Article, Emergency Medicine

Effects of therapeutic hypothermia on coagulopathy and microcirculation after cardiopulmonary resuscitation in rabbits

Unlabelled imageAmerican Journal of Emergency Medicine (2011) 29, 1103-1110

Original Contribution

Effects of therapeutic hypothermia on coagulopathy and microcirculation after cardiopulmonary resuscitation in rabbits

Hu Chun-Lin MD a, Wen Jie MD b, Liao Xiao-Xing PHD a, Li Xing MD a,

Li Yu-Jie PHD a, Zhan Hong a, Jing Xiao-Lia,?, Wu Gui-Fu PHD c

aDepartment of Emergency Medicine, the First Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510080, China

bDepartment of the Pharmacy Development Centre, Sun Yat-Sen University, Guangzhou 510080, China.

cDepartment of Assisted Circulation Laboratory, Sun Yat-Sen University, Guangzhou 510080, China.

Received 23 April 2010; revised 26 June 2010; accepted 30 July 2010

Abstract

Objective: The aim of this study was to investigate the effects of Therapeutic hypothermia on coagulopathy and cerebral microcirculation disorder after cardiopulmonary resuscitation (CPR) in rabbits. Methods: Cardiac ventricular fibrillation was induced by alternating current in 24 New Zealand rabbits, and hypothermia was induced by Surface cooling or normothermia (NT) was maintained for 12 hours after the return of spontaneous circulation (ROSC). Several physiologic indexes were measured before CPR and at 4, 8, and 12 hours after ROSC. The microcirculation flow in the cerebral cortex was measured with a PERIMED Multichannel Laser Doppler system (Perimid, Sweden), and glomerular fibrin deposition was determined by microscopy.

Results: Compared with the NT group, the prothrombin time, Activated partial thromboplastin time, and international normalized ratio in the TH group were increased; there were no differences in anti-thrombin- III, protein C, and D-dimer indexes. The microcirculation flow in the cerebral cortex before CPR and after ROSC at 4, 8, and 12 hours was 401.60 +- 11.76, 258.86 +- 34.58, 317.59 +- 23.36, and 371.98 +- 5.79 mL/

min, respectively, in the NT group, and 398.18 +- 12.91, 336.19 +- 19.27, 347.76 +- 13.80, and 383.78 +-

3.29 mL/min, respectively, in the TH group. There were apparent disparities at each checkpoint after ROSC in these 2 groups (4 hours: P = .001; 8 hours: P = .011; 12 hours: P = .009). The Pearson correlation test showed that the microcirculation flow in the cerebral cortex was positively correlated with activated partial thromboplastin time after ROSC (4 hours: r = 0.503, P = .033; 8 hours: r = 0.565, P =

.035; 12 hours: r = 0.774, P = .009), but not with other coagulation parameters.

Conclusions: Therapeutic hypothermia might cause coagulant dysfunction but concomitantly improves the microcirculation flow in the cerebral cortex, which might be an effect of TH that results in Cerebral protection.

(C) 2011

Introduction

* Corresponding author. Tel.: +86 13610181339.

E-mail address: [email protected] (J. Xiao-Li).

Cerebral reperfusion is impaired after cardiac arrest , partly as a result of blood coagulation activation without

