Article, Cardiology

Effect of valproic acid combined with therapeutic hypothermia on neurologic outcome in asphyxial cardiac arrest model of rats

a b s t r a c t

Backgrounds: valproic acid has been reported to have survival and Neuroprotective effects in a cardiac ar- rest Rat model. This study was designed to investigate the effect of VPA combined with therapeutic hypothermia (HT) in an Asphyxial cardiac arrest rat model.

Methods: Rats were subjected to 6 minutes of asphyxial cardiac arrest. Cardiopulmonary resuscitation was per- formed and then the randomly allocated to 1 of 4 groups (normal saline [NS]/normothermia [NT], VPA/NT, NS/ HT, and VPA/HT). Hypothermia (32.5?C +- 0.5?C, 4 hours of HT and 2 hours of rewarming) or NT (37?C +- 0.5?C for 6 hours) was applied, and VPA (300 mg/kg) or NS was administered immediately after the return of sponta- neous circulation. neurologic deficit score was measured, and a tape removal test was performed for 3 days. His- tologic injury of hippocampus was evaluated.

Results: Valproic acid significantly improved neurologic deficit score at 48 and 72 hours in the NT-treated rats and at 72 hours in the HT-treated rats (all P b .05). Although the latency and success rate were not significantly dif- ferent between the VPA/NT and NS/NT groups, the VPA/HT group showed significantly lower latency and higher success rates compared to the NS/HT group (P b .05). The histologic Injury score in the hippocampal CA1 sector was significantly lower in the VPA/NT group than the NS/NT group (P b .05) and showed a tendency to be de- creased in the VPA/HT group compared with the NS/HT group (P = .06).

Conclusion: In an asphyxial cardiac arrest rat model, administration of VPA improved neurologic outcomes and added a neuroprotective effect to HT.

(C) 2015

Introduction

Cerebral injury after cardiac arrest remains as a significant health care problem [1]. Although an understanding of pathophysiology and improvement of Resuscitation techniques have led to significantly im- proved outcomes in cases of out-of-hospital cardiac arrest [2], therapeu- tic intervention for cerebral injury after cardiac arrest remains suboptimal. Therefore, a more sophisticated therapeutic intervention is needed to improve outcomes in patients with cardiac arrest.

It has been reported that valproic acid (VPA) as a histone deacetylase inhibitor has neuroprotective effects in animal models of ischemic cere- bral injury [3-5]. In addition, therapeutic hypothermia (HT) is a clinical- ly proven therapeutic measure to decrease cerebral injury and is recommended in practice guidelines [6-9]. Recently, it has been report- ed that VPA and therapeutic HT showed a synergistic neuroprotective

? Source of support: This research was supported by Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (NRF-2012R1A1A2006965).

* Corresponding author at: Department of Emergency Medicine, Seoul National Univer- sity Bundang Hospital, 300 Gumi-dong, Bundang-gu, Seongnam-si, Gyeonggi-do, 463-707, Republic of Korea. Tel.: +82 31 787 7579; fax: +82 31 787 4055.

E-mail address: [email protected] (K. Kim).

effect on hypoxic hippocampal cell (HT-22 cells) injury [10]. However, the combined effect of these Neuroprotective treatments in cases of car- diac arrest-induced brain injury has not been reported.

This study was designed to evaluate the neuroprotective effect of VPA combined with therapeutic HT in an asphyxial cardiac arrest rat model.

Materials and methods

The institutional animal care and use committee approved the study.

Animal preparation

Male Sprague-Dawley rats weighing 300 to 350 g were used in the experiments. Before the experiments, the rats were housed in a con- trolled environment with free access to water and food. The method of animal preparation has been described previously [5]. Briefly, rats were anesthetized using Intramuscular injection of zoletil (30 mg/kg) and xylazine (15 mg/kg) and intubated with a 16-gauge catheter (BD Insyte Autoguard, Franklin Lakes, NJ). Rats were mechanically ventilat- ed with a rodent ventilator (tidal volume, 2 mL; respiratory rate, 55 per minute; fraction of inspired oxygen, 0.21; Harvard rodent ventilator model 645, Harvard apparatus, Holliston, MA), and minute ventilation

http://dx.doi.org/10.1016/j.ajem.2015.08.036

0735-6757/(C) 2015

was adjusted to ensure PaCO2 between 35 and 40 mm Hg according to the result of blood gas analysis. Intravascular catheters (24 gauge; BD Insyte Autoguard) were inserted in the left femoral artery for Blood pressure monitoring and blood sampling (for blood gas analysis) and in the tail vein for drug administration.

Cardiac arrest and cardiopulmonary resuscitation

Induction of asphyxial cardiac arrest and cardiopulmonary resuscita- tion (CPR) has been described previously [5]. Briefly, vecuronium was administered, and the mechanical ventilator was disconnected to in- duce respiratory arrest. circulatory arrest was defined as the onset of mean arterial pressure decline to 20 mm Hg and was maintained for 6 minutes.

