acra Cut Inc., Acton MA, USA). Sagittal sinus was cannulated through dura mater with 22G 0.8 · 25 mm (22 G x 25 mm BD Venflon™ Peripheral IV Catheter with Injection port) cannula for blood sampling of the returning blood of the brain. After the experiment, the sagittal sinus was decannulated and the cannula enter wound was sutured with 7-0 non-absorbable polypropylene sutures to avoid subdural hematoma.

Remote Ischemic Preconditioning A pediatric blood pressure cuff was used for preconditioning in the intervention group of the right hind leg by applying a static pressure of 250 mmHg. 4 cycles of 5-minute ischemia followed by 5minute reperfusion were completed. The control group had the blood pressure cuff wrapped around

 

the right hind leg, but not pressurised, for the same time period. Cardiopulmonary bypass (CPB) was initiated 40 minutes after the intervention.

Cardiopulmonary Bypass Preoperatively, a membrane oxygenator (D905 Eos, Dideco, Mirandola, Italy) was primed with 800 ml of Ringer acetate solution and heparin (15,000 IU). Donor blood was also used in priming to maintain adequate hematocrit and hemoglobin levels in the experimental animals. The donor piglets were sedated, intubated, and kept under anesthesia as previously described, and euthanised after the procedure using pentobarbital (60 mg/kg) while anaesthetised. The donor and receiver blood were tested for cross reactions prior to cardiopulmonary bypass. The right anterolateral thoracotomy was performed in the 4th intercostal space, and the right atrium was exposed. After systemic heparinisation of 500 IU/kg, the right atrial appendage was cannulated using a 24F venous cannula, and an 18F arterial straight tip cannula was used for aortic cannulation. CPB was initiated, and cooling started immediately using the pH-stat strategy. The flow was adjusted to maintain mean arterial pressure of 55-60 mmHg with no vasoactive drugs. The core temperature was lowered to the target temperature of 18 ࢓C in 30 minutes using a heat exchanger. After the cooling phase, the perfusion was stopped and HCA was initiated for 60 minutes. Potassium chloride (40 mmol) was mixed with 40 ml of blood and used for cardioplegia to stop the heart. The arterial and rectal temperatures were monitored continuously and maintained at 18 ࢓C with topical ice packs surrounding the subject’s body and the head during HCA. Reperfusion was initiated 60 minutes after the beginning of the HCA and during the first 5 minutes was circulated at 18 ࢓C. Furosemide (40 mg), lidocaine (40 mg), methylprednisolone (40 mg), and calcium glubionate (90 mg) were administered to the CPB circulation at the beginning of the perfusion, and mannitol 100 ml (15 mg/100 ml) was administered after the blood samples at 10 minutes of rewarming. The piglets were warmed to 37 ࢓C during 45 minutes of reperfusion prior

 

to decannulation. The arterial pressure was maintained at 60 mmHg during reperfusion. The heart was defibrillated, if necessary, at 30 ࢓C. Ventilation was started 10 minutes before weaning from CPB. The metabolic follow-up under anesthesia lasted 6 hours after the HCA, and the total followup period was 7 days.

Biochemical, Metabolic, and Blood Count Data Baseline measurements were taken after anesthesia induction, cannulation of sagittal sinus, and invasive hemodynamic measurements had begun and the thoracotomy was ready for cannulation. The amount of fluids infused and donor blood were recorded; urine amount and central temperatures were monitored. Blood gas values, electrolytes, plasma lactate levels, pH, serum ionised calcium, venous glucose, hematocrit and hemoglobin levels, and blood count, as well as differential cell count, were measured at baseline, 15 minutes after intervention and sham treatment, after 20 minutes of cooling, and at 10 m, 1 h, 2 h, 4 h, and 6 h after the start of rewarming (i-STAT Analyzer; i-STAT Corporation, East Windsor, NJ, USA).

Enzyme-Linked Immunosorbent Assay (ELISA) The serum values of 8-OHdG were measured from both vena cava serum samples and sagittal sinus serum samples at baseline, post-intervention, and post-sham treatment, and 1 h and 6 h after the start of rewarming. The serum samples were stored in polypropylene tubes at -80-88 ࢓C until analysed. Highly Sensitive 8-OHdG Check ELISA kits were used to analyse the samples (from the Japan Institute for the Control of Aging, Fukuroi, Japan), following the manufacturer’s instructions.