0735-6757/$ – see front matter (C) 2011 doi:10.1016/j.ajem.2010.07.016

adequate concomitant activation of endogenous fibrinolysis [1,2]. Intravascular clotting may hinder blood flow and oxygen delivery to vital organs during chest compression, influencing resuscitability. In support of this hypothesis, intravital microscopy has shown the presence of microthrombi in cerebral vessels as early as 5 to 10 minutes after CA [3]. This suggests that intravascular fibrin formation and micro- vascular thrombosis after CA may contribute to organ dysfunctions, including neurologic impairment. Consistent with this hypothesis, thrombolytic therapy after cardiopulmo- nary resuscitation (CPR) has been shown to improve microcirculatory reperfusion, to improve survival in experi- mental models of induced CA [4,5] and allow the return of spontaneous circulation (ROSC) after failed initial CPR [6]. Even though the use of fibrinolytics has not improved outcomes in large clinical trials [7], the coagulopathy actually plays a role in the process of pathophysiology after ROSC. Recent studies have shown that application of mild hypother- mia (32-33?C) can improve neurologic outcome in patients with CA [8,9]. However, hypothermia affects hemostasis, and even Mild hypothermia is associated with bleeding and increased Transfusion requirements in patients undergoing surgery [10]. Until now, little has been known about whether the effect of Therapeutic hypothermia on hemostasis is detrimental or beneficial to cerebral microcirculation flow after ROSC. We hypothesize that hypothermia might increase the tendency of bleeding after CA, as well as decrease fibrin formation in the microcirculation, then subsequently improve cerebral microcirculation flow.

In this study, we investigated the relationship between hemostasis and cerebral microcirculation flow and the effect of TH on hemostasis and cerebral microcirculation flow after CA.

Materials and methods

This study was approved by the Animal Investigation Committee of Sun Yat-Sen University and was performed in accordance with the National Institutes of Health Guidelines for ethical animal research.

Animal preparation

This prospective laboratory study was conducted in the Assisted Circulation Laboratory of Sun Yat-Sen University according to the Utstein-style guidelines [11]. After an overnight fast (with free access to water), 24 adult New Zealand rabbits of both sexes (49% male/51% female), weighing 2.2 to 3.1 kg were anesthetized by ear vein injection with 30 mg/kg pentobarbital, placed in a supine position on a surgical board and had their extremities immobilized. The proximal trachea was orally intubated with a 3.0 endotracheal catheter (Becton Dickinson Medical Devices Co Ltd, Suzhou, China) mounted on a blunt needle with a 145? angle tip. The tracheal tube was fixed at the

mouth, and mechanical ventilation was initiated (Harvard Rodent Ventilator; Harvard Apparatus, Holliston, MA) with room air. Ventilation parameters were adjusted using arterial blood gas to maintain the pCO2 in a range of 35 to 45 mm Hg. A conventional single-lead II electrocardiography device was attached using subcutaneous needles. The ear artery was cannulated using a small retained needle (Becton Dickinson Medical Devices Co Ltd) filled with physiologic saline solution containing 5 IU/mL of bovine heparin. Arterial blood pressure (MAP) was continuously measured with a high sensitivity pressure transducer (Kombidyn Monitoring Set; Braun, Melsungen, Germany). All data were recorded for subsequent analysis with a BIOPAC MP150 data acquisition system (model MP150, version 3.8.1; Goleta, CA). A bone window beside the bregmatic fontanel 1.5 cm wide was opened to monitor real-time cerebral cortex microcirculation flow with a Multichannel Laser Doppler system (Peri-Flux 4001 Master, Perimid, Sweden).

Experimental protocol

Twenty-four rabbits were randomly divided into 2 groups: normothermic (NT) and TH. Two Acupuncture needles were inserted, one subcutaneously and the other into the epicardium. Ventricular fibrillation (VF) was induced by an external transthoracic alternating current (AC) applied to the needles. Initially, 50-Hz, 6-V AC was delivered for 10 seconds. If VF spontaneously reverted to sinus, rhythm stimulation was repeated. Ventricular fibrillation resulted in the disappearance of an arterial pulse and blood pressure falling to approximately 0 mm Hg [10]. After successful induction of VF, the ventilator was disconnected. After untreated VF for 3 minutes, manual chest compression and mechanical ventilation were initiated. Ventilation was performed with room air at 45 breaths/min, and tidal volume was adjusted to 8 mL/kg. Manual chest compression at a rate of 200 compressions/min with equal compression-relaxation duration was performed by one investigator who was masked from the hemodynamic monitor tracings and guided only by audio tones. Compression depth at maximal compression was approximately 30% of the anterior-posterior chest diameter. If resuscitation was not achieved after 2 minutes of CPR, a single dose of epinephrine 20 ug/kg was given for 3 minutes, and precordial compression was resumed for several minutes. restoration of spontaneous circulation was defined as the return of supraventricular rhythm with a mean aortic pressure of 60 mm Hg or higher for a minimum of 10 minutes [10]. If ROSC was not achieved 15 minutes after resuscitation was commenced, resuscitation efforts were discontinued.

