A novel small animal extracorporeal circulation model for studying pathophysiology of cardiopulmonary bypass

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  • ORIGINAL ARTICLE Cardiopulmonary Bypass

    A novel small animal extracorporeal circulation modelfor studying pathophysiology of cardiopulmonary bypass

    Yutaka Fujii Mikiyasu Shirai Shuji Inamori

    Yoshiaki Takewa Eisuke Tatsumi

    Received: 23 May 2014 / Accepted: 21 October 2014

    The Japanese Society for Artificial Organs 2014

    Abstract Extracorporeal circulation (ECC) is indispens-

    able for cardiac surgery. Despite the fact that ECCcauses

    damage to blood components and is non-physiologic, its

    pathophysiology has not been fully elucidated. This is

    because difficulty in clinical research and animal experi-

    ments keeps the knowledge insufficient. Therefore, it is

    desirable to have a miniature ECC model for small ani-

    mals, which enables repetitive experiments, to study the

    mechanism of pathophysiological changes during ECC.

    We developed a miniature ECC system and applied it to

    the rat. We measured changes in hemodynamics, blood

    gases and hemoglobin (Hb) concentration, serum cytokines

    (TNF-a, IL-6, IL-10), biochemical markers (LDH, AST,ALT), and the wet-to-dry weight (W/D) ratio of the lung

    for assessing whether the rat ECC model is comparable to

    the human ECC. The ECC system consisted of a mem-

    branous oxygenator (polypropylene, 0.03 m2), tubing line

    (polyvinyl chloride), and roller pump. Priming volume of

    this system is only 8 ml. Rats (400450 g) were divided

    into the SHAM group (n = 7) and the ECC group (n = 7).

    Blood samples were collected before, 60 and 120 min after

    initiation of ECC. During ECC, blood pressure and Hb

    were maintained around 80 mmHg and 10 g/dL, respec-

    tively. The levels of the inflammatory and biochemical

    markers and the W/D ratio were significantly elevated in

    the ECC group, indicating some organ damages and sys-

    temic inflammatory responses during ECC. We success-

    fully established the ECC for the rat. This miniature ECC

    model could be a useful approach for studying the mech-

    anism of pathophysiology during ECC and basic assess-

    ment of the ECC devices.

    Keywords Extracorporeal circulation Rat ECC model Inflammatory response Biological reaction

    Introduction

    Extracorporeal life support (ECLS) devices, such as the

    cardiopulmonary bypass, preserve the patients life by

    providing adequate oxygen supply and blood flow to vital

    organs [1]. However, cardiac surgery with the use of

    extracorporeal circulation (ECC) is often accompanied by

    the systemic inflammatory response, influencing signifi-

    cantly the morbidity and mortality after ECC [2]. Further

    studies are needed to elucidate the pathophysiology during

    ECC. However, difficulty in clinical research and animal

    experiments keeps its elucidation insufficient. Therefore, it

    is desirable to have a miniature ECC model for small

    animals, which enables repetitive experiments, to study the

    mechanism of pathophysiological changes during artificial

    perfusion.

    In this study, we developed a miniature ECC model and

    applied the system to the rat. For assessing whether the rat

    ECC model is comparable to the human ECC, we mea-

    sured changes in the hemodynamics, blood gases and Hb,

    Y. Fujii (&) Y. Takewa E. TatsumiDepartment of Artificial Organs, National Cerebral and

    Cardiovascular Center Research Institute, 5-7-1, Fujishiro-dai,

    Suita, Osaka 565-8565, Japan

    e-mail: yfujii@ncvc.go.jp

    M. Shirai

    Department of Cardiac Physiology, National Cerebral and

    Cardiovascular Center Research Institute, 5-7-1, Fujishiro-dai,

    Suita, Osaka 565-8565, Japan

    S. Inamori

    Department of Clinical Engineering Faculty of Health Sciences,

    Hiroshima International University, 555-36, Kurose-gakuendai,

    Higashi-hiroshima, Hiroshima 739-2631, Japan

    123

    J Artif Organs

    DOI 10.1007/s10047-014-0804-y

  • serum cytokines: tumor necrosis factor-a (TNF-a), inter-leukin-6 (IL-6), and interleukin-10 (IL-10), and biochem-

    ical markers: lactate dehydrogenase (LDH), aspartate

    aminotransferase (AST), and alanine aminotransferase

    (ALT), and the wet-to-dry weight (W/D) ratio of the lung.

    Materials and methods

    Animal

    The study was approved by the National Cerebral and

    Cardiovascular Center Research Institute Animal Care and

    Use Committee, and all procedures met the National

    Institutes of Health guidelines for animal care.

    SpragueDawley rats (male 400450 g) were housed

    three per cage under a 12-h lightdark cycle with food and

    water available ad libitum.