 

Immunohistochemistry and Histopathological Analysis The brain, right ovary, and 2 cm × 2 cm biopsies from the right ventricle of the heart and right kidney were fixed in buffered 10% formalin solution for 2 weeks prior to paraffin-embedding and staining with hematoxylin-eosin (HE), as described in our earlier study (20). Table 1 shows the immunohistochemical methods used in this study. After staining, the samples were analysed by an experienced blinded pathologist. Samples from the right ventricle, ovary, kidney, and 3 regions of the CNS were scored. Scoring of the HE samples was based on the presence of edema (0 to 3), neuron degeneration (0 to 1), hemorrhages (0 or 2), and presence of infarcted tissue (0 or 3). The CNS total regional score was the sum of the scores in each specific brain area (cortex, thalamus, hippocampus, brainstem, cerebellum). A total histopathological score was calculated by summing all regional scores to allow semiquantitative comparison between the animals, as in our previous study (20). The immunohistochemical stainings were scored for DJ-1, 8-OHdG, HIF-1-Į, Nrf2, and Ogg1 using the following semiquantitative protocol: 0 = negative, 1 = positive, 2 = strongly positive, 3 = very strongly positive. All neurons from cortex and hippocampus and Purkinje neurons from the cerebellum were chosen for regions of the CNS. The heart, kidneys, and ovaries were also chosen for immunohistochemical staining (21).

Statistical Analysis Summary measurements are expressed as mean and standard deviation (SD) or as median and 25th – 75th percentiles. Either the Student t-test or Mann-Whitney U test was used to assess the distribution of continuous and ordinal variables between the study groups. The repeatedly measured data were analysed using a linear mixed model (LMM) with patients fitted as random, and the covariance pattern was chosen according to Akaike’s information criteria (22). P-values reported for the LMM are as follows: pt indicates change over time, pg indicates a level of difference between groups, and pt*g indicates interaction between groups and time. Univariate comparison between groups for



 

categorical variables was made using the Ȥ2-test and Fisher`s exact test, when appropriate. Twosided p-values are reported. Analyses were performed using SPSS (IBM Corp., Released 2012., IBM SPSS Statistics for Windows, Version 21.0. Armonk, NY: IBM Corp.) and SAS (version 9.3, SAS Institute Inc., Gary, NC., USA).

RESULTS Comparison of Study Groups One piglet from the control group was excluded due to failed cannulation of sagittal sinus and damage to the brain, reducing the control group to 5. The median weight of the piglets was 20.1 kg (18.1-21.7 kg). The median amount of donor blood used was 59.91 ml/kg (53.66-66.30 ml/kg). There were no statistically significant differences between the groups, considering the weight or the donor blood.

Metabolic and Hemodynamic Data The baseline electrolyte content and hemoglobin were comparable, with no significant differences between the groups. The hemodynamic data were comparable, as there were no significant differences in the mean arterial pressure, pulmonary capillary wedge pressure, or central venous pressure between the groups. There were no differences in cardiac index, arterial O2, infused amount of fluids, inotropes used, or temperatures between the groups throughout the experiment.

Blood 8-OHdG Concentrations The level of 8-OHdG measured from sagittal sinus tended to rise during the operating period in both groups. In the RIPC group, the measured 8-OHdG was returned to baseline 360 minutes after the initiation of reperfusion, whereas the 8-OHdG in the control group remained elevated. This


 

difference, however, did not reach a statistically significant difference. In vena cava samples, the behaviour of measured 8-OHdG was similar between the groups throughout the experiment. The measured 8-OHdG level referred to baseline, however, differed significantly in the sagittal sinus samples at 360 minutes. The compared value in the control group at 360 minutes after the reperfusion was 1.08 (1.01-1.10) and was 0.93 (0.89-0.93) in the RIPC group (p = 0.018) (Tables 2 and 3).