After ROSC, the rabbits in the NT group were put on electrically heated blanket in Room temperature to maintain the temperature in the physiologic range of 38 to 39?C after ROSC for 12 hours, whereas the rabbits in the TH group underwent induction of hypothermia by surface cooling performed by putting ice bags around the animal’s body, and depending on the change of tympanic temperature (33-35?C), the number of ice

bags was increased or decreased and then maintained for 12 hours after attaining the target temperature. During the stage of maintain hypothermia, we scrupulously monitored the change of temperature, to avoid higher or lower than the target temperature. Before CPR and after ROSC at 4, 8, and 12 hours, blood samples were taken for measurements of coagulation. prothrombin time , Activated partial thromboplastin time , and International normalized ratio were measured using the Behnk Electronic Thrombotimer 4 system (Behnk Electronic, Norderstedt, Germany). platelet counts were measured from sodium citrated blood using an ABX Pentra 120 Hematology Analyzer (ABX Diagnostics, Irvine, CA). Plasma antithrombin III (AT-III) activity was measured before and after ROSC using chromogenic assay (Coamatic Antithrombin kit; Diapharma Group, Inc, West Chester, OH). Protein C (PC) activity was measured in a microtiter plate by chromogenic assay (substrate S-2366; Chromogenix, Molndal, Sweden) using Protac (Alpha Laboratories, Hampshire, UK) as the activator and an end point detection method. The activity of AT- III and PC was expressed as the percent of baseline. The levels of D-dimer (DD) were tested by an enzyme-linked immunosorbent assay

(Uscn Life Science Inc. Wuhan, China).

The animals were killed while they were under deep anesthesia immediately after blood sampling at 12 hours. Renal tissue specimens were fixed in formalin. The percentage of glomerular fibrin deposition (%GFD) was determined by microscopy after staining the renal tissue specimens with phosphotungstic acid hematoxylin. One hundred glomeruli were examined in each sample. The number of fibrin-containing thrombi observed was expressed coagulation biomarkers after ROSC”>as a percentage [12]. The impact of hypothermia on coagulant is systemic, and the glomerulus is independent one by one, easily to be identified, so it has unique superiority to quantitative the fibrin deposition. We selected it as a representative for fibrin deposition in whole body.

Statistics

All data are expressed as mean +- SD. Kruskal-Wallis test was used for between-group differences, and Friedman’s test was used for combined within-group differences over time. The spearman correlation was used for evaluating the correlation between changes in the microcirculation flow in cerebral cortex and values of coagulation parameters (PT, aPTT, INR, DD, activity of AT-III, and PC). A 2-tailed value of P b .05 was considered statistically significant.

Results

Outcome of induction of VF and CPR

Ventricular fibrillation was successfully induced by AC stimulation in all 24 rabbits, 12 rabbits in each group. Ten of 12 rabbits in the TH group achieved ROSC; 10, 7, and 6 animals survived 4, 8, and 12 hours, respectively, after ROSC. Nine of 12 animals in the NT group achieved ROSC; 8, 7, and

5 rabbits survived 4, 8, and 12 hours, respectively, after ROSC. There were no differences in ROSC rate and survival rate at each checkpoint after ROSC between the 2 groups. The baseline physiologic variables were not statistically different between the 2 groups (Table 1). The base life support time and defibrillation time were 3.46 +- 1.58 minutes and 3.16 +- 1.92

times in group NT and 3.61 +- 2.98 minutes and 2.33 +- 0.87 times in group TH; there were no differences between the 2 groups. At each checkpoint after ROSC, there was no difference in mean MAP between the 2 groups (Table 2).