After 6 minutes of circulatory arrest, CPR was performed. Resuscita- tion consisted of resuming mechanical ventilation (tidal volume, 2 mL; fraction of inspired oxygen, 1.0; respiratory rate, 55 per minute), admin- istering intravenous epinephrine (0.01 mg/kg) and bicarbonate (1.0 mEq/kg), and performing continuous external chest compressions at a rate of 200 compressions per minute using a mechanical thumper (cus- tom-made device, Compressed air-driven, rate was set at 200 cycles/ min) until Spontaneous pulse was observed in arterial tracing and mean arterial pressure greater than 50 mm Hg was reached. After the return of spontaneous circulation (ROSC), rats were observed and main- tained using mechanical ventilation.

Inclusion and exclusion criteria

The objective of this study is to evaluate the neuroprotective effect of VPA administered just after ROSC and combined with HT in asphyxia cardiac arrest. Therefore, rats that were successfully resuscitated from cardiac arrest and weaned from mechanical ventilation after 6 hours of postresuscitation care were included; rats were excluded if cardiac arrest induction time was longer than 3 minutes to control hypoxia du- ration and if mechanical ventilation was not weaned after 6 hours of postresuscitation care. In our previous reports, 6 minutes of cardiac ar- rest resulted in successful weaning of mechanical ventilation [5].

Study group allocation and postresuscitation care

After ROSC, rats were randomly allocated to 1 of 4 groups (normal saline [NS] + normothermia [NT] group; VPA + NT [VPA/NT] group; NS + HT [NS/HT] group; VPA + HT [VPA/HT] group) (Fig. 1). Just after the ROSC, VPA (300 mg/kg) or vehicle (same amount of NS) was admin- istered via tail vein and HT (target temperature, 33?C +- 0.5?C) was in- duced using ice slurry and a fan and maintained for 4 hours. Thereafter, rewarming was performed actively for 2 hours using a heating fan. Body temperature of rats in the NT group was maintained at 36.5?C to 37.0?C during 6 hours of postresuscitation care. If adequate spontaneous respiration could be recovered after 6 hours of postresuscitation care, the mechanical ventilator was disconnected.

Fig. 1. Timeline of experiment.

Adequate spontaneous ventilation was defined as the PaO2 greater than 60 mm Hg on room air and no decrease in mean arterial pressure 5 mi- nutes after disconnecting the mechanical ventilator. Subsequently, in- travascular catheters were removed, and the groin wound was closed. The endotracheal tube was then removed, and rats were returned to their cages. To ensure nutritional support and hydration, fluid (5% dex- trose in saline, 50 mL/kg) was administered subcutaneously every 24 hours during the 72-hour observation periods.

Assessment of neurologic outcomes

Neurologic deficit scale score

General neurologic status was assessed using the validated neuro- logic deficit scale (NDS) [11] at 24, 48, and 72 hours after cardiac arrest. The NDS ranges from 80 indicating a functionally normal rat to a score of 0 for brain or cardiac death.

Sensorimotor function

Sensorimotor function was evaluated using a sticky tape removal test modified from the methods proposed by Schallert et al [12]. The original test was used to lateralize sensorimotor deficit. However, cardiac arrest causes global cerebral ischemia and is not expected to cause a unilateral deficit. Therefore, the test was modified. After a rat was removed from the cage, a piece of adhesive tape approximately 1 x 1.5 cm was attached to the radial side of one of forepaw; the rat was returned to the cage. The time required to remove the tape was recorded and defined to latency. Preliminary experiments had shown that there was no significant differ- ence in performance between the right and left forepaws in rats at base- line and after cardiac arrest. Therefore, the test was performed in one of both forepaws randomly, and the mean value of 10 repeats was used for comparison. The test was truncated at 180 seconds in a manner similar to previous reports [13]. In addition, the removal success rate over 10 re- peats of the test was measured and compared among groups.

Assessment of histologic hippocampal injury

After 72 hours of observation and neurologic outcome assessment, rats were sacrificed by isoflurane overdose and perfused transcardially

with 4% paraformaldehyde. Brains were removed and fixed in 4% para- formaldehyde. Fixed brain tissues were cut into coronal blocks on a ro- dent brain matrix and embedded in paraffin. The paraffin block was sectioned at 4 um thickness and stained with hematoxylin and eosin. Histologic evaluation was performed by a pathologist who was blinded to the treatment group. The hippocampus (CA1, CA2, CA3, and CA4) was examined under a light microscope using magnifications x20 for ische- mic neuronal damage (eosinophilic cytoplasm, pyknotic nuclei, nuclear karyorrhexis, necrotic change) and scored semiquantitatively using a 6-point ordinal scale (0, none; 1, minimal, b 5%; 2, mild, 6%-20%; 3, mod- erate, 21%-50%; 4, marked, 51%-75%; and 5, severe, 76%-100%) [14].