Systemic Complete Blood Count There were no differences between the groups with respect to white blood cell count and differential white cell count at baseline. During the experiment, the differential white cell count differed significantly, as the basophil count was significantly higher in the control group (pg = 0.023). The lymphocyte count seemed to rise rapidly in the control group after the rewarming phase, whereas the lymphocyte count of the RIPC group rose at much slower pace. This difference did not reach a statistically significant difference between the groups. The total white blood count is shown in Table 4.

Histopathology and Immunohistochemistry The CNS’s HE-stained regional and total scores did not reveal statistically significant differences between the groups. The total histopathological score was 3.8 (1.8-6.0) in the RIPC-group and 4.4 (2.5-6.5) in the control group. The difference was observed in cortex and thalamus regions. HIF-1-Į of the nucleus had strong and very strong positive staining in the CNS samples. All regional scores of CNS were higher in the control group, summing the total score to 7.2 (7.0-8.0), whereas the sum in the RIPC group was 5.3 (3.0-9.0); however, statistical significance was not reached (p = 0.41). HIF-1-Į differed significantly in the heart; however, the control group scored 2.8 (2.5-3), whereas the RIPC group scored 1.3 (0.8-2.3) (p = 0.026). The cytoplasmic Nrf2


 

staining was higher in the CNS samples of the RIPC group, but did not reach a statistically significant difference. DJ-1, 8-OHdG, and OGG1 stainings had no statistically significant differences between the groups. The histopathological and immunohistochemical data are presented in Tables 6 and 7. The findings in relation to the protocol, is presented in figure 1.

DISCUSSION The neuroprotective effect of remote ischemic precondition is not yet well-documented, but our findings are in line with previous reports. We also collected samples from the sagittal sinus to measure the cerebral oxidative stress accurately, as the metabolically active cerebral tissue is more sensitive to ischemic damage. The main finding of this study is that the reliable marker for oxidative stress 8-OHdG referred to baseline was reduced in the sagittal sinus’s samples in the RIPC group after a prolonged deep hypothermic circulatory arrest. The difference can be seen at 360 minutes after reperfusion, when the inflammatory response of ischemia reperfusion has resulted in more hydroxyl radical production. This could indicate the mitigation of cerebral oxidative stress and ischemia reperfusion injury in the RIPC group (23). The timing of the difference refers to the reduction in inflammatory response and supports our earlier findings (18). Such difference could not be seen in the samples of systemic blood flow. Another interesting finding relates to the complete blood count, in which it seems that the lymphocyte count tends to be higher in the control group at 60 minutes and 120 minutes after the initiation of reperfusion. This difference does not reach statistical power, however. The Tlymphocytes play a role in stroke patients producing and secreting cytokines that contribute to cerebral inflammation. The mechanism of how RIPC reduces oxidative stress in the central nervous


 

system after ischemia reperfusion injury can be partially explained by the alteration of immune response (24). There were no statistically significant differences in the total histopathological score between the groups. The expression of nucleus’s HIF-1-Į was statistically non-significantly higher in the CNS samples of the control group, after 7 days of follow-up. In normoxic cells, HIF-1-Į is rapidly degraded via the ubiquitin-dependent proteasome. HIF-1-Į also stabilises when ROS is present in the cytoplasm, following neuronal ischemia-reperfusion injury and damage to the bloodbrain-barrier (25, 26). Interestingly, the heart was observed with systematic statistically significant difference of HIF-1-Į expression. HE staining did not reveal structural differences between the groups. Remote ischemic preconditioning 40 minutes prior to ischemia-reperfusion injury seems to have long-lasting effects, as it lowers HIF-1-Į expression after 7 days of ischemic insult. As all of the heart samples observed demonstrated fibrinous pericarditis but no structural damage, RIPC might reduce inflammatory response and thus ROS and HIF-1-Į production. Cytoplasmic Nrf2 was observed in all of the samples of the CNS, and it was higher in cortex and hippocampus regions of the RIPC group. Nrf2 expression is associated with neuronal cell protection, as it responds to oxidative stress. However, we could not observe any presence of Nrf2 in the nucleus, where it binds to antioxidant response element leading to induction of cytoprotective genes (27). HIF-1-Į also downregulates Nrf2, which can explain our findings of difference in cytoplasmic Nrf2 expression between the groups (28). There were no differences in the immunohistochemical expression of OHdG, OGG1, and DJ-1 between the study groups. This may be due to the fact that 8-OHdG, OGG1, and DJ-1 are rapidly induced acute phase enzymes and adducts, but the samples were collected after 7 days of follow-up.