Core temperature regulation

The animals in the NT group were put on electrically heated blanket in room temperature and were not hypother- mia induced; their tympanic temperature was maintained in the range of 38 to 39?C after ROSC. The tympanic temperature of animals in the TH group decreased quickly after surface cooling, attaining the target temperature at approximately 60 minutes and then maintaining the temperature in the range of 33 to 35?C for 12 hours (Fig. 1).

Coagulation biomarkers after ROSC

coagulation abnormalities occurred after ROSC in the NT group animals, including increased coagulation activation and reduced anticoagulation. The values of PT, aPTT, and INR gradually became shorter after ROSC in the NT group. Activated partial thromboplastin time at 12 hours after ROSC was significantly shorter than that at baseline (23.32 +- 5.19 vs 29.53 +- 5.10, P = .025), and the activity of AT-III and PC was decreased significantly. Compared with the NT group, the PT, aPTT, and INR in the TH group were increased significantly (Fig. 2), whereas there were no differences between the 2 groups in activity of AT-III and PC and plasma concentration of DD at specific time intervals after ROSC (Table 3).

Table 1 Baseline physiologic variables between 2 groups

Group

BW (kg)

HR (times/min)

MAP (mm Hg)

Tympanic temperature

NT (n = 12)

.51 +- 0.54

281.09 +- 33.30

94.20 +- 4.46

38.79 +- 0.54

TH (n = 12)

2.65 +- 0.32

281.91 +- 30.69

95.13 +- 6.36

38.84 +- 0.66

P

.798

.791

.909

.852

BW, body weight; HR, heart rate; MAP, mean arterial pressure.

ROSC

Group

4 h

8 h

12 h

NT

84.67 +- 5.75

74.13 +- 4.74

67.87 +- 5.45

(n = 8)

(n = 7)

(n = 5)

TH

70.27 +- 2.65

67.67 +- 7.74

65.40 +- 13.10

(n = 10)

(n = 7)

(n = 6)

P

.086

.167

.865

Cortical cerebral blood flow before CPR and after ROSC

After ROSC, cerebral blood flow rapidly decreased, even at 12 hours, and the mean cortical cerebral blood flow was not equal to the prearrest flow level. The microcirculation flow in the cerebral cortex before CPR and after ROSC at 4, 8, and 12 hours was 401.60 +- 11.76,

258.86 +- 34.58, 317.59 +- 23.36, and 371.98 +- 5.79 mL/

min, respectively, in the NT group and 398.18 +- 12.91,

336.19 +- 19.27, 347.76 +- 13.80, and 383.78 +- 3.29 mL/

min, respectively, in TH group. There were differences at each checkpoint after ROSC between the 2 groups (4 hours: P = .001, 8 hours: P = .011; 12 hours: P = .009;

Fig. 3).

The Pearson correlation test for the cortical cerebral blood flow and coagulation biomarkers at specific time intervals

after ROSC demonstrated that the microcirculation flow in the cerebral cortex was positively correlated with aPTT after ROSC (4 hours: r = 0.503, P = .033; 8 hours: r = 0.565, P =

.035; 12 hours: r = 0.774, P = .009); the other coagulation biomarkers were not correlated with it.

Table 2 MAP (mm Hg) at each time point after ROSC, no differences between 2 groups

Pathologic examination

Glomerular fibrin deposition is presented as %GFD. Marked and continued fibrin deposition was a specific finding in the rabbits in both groups after ROSC (Fig. 4). There were significant differences in the %GFD between the 2 groups (54.6% +- 4.77% in the NT group and 36.67% +- 4.97% in the TH group, P = .000).