    Anesthesia, surgical preparation, and extracorporeal

    circulation

    The animals were anesthetized with pentobarbital sodium

    (50 mg/kg body weight intraperitoneal injection), placed in

    the supine position and rectal thermocouple probe kept in

    place. Then, orotracheal intubation was performed using a

    14G cannula (Insyte BD Medical, Sandy, Utah) and rats

    were ventilated with a respirator (Model SN-480-7, Shi-

    nano Seisakusho Co., Ltd, Tokyo, Japan). Ventilation was

    volume-controlled at a frequency of 70/min, a tidal volume

    of 810 ml/kg body weight and 100 % of inspired oxygen

    fraction. Rectal temperature was maintained at 36 Cthroughout the experiment. Arterial blood pressure was

    monitored (Model 870, PowerLab system, AD Instruments,

    Castle Hill, Australia) via the femoral artery, which was

    cannulated with polyethylene tubing (SP-31 Natsume Sei-

    sakusho Co., Ltd, Tokyo, Japan). The left common carotid

    artery was cannulated with a polyethylene tubing (SP-55

    Natsume Seisakusho Co., Ltd, Tokyo, Japan) to serve as

    the arterial inflow cannula for the ECC circuit. 500 IU/kg

    heparin sodium was administered after placement of this

    cannula. A 16G cannula (Insyte BD Medical, Sandy, Utah)

    was advanced through the right external jugular vein into

    the right atrium and served as a conduit for venous outflow.

    The small animal ECC system (Fig. 1) consisted of a

    membranous oxygenator (polypropylene, 0.03 m2: Senko

    Medical Co., Ltd, Osaka, Japan), tubing line (Senko

    Medical Co., Ltd, Osaka, Japan) and roller pump (Micro

    tube pump MP-3 Tokyo Rikakikai Co., Ltd, Tokyo, Japan)

    was primed by 5 ml of Ringers solution, 1 ml of mannitol,

    1 ml of sodium bicarbonate, and 1 ml (1000 IU) of hepa-

    rin. Total priming volume of this system was 8 ml.

    Fig. 1 The small animal ECCsystem. Polypropylene

    membranous oxygenator with

    membrane area of 0.03 m2 and

    polyvinyl chloride tubing line

    (Senko Medical Co., Ltd,

    Osaka, Japan), and roller pump

    (MP-3 Tokyo Rikakikai Co.,

    Ltd, Tokyo, Japan) are shown

    J Artif Organs

    123

  • Experimental design

    The animals were divided into 2 groups: SHAM group

    (n = 7) and ECC group (n = 7). The SHAM group

    received surgical preparation only without CPB. In the

    ECC group, ECC was initiated and maintained at 70 ml/kg/

    min for 60 min.

    Partial pressure of arterial carbon dioxide (PaCO2) and

    partial pressure of arterial oxygen (PaO2) were maintained

    at 3545 mmHg and 300400 mmHg. Blood samples were

    collected at three defined time points, before ECC (pre-

    ECC), 60 min after initiation of ECC. and 120 min after

    initiation of ECC (end-ECC).

    To evaluate the inflammatory responses [3], TNF-a, IL-6, IL-10 were measured by enzyme-linked immunosorbent

    assay (ELISA kit, R&D systems, MN, USA). The con-

    centrations of LDH, AST, and ALT which are used as

    biochemical markers for evaluating organ damage [4] were

    measured (DRI-CHEM 7000 Analyzer, FUJIFILM,

    Kanagawa, Japan).

    Blood gases, pH, hemoglobin concentration, and elec-

    trolytes were also measured (ABL800 FLEX system,

    RADIOMETER, Copenhagen, Danmark). Animals in

    which the hemoglobin level declined to less than 8 g/dL at

    any point were excluded from the study. In general, when

    the hemoglobin becames 78 g/dL in clinical site, we

    consider blood transfusion [5, 6]. In this study, the purpose

    was to perform extracorporeal circulation without blood

    transfusion. All animals were killed at the end of ECC by

    potassium chloride injection and the left lung was harvested

    and divided into three parts. The superior third was used for

    the calculation of W/D ratio. The lung block was weighed

    before and after desiccation for 72 h in a dry oven at 70 C.

    Statistics

    All data are expressed as mean standard deviation (SD).

    The Students t test was used for subsequent comparison

    between groups at the same time points. All statistical

    analyses were performed using Stat-View 5.0 (Abacus

    Concepts, Berkeley, CA). Significance was set at P \ 0.05.

    Results

    Table 1 shows the changes in hemodynamic variables, Hb

    concentration, PaO2, PaCO2, and level of electrolyte in the

    SHAM and ECC groups during experiments. During ECC,

    MAP and Hb were significantly decreased but were

    maintained around 80 mmHg and 10 g/dL, respectively.

    All rats hemoglobin level did not fall below 8 g/dL at any

    point. There was no exclusion in the both groups. There

    were no significant changes in the value of the electrolyte

    in the both groups. However, in the ECC group, it tended to

    high potassium during ECC.

    Before ECC, the serum levels of inflammatory and

    biochemical markers were not statistical different between

    the SHAM and ECC groups. Serum inflammatory and

    biochemical markers remained unchanged during experi-

    ment periods in the SHAM group. In the ECC group, all the

    systemic inflammatory markers increased significantly,

    reaching a maximum (TNF-a 1129 137 pg/ml, IL-61157 150 pg/ml, IL-10 385 55 pg/ml) at the end of

    ECC (Fig. 2ac). Additionally, in the ECC group, the

    levels of biochemical markers significantly increased

    (LDH 425 65 U/L, AST 113 6 U/L, ALT 55 8

    U/L) 60 min after the ECC initiation and increased further

    (LDH 708 126 U/L, AST 76 7 U/L, ALT 159 14

    U/L) 120 min after the ECC initiation (Fig. 2df).