 

CONCLUSIONS This study demonstrates that the remote ischemic preconditioning before deep HCA seems to lower ischemia reperfusion-related oxidative stress in the brain 6 hours after the initiation of reperfusion. It also shows a long-term statistically non-significant difference in markers of oxidative stress and in the regulators of cellular oxidative balance in a chronic animal model. However, with this study group size, we could not demonstrate differences in the neurological outcome after 7 days of follow-up period. Further investigation is needed to evaluate the role of white blood cells in mitigating ischemia reperfusion injury in remote ischemic preconditioned patients.

ACKNOWLEDGEMENTS We show our gratitude to Seija Seljanpera RN for help in caring for the animals. We also thank biostatistician Pasi Ohtonen, MSc, for his expertise with statistical analysis.

Figure 1. Remote ischemic preconditioning alters cellular oxidative balance after following ischemia


 

Table 1. Immunohistochemical methods used in this study Antibody (clone/product

Dilution

Immunostaining

code) Nrf-2 (C-20)

Source of Primary Antibody

Method 1:300

Dako Envision Kit

Santa Cruz Biotechnology, Santa Cruz, USA

Ogg-1

1:500

Dako Envision Kit

Novus Biologicals

1:1000

Novo Link kit

Neo Markers

1:50

Invitrogen kit

Japan Institute For the Control of

(pAb anti Ogg-1 Antibody) HIF-1-Į (Ab-4 H1alpha67) 8-OHdG

Aging DJ-1/ PARK 7 (ab 18257)

1:20000

Envision kit

 




Table 2. 8-OHdG concentration measured a Baseline 8-OHdG in sagittal sinus blood (ng/ml)

Post RIPC

2.43 (2.143.42) 2.84 (2.76Control 2.99) 0.855 P-value 8-OHdG in vena cava blood (ng/ml) 2.76 (2.16RIPC 3.34) 3.06 (3.02Control 3.54) 0.361 P-value

2.71 (2.213.26) 3.33 (2.613.78) 0.465

RIPC

a

2.59 (2.123.28) 3.04 (2.823.47) 0.465

60 minutes 360 minutes After the initiation of warming phase 2.68 (2.292.77)

1.000

2.44 (2.003.03) 3.03 (2.693.45) 0.234

2.82 (2.662.96) 2.92 (2.853.47) 0.361

2.47 (2.292.72) 2.58 (2.583.21) 0.272

2.59 (2.4-2.94)

pg

0.50 0.46 7 9

0.47 0.85 4 9

Values are shown as medians and 25th and 75th percentiles;

pg = p value between the groups (level of difference between the groups); pg*t = value time * group (behaviour between groups over time); RIPC = remote ischemic preconditioning group; Control = Control group.

pg*t


 

Table 3. 8-OHdG concentration compared with baseline a Baseli ne

Post RIPC

60 minutes

360 minutes

After the initiation of warming phase

8-OHdG in sagittal sinus blood RIPC

1.00

Control

1.00

P-value 8-OHdG in vena cava blood

1.06 (1.001.13) 1.13 (1.071.17) 0.465

1.03 (0.881.15) 0.94 (0.851.07) 0.715

0.93 (0.890.93) 1.08 (1.011.10) 0.018

pg

pg*t

0.30 0

0.56 7

0.98 (0.901.01 (0.910.87 (0.801.01) 1.16) 0.96) 1.01 (0.980.93 (0.880.91 (0.850.85 0.97 1.00 Control 1.02) 1.01) 0.91) 6 8 0.465 0.584 0.584 P-value a Values are shown as medians and 25th and 75th percentiles; pg = p value between the groups (level of difference between the groups); pg*t = value time * group (behaviour between groups over time); RIPC = remote ischemic preconditioning group; Control = Control group. RIPC