Discussion

Consistent with previous studies [1,13], in the present study, we found the activation of coagulation after CA and CPR. The coagulopathy was consistently present in successfully resuscitated rabbits in the NT group. Coagula- tion was activated, mainly via the endogenous coagulation system, with anticoagulant factors diminished and the activity of AT and PC decreased after ROSC. Previous study [1] also showed a marked activation of blood coagulation and fibrin formation after prolonged CA and CPR in humans that were not balanced adequately by concomitant activation of endogenous fibrinolysis. These

Fig. 1 The tympanic temperature of animals in the NT and TH groups changed after ROSC: animals in the NT group maintained temperature in the range of 38 to 39?C, whereas in the TH group, temperature decreased quickly after surface cooling, and the target temperature was attained at approximately 60 minutes (left) and then maintained in the range of 33 to 35?C for 12 hours (right).

Effects of TH on coagulopathy and microcirculation after CPR

1107

Fig. 2 Comparisons of PT, aPTT, and INR between the 2 groups of rabbits at all time points after the ROSC.

Table 3 Coagulation biomarkers at baseline and at specific time intervals after ROSC

Group

Basal

ROSC

4 h

8 h

12 h

AT-III (%)

NT

132.90 +- 21.42

97.00 +- 20.35 ?

76.36 +- 21.79 ?

81.84 +- 49.09 ?

TH

112.82 +- 26.65

90.98 +- 24.14 ?

77.50 +- 35.63 ?

54.30 +- 41.88 ?

PC (%)

NT

66.79 +- 11.65

45.91 +- 9.93 ?

35.09 +- 8.87 ?

32.08 +- 20.94 ?

TH

60.38 +- 17.11

42.41 +- 13.47 ?

27.11 +- 11.91 ?

19.20 +- 7.40 ?

BPC x 109

NT

323.88 +- 124.44

281.00 +- 96.06

261.14 +- 71.93

267.00 +- 14.56

TH

280.80 +- 127.97

219.70 +- 94.81

209.71 +- 89.79

206.80 +- 64.33

DD (mg/L)

NT

34.75 +- 9.79

42.88 +- 10.29

44.57 +- 14.72

43.80 +- 17.44

TH

74.00 +- 95.20

60.00 +- 65.31

25.71 +- 13.45

27.40 +- 26.51

BCP, blood platelets count.

* Comparing each time point vs baseline in each group, respectively, P b .05.

changes may contribute to reperfusion disorders, such as the cerebral “no-reflow” phenomenon, by inducing fibrin deposition and formation of microthrombi. Concomitant with these phenomena, the cerebral circulation was impaired after ROSC; and even at 12 hours after ROSC, it was not restored to the normal range in current study, whereas TH prolonged the PT, aPTT, and INR after ROSC as well as increased the cerebral microcirculation. There were no differences between the 2 groups in activity of AT and PC after ROSC. The Pearson correlation test showed that the microcirculation flow in the cerebral cortex was positively correlated with aPTT after ROSC. The current study demonstrated that TH prolongs the PT, aPTT, and INR and decreases microfibrin deposition after ROSC as well as increases the cerebral microcirculation.

Fig. 3 There were significant differences between the NT and TH groups in cortical cerebral blood flow at specific time intervals after the ROSC (NT vs TH, 4 hours: P = .001; 8 hours: P = .011; 12 hours: P = .009).

The activation of blood coagulation after CA leads to formation of microvascular fibrin and microthrombosis. This is believed to impair microcirculatory blood flow during reperfusion. In the present study, we used laser Doppler flowmetry to assess the cerebral microcirculation. This is a highly suitable method for continuous measurement of cerebral cortical blood flow [14]. The cerebral circulation was impaired and was not restored to normal range even at 12 hours after ROSC. Therapeutic hypothermia can reduce the disturbance of cerebral microcirculation after CPR, mainly by contributing to a decrease in fibrin formation in capillary vessels. It is not clear how this expected reduction in procoagulant and anticoagulant protein activity affects the complex, multicomponent process of blood coagulation. Many factors are involved. Hypothermia-associated coagu- lopathy has been hypothesized to result, at least in part, from reduced activity of coagulation enzymes, including both procoagulant and anticoagulant activities. It is well recog- nized that, in general, the activity of an enzyme is reduced by approximately 50% for every 10?C drop in temperature [15]. Several studies, however, have suggested that quantitative and qualitative platelet abnormalities are involved in hypothermia-related coagulopathy [15,16]. Most of the studies on the effects of hypothermia on Platelet functions were carried out at temperatures below 30?C [16] and therefore may not reflect platelet function at degrees of hypothermia in the current study.