    The ECC group showed significantly higher W/D ratio

    of the lung than the SHAM group (SHAM 4.68 0.18,

    ECC 5.46 0.23) (Fig. 3).

    Discussion

    In this study, our small animal ECC system was able to

    maintain adequate levels of blood gases and Hb, and blood

    pressure. Furthermore, our model offers the advantage of a

    low priming volume not requiring transfusion in ECC

    group rats.

    Table 1 Hemodynamic variables, Hb and blood gas partial pres-sures, and level of electrolyte before and during ECC

    Group Pre-ECC ECC 60 min ECC

    120 min

    MAP

    (mmHg)

    SHAM 103 11 100 13 105 11

    ECC 102 5 94 24 87 19*

    HR (beat/

    min)

    SHAM 387 38 373 38 389 26

    ECC 395 25 366 30 365 17

    PaO2(mmHg)

    SHAM 110 17 106 16 105 14

    ECC 112 12 421 40* 412 34*

    PaCO2(mmHg)

    SHAM 38 3 37 2 40 2

    ECC 41 3 40 3 39 3

    Hb (mg/dL) SHAM 14.7 1.1 14.5 0.9 14.2 0.9

    ECC 15.1 1.0 11.8 1.1* 11.6 1.0*

    Na (mEq/L) SHAM 139.6 1.1 140.6 1.2 141.0 0.9

    ECC 138.9 0.9 141.2 1.0 142.0 1.3

    K (mEq/L) SHAM 5.2 0.2 5.4 0.3 5.5 0.3

    ECC 5.1 0.2 5.7 0.4 5.9 0.5

    Cl (mEq/L) SHAM 105.6 1.5 108.6 1.4 107.3 2.1

    ECC 106.1 1.8 108.9 2.2 108.7 2.7

    Variables are expressed by mean standard deviation

    * P \ 0.05 versus SHAM group at the same time

    J Artif Organs

    123

  • The significant systemic inflammatory responses

    occurred, reaching a maximum at the end of ECC. Addi-

    tionally, the biochemical markers reflecting organ damages

    significantly increased 60 min after the ECC initiation and

    increased further 120 min after the ECC initiation. The

    significant increase in the W/D ratio of the lung which

    suggests pulmonary edema [7, 8] is consistent with the

    previous study data [9]. From these data, our rat ECC

    model is considered useful for studying mechanism of

    pathophysiology during ECC, as an alternative to the

    established human ECC, which is often associated with

    systemic inflammation and organ damage [10].

    It has been suggested that the factors responsible for the

    inflammatory response during ECC are blood contact with

    the surface of the extracorporeal circulation unit, endo-

    toxemia, surgical trauma, ischemic reperfusion injury, and

    blood loss [10, 11]. Many studies showed the blood con-

    tacting surface of the ECC circuit activates white cells,

    platelets, and the complement system. The increase in

    cytokines, such as interleukins and necrosis factor [12],

    aggravates the inflammatory response [13]. These complex

    interactions during ECC lead to further inflammation [13].

    In our rat ECC models, the insufflation of hydrogen which

    selectively reduces the hydroxyl radical could decrease the

    levels of serum cytokines and biochemical markers, and the

    Fig. 2 Serum tumor necrosisfactor (TNF)-a (a), interleukin(IL)-6 (b), interleukin (IL)-10(c), lactate dehydrogenase(LDH) (d), aspartateaminotransferase (AST) (e),alanine aminotransferase (ALT)

    (f). *P \ 0.05 versus SHAMgroup at the same time periods

    Fig. 3 Wet-to-dry ratio of the left lung at the end of CPB. *P \ 0.05versus SHAM group

    J Artif Organs

    123

  • W/D ratio of the lung [7, 8]. These findings suggest that

    hydroxyl radical contributes toward promoting the sys-

    temic inflammatory responses and organ damages during

    ECC [7, 8].

    In the current study, we have not been able to perform

    an analysis of hemolysis. The possibility of hemolysis in

    the ECC group cannot be denied. Therefore, in the next

    study, we are going to analyze for damage of blood cells.

    Furthermore, in the future, we will conduct research on

    pathophysiology of cardiopulmonary bypass by using this

    novel small ECC model.

    Conclusion

    In this study, we developed a novel small ECC model and

    applied the system to the rat. In our rat ECC models, we

    demonstrated that adequate levels of blood gases and Hb,

    and blood pressure were maintained and that the systemic

    inflammatory response and organ damages including pul-

    monary edema were induced associated with the produc-

    tion of cytokines. This novel small ECC model could be a

    useful approach for studying the mechanism of patho-

    physiology (systemic inflammation and organ damage)

    during ECC and basic assessment of the ECC devices.

    Conflict of interest The authors have no conflict of interest directlyrelevant to the content of this article.

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