1.00


 

Table 4. Complete blood count

Variable

Total leukocyte count (109/l) RIPC

Control

Neutrophi ls (109/l) RIPC

Control

Lymphocy tes (109/l) RIPC

Control

Monocytes (109/l) RIPC

Control

Baselin e

Post Coolin Warmi 60 120 240 360 RIP g 20 ng minut minut minut minut C 10 es es es es minute s End of HCA after the initiation of warming phase pg

15.7 (14.818.6) 15.3 (15.223.0) (p=0.85 5)

16.1 6.6 (14.4- (4.818.0) 10.3) 17.5 9.0 (16. (7.088.6) 21.4 )

5.0 (4.17.1) 8.3 (7.08.6)

13.1 (9.219.5) 18.8 (12.128.1)

24.9 (23.735.4) 35.1 (25.844.4)

35.7 (26.243.6) 39.7 (29.23 -45.3)

30.4 (25.935.9) 42.4 (27.049.2)

3.0 (2.64.3) 3.3 (2.97.1) (p=0.53 7)

3.1 (2.34.4) 4.6 (4.55.3)

1.6 (0.92.3) 1.5 (1.02.0)

0.8 (0.51.7) 1.2 (0.81.5)

2.7 (1.48.2) 3.9 (3.66.0)

11.9 (8.619.1) 10.2 (6.012.0)

14.3 (5.524.2) 12.1 (6.117.8)

9.4 (6.118.4) 4.8 (3.422.4)

12.1 (10.113.2)

4.7 (3.87.2)

3.8 (3.44.5)

9.4 (7.012.4)

13.5 (11.514.4)

19.3 (18.220.8)

18.8 (11.628.0)

11.6 (11.314.6) (p=0.93 1)

11.7 (10. 913.7 ) 11.8 (11. 714.9 )

5.8 (5.27.0)

6.2 (5.06.6)

16.4 (11.322.1)

19.9 (12.828.0)

22.2 (20.725.9)

21.6 (20.729.2)

0.9 (0.71.0) 0.7 (0.6-

0.6 (0.51.0) 0.8 (0.6-

0.2 (0.10.3) 0.4 (0.2-

0.1 (0.10.3) 0.2 (0.2-

0.5 (0.30.9) 0.4 (0.2-

0.8 (0.61.0) 0.8 (0.8-

1.1 (1.01.3) 1.3(1. 0-1.6)

1.0 (0.91.7) 1.2 (1.0-

pg*t

0.237 0.59 3

0.993 0.94 4

0.116 0.77 0

0.823 0.67 6


 

Eosinophil s (109/l) RIPC

Control

Basophils (109/l) RIPC

Control

0.8) (p=0.66 2)

1.0)

0.4)

0.2)

1.0)

1.4)

0.1 (00.1)

0.1 (0.10.3) 0.06 (00.15 )

0.1 (00.1) 0.08 (0.050.15)

0.03 (0.010.04) 0.04 (0.040.07)

0.06 (0.040.09) 0.14 (0.030.19)

0.07 (0.050.1) 0.11 (0.070.16)

0.02 (0.020.04) 0.05 (0.040.07)

0.01 (0.010.02) 0.02 (0.015 0.025)

0.04 (0.0 3006) 0.06 (0.0 50.08 )

0.02 (0.020.03)

0.025 (0.020.03)

0.035 (0.030.04)

0.045 (0.040.05)

0.045 (0.040.06)

0.04 (0.030.04)

0.04 (0.030.04)

0.03 (0.020.04)

0.06 (0.040.09)

0.08 (0.050.09)

0.08 (0.070.1)

0.09 (0.050.09)

0.11 (0.090.22) (p=0.42 9)

0.06 (0.060.06) 0.05 (0.040.11) (p=0.93 1)

1.8)

0.312 0.24 4

0.023 0.46 * 1

Table 4, Values are shown as medians and 25th and 75th percentiles; p = p value between groups at baseline; pg = p value between the groups (level of difference between the groups); pg*t = value time * group (behaviour between groups over time); RIPC = remote ischemic preconditioning group; Control = Control group