Antithrombin (AT) is an anticoagulant protein that inhibits nonclot bound thrombin and activates factor X in plasma. Its importance in regulating coagulation has been demonstrated by embryonic lethality in AT knockout mice [17], as well as by the association between low AT levels and venous thrombosis [18]. However, no improvement in the cerebral circulation during reperfusion was observed after the administration of AT; therefore, it is possible that the physiologic levels of AT are sufficient to meet the needs after CA and that the sufficiency of AT plays only a minor role in the disturbance of cerebral microcirculation in the CA model [19].

Activated PC (APC) is an endogenous protein that enhances fibrinolysis, limits thrombin generation, and

Effects of TH on coagulopathy and microcirculation after CPR 1109

Fig. 4 Pathologic features of kidneys stained with phosphotungstic acid hematoxylin; fibrin is indicated by the arrows. A, NT group. B, TH group (x40 field).

modulates inflammation. It is converted from its inactive precursor, PC, by thrombin coupled to thrombomodulin. Low levels of APC after CA have been found to be related to higher mortality, an association that also exists in severe sepsis [20]. Activated PC was low at admission and remained low over the 2-day study period in a clinical study [21]. After CA, the conversion of endogenous PC to APC may be impaired because of endothelial dysfunction with down- regulation of thrombomodulin and endothelial PC receptor [21]. Interestingly, APC has been shown to minimize ischemia/reperfusion injury to the damaged spinal cord and to the brain in stroke models [22-24], but not in CA animal models [25,26], so PC may play a minor role in the protection of cerebral microcirculation after ROSC. In the present study, there were no differences in AT and PC activity between the 2 groups, so the influence of hypothermia on coagulation was mainly through prolonging aPTT, rebalancing procoagulation and anticoagulation, subsequently decreasing microfibrin formation and improv- ing the cerebral circulation. Many studies have shown that the effect of hypothermia on coagulation is temperature dependent [27,28]. The effect of TH on coagulation is limited to a mild increase in the risk of mucous bleeding [29]. Other factors may also contribute to the effect of hypothermia on cerebral microcirculation. Hypothermic preconditioning significantly reduces tissue necrosis of pedicled flaps by improving the microcirculation through heme oxygenase-1-mediated dilation of nutritive capillar- ies [30]. The decrease in endothelin-1 induced by mild hypothermia contributes to the improvement of the cerebral microcirculation after ischemia [31]. The function of adhesion molecules is temperature dependent, and there- fore, since the expression of intercellular adhesion molecule 1 protein was attenuated by hypothermia, the protection may be related to the reduction of leukocyte

adhesion [32].

An important limitation of this study is that this is an animal study; the results may not reflect the real scenario in human subjects. Hence, it can be suggested that it is far from further application on human beings, but the animal study often gives clue for clinical study. The other limitation of our study is the ear artery cannulated using a small retained

needle filled with physiologic saline solution containing 5 IU/mL of bovine heparin; it would impact the coagulant system, which is unavoidable. However, the total volume of physiologic saline-heparin solution was small, which was about 0.3 mL, the same protocol used in the 2 groups, so we think that the impact was mild.

Conclusions

Therapeutic hypothermia can influence the delicate balance between coagulant-anticoagulant, reduce microfi- brin formation, and allow restoration of more cerebral cortex blood flow after ROSC, which may be one of the mechanisms through which TH provides cerebral protection.

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