  Table 5. Hemodynamic data

Variable

Baselin e

Post RIPC

Coolin Warmi 60 120 240 360 g 20 ng 10 minut minut minut minut minutes es es es es End of HCA after the initiation of warming phase

CI (l/min/m2) RIPC

Control

5.22 (4.685.27)

4.17 (84.0 24.82) 4.24 (4.164.8)

3.17 (2.73.36)

3.48 (2.824.9)

4.5 (2.686.3)

3.71 (2.994.13)

3.6(3.2 -4.02)

4.04 (2.955.19)

2.82 (2.593.26)

3.6 (3.283.95)

4.16 (3.74.37)

3.49 (3.273.54)

4.05 (44.44)

3.67 (3.164.38)

73 (69102) 68 (6488)

57 (5758) 61 (5562)

60(5362)

74 (6980) 79 (7384)

78 (7378) 57 (5558)

62 (5767) 60 (5867)

57 (5669) 55 (5459)

7.51 (7.477.52) 7.53 (7.497.55) (p=0.52 2)

7.51 (7.487.52) 7.48 (7.487.52)

7.46 (7.387.62) 7.39 (7.357.41)

7.39 (7.307.54) 7.36 (7.317.39)

7.42 (7.377.45) 7.34 (7.337.38)

7.47 (7.457.50) 7.44 (7.447.44)

7.51 (7.487.52) 7.46 (7.447.47)

7.49 (7.477.50) 7.47 (7.527.49)

40.8 (38.642.9)

37.9 (36.442,0)

83.3 (82.685)

33.6 (29.338.7)

34.0 (29.338.7)

33.1 (29.235.2)

33.4 (27.837.9)

41.9 (37.244.3) (p=0.85 5)

40.9 (3743.3)

106.6 (106.5 106.5) 106.6 (106.5 106.5)

86.5 (85.690.4)

32.3 (2.434.5)

33.8 (23.934.1)

34.1 (24.635.4)

31.1 (25.136.1)

82 (7882)

82 (8285)

73 (5485)

78 (7182)

84 (7888)

73 (6175)

80 (7287)

5.27 (4.315.63) (p=0.58 4)

pg

pg*t

0.80 9

0.92 3

0.58 9

0.19 6

0.09 9

0.30 1

0.87 5

0.63 8

MAP (mmHg) RIPC

90 (81107)

Control

93 (6999) (p=0.64 7)

62 (6063)

Arterial pH RIPC

Control

Arterial pO2 (kPa) RIPC

Control

Hemoglobin (g/l) RIPC

80 (7182)


  Control

Rectal temperature (centigrade) RIPC

Control

Blood temperature (centigrade) RIPC

Control

78 (7882) (p=0.55 5)

85 (8285)

78 (7878)

75 (7178)

78 (7585)

88 (6892)

75 (6175)

65 (4468)

38.0 (37.338.3) 37.2 (37.137.6) (p=0.12 0)

38.5 (37.538.9) 37.3 (37.137.5)

23.5 (18.425.9) 21.9 (18.324.7)

21.0 (20.522.4) 23.4 (21.523.8)

35.6 (34.335.7) 35.1 (34.835.6)

36.0 (35.536.7) 36.6 (35.936.6)

37.9 (37.338.4) 37.4 (37.338.0)

37.5 (37.338.5) 37.1 (37.137.9)

38.1 (37.338.3) 37.2 (37.137.6) (p=0.11 9)

38.6 (37.838.7) 37.3 (37.137.6)

17.5 (17.518.2) 17.5 (17.517.5)

22.6 (19.723.8) 24.8 (23.925.4)

35.8 (34.936.1) 35.9 (34.836.2)

36.6 (34.537.1) 36.8 (36.636.8)

38.4 (37.739.3) 37.4 (37.038.0)

38.5 (3838.7) 37.4 (37.438.8)

0.29 1

0.10 6

0.65 3

0.66 4

0.78 2

0.36 7

Table 5. Values are shown as medians and 25th and 75th percentiles; p = p value between groups at baseline; pg = p value between the groups (level of difference between the groups); pg*t = value time * group (behaviour between groups over time); RIPC = remote ischemic preconditioning group; Control = Control group; MAP = Mean arterial pressure; CI = Cardiac index



Table 6. Histopathology Protocol

Cortex Score

Thalamus Score

Hippocampus Score

Brainstem Score

Cerebellum Score

Total Score

RIPC mean 2.0 (1.0-3.0) 0.7 (0.0-1.0) 0.8 (0.0-1.3) 0.2 (0.0-0.3) 0.2 (0.0-0.3) 3.8 (1.8-6.0) Control mean 2.2 (1.0-3.5) 1.0 (0.5-1.5) 0.8 (0.5-1.0) 0.2 (0.0-0.5) 0.2 (0.0-0.5) 4.4 (2.5-6.5) 0.82 0.41 0.93 0.90 0.90 0.72 P-value RIPC n = 6, Control n=5. Histopathological score of HE stained brain samples gathered after the 7th postoperative day. Score; mean (interquartile range), Mann-Whitney U.

 

Table 7. Immunohistochemistry Immunosta ining

Proto col

DJ-1

RIPC mean

CRTL mean

8-OHdG

Pvalue RIPC mean

CRTL mean

HIF-1-Į

Pvalue RIPC mean

CRTL mean

Nrf2

Pvalue RIPC mean

Cortex Score

Hippoca mpus Score

Cerebel lum Score

Tot al Sco re

Ovary

Kidney

Heart

0.0 (0.00.0)

0.0 (0.00.0)

0.2 (0.00.3)

0.2 (0.0 0.3)

0.7 (0.01.0)

0.8 (0.751.0)

0.0 (0.00.0)

0.0 (0.00.0)

0.0 (0.00.0)

0.0 (0.00.0)

1.0 (1.01.0)

1.0 (1.01.0)

0.0 (0.00.0)

-

-

0.36

0.0 (0.0 0.0) 0.3 6

0.18

0.36

-

0.0 (0.00.0)

0.0 (0.00.0)

0.2 (0.00.3)

0.2 (0.0 0.0)

0.7 (0.01.5)

0.3 (0.01.0)

0.8 (0.02.3)

0.4 (0.01.0)

0.4 (0.01.0)

0.0 (0.00.0)

0.4 (0.01.0)

0.4 (0.01.0)

0.8 (0.01.5)

0.18

0.18

0.36

0.8 (0.0 2.0) 0.2 9

0.66

0.89

0.96

2.0 (1.03.0)

1.5 (0.01.0)

1.8 (0.83.0)

5.3 (3.0 9.0)

0.2 (0.00.3)

0.8 (0.751.0)

1.3 (0.82.3)

2.6 (2.03.0)

2.2 (1.02.0)

2.4 (2.03.0)

0.0 (0.00.0)

0.4 (0.01.0)

2.8 (2.53.0)

0.34

0.29

0.32

7.2 (7.0 8.0) 0.4 1

0.36

0.19

0.026*

2.3 (1.03.0)

2.2 (1.83.0)

1.7 (0.03.0)

6.2 (5.0 7.0)

0.3 (0.01.0)

1.0 (1.01.0)

1.2 (0.81.5)

 

CRTL mean

Ogg1

Pvalue RIPC mean

CRTL mean

1.2 (0.01.0)

1.4 (0.02.5)

1.6 (0.53.0)

0.16

0.30

0.94

0.0 (0.00.0)

0.0 (0.00.0)

0.3 (0.00.5)

0.0 (0.00.0)

0.0 (0.00.0)

0.0 (0.00.0)

4.2 (3.0 6.0) 0.2 3

0.0 (0.00.0)

0.8 (0.51.0)

1.8 (1.52.0)

0.18

0.37

0.20

0.3 (0.0 0.0)

0.3 (0.01.0)

0.2 (0.00.3)

0.0 (0.00.0)

0.0 0.6 (0.0- 0.0 (0.0- 0.0 (0.0(0.0 1.0) 0.0) 0.0) 0.0) P0.36 0.3 0.43 0.36 value 6 RIPC n = 6, Control n=5. Imunohistochemical data after the 7th postoperative day. Score; mean (interquartile range), Mann-Whitney U.

 

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