Statistical analysis

The Kolmogorov-Smirnov test was used to assess the normality of the data. Normally distributed data were expressed as the mean (SD), and nonnormally distributed data were expressed as the median and range. The Kruskal-Wallis test with multiple comparisons was per- formed to compare among groups (histologic injury score). Repeated- measures analysis of variance using Bonferroni post hoc comparison (for NDS) or the Friedman test with post hoc comparisons (for the tape removal test) was performed. P b .05 was considered significant. Statistical analyses were performed using STATA 10.0IC (StataCorp LP, College Station, TX), and graphs were drawn using GraphPad Prism

5.0 (GraphPad Software, Inc, La Jolla, CA).

Results

A total of 44 rats were subjected to asphyxial cardiac arrest. Of those, 4 rats were excluded before randomization because cardiac arrest in- duction took longer than 3 minutes. Thus, 40 rats (10 in each group) were included and randomly assigned to 1 of 4 treatment groups. All rats were successfully resuscitated. However, 5 rats were not weaned from mechanical ventilation and were, therefore, excluded (1 in the NS/NT group, 2 in the VPA/NT group, 1 in the NS/HT group, and 1 in the VPA/HT group). Finally, 8 or 9 rats in each group were included for analysis; all rats survived for 72 hours.

Table

Experimental characteristics of cardiac arrest and resuscitation.

Variables

NT

HT

P

NS (n = 9)

VPA (n = 8)

NS (n = 9)

VPA (n = 9)

baseline values

Body weight (g)

313.1 +- 3.7

312.4 +- 3.1

308.9 +- 3.0

309.6 +- 5.5

.70

pH

7.38 +- 0.01

7.41 +- 0.02

7.38 +- 0.01

7.42 +- 0.01

.08

PaCO2 (mm Hg)

35.7 +- 1.8

33.7 +- 2.5

35.3 +- 1.2

34.1 +- 6.3

.14

PaO2 (mm Hg)

73.4 +- 3.4

77.6 +- 2.3

75.1 +- 3.1

82.9 +- 3.6

.24

HCO (mEq/L)

21.2 +- 0.6

21.1 +- 0.8

21.5 +- 0.7

21.7 +- 3.3

.20

Base excess

-2.9 +- 0.5

-2.4 +- 0.6

-2.5 +- 0.7

-1.6 +- 2.8

.56

Lactate (mmol/L)

0.8 +- 0.2

1.1 +- 0.3

0.8 +- 0.1

0.9 +- 0.1

.88

Mean arterial pressure (mm Hg)

82.7 +- 3.3

96.8 +- 8.3

79.4 +- 11.1

93.4 +- 3.8

.35

Heart rate (beat/min)

305.2 +- 5.3

287.5 +- 13.6

303.5 +- 9.4

286.6 +- 6.3

.37

Body temperature (?C)

36.2 +- 0.2

36.0 +- 0.2

36.4 +- 0.2

35.8 +- 0.3

.24

Cardiac arrest and CPR

Induction time

51.4 +- 5.9

69.9 +- 5.6

55.7 +- 4.1

66.7 +- 2.1

.06

CPR duration (s)

32.0 +- 4.2

30.6 +- 3.6

27.4 +- 2.4

29.1 +- 2.6

.80

Total ischemia time (s)

443.4 +- 6.2

460.4 +- 5.8

443.1 +- 5.3

455.9 +- 2.6

.12

Lactate after ROSC (mmol/L)

7.3 +- 0.5

7.2 +- 0.5

7.0 +- 0.5

7.0 +- 0.6

.94

6-h postresuscitation care

pH

7.31 +- 0.02

7.39 +- 0.03

7.31 +- 0.02

7.32 +- 0.02

.09

PaCO2 (mm Hg)

35.1 +- 2.7

31.3 +- 1.2

36.6 +- 1.7

33.7 +- 2.8

.62

PaO2 (mm Hg)

67.8 +- 1.0

72.2 +- 5.0

74.4 +- 3.9

82.1 +- 2.8

.04*

HCO (mEq/L)

17.7 +- 1.0

17.5 +- 0.7

18.5 +- 0.6

16.1 +- 1.2

.33

Base excess

-7.1 +- 0.9

-6.1 +- 0.9

-6.5 +- 0.6

-8.4 +- 1.1

.35

Lactate (mmol/L)

1.0 +- 0.3

1.4 +- 0.4

0.8 +- 0.1

1.1 +- 1.1

.49

Mean arterial pressure (mm Hg)

72.3 +- 2.3

74.0 +- 2.0

83.3 +- 7.1

76.3 +- 2.5

.31

Heart rate (beat/min)

299.4 +- 10.4

312.7 +- 3.5

263.0 +- 11.7

286.3 +- 22.4

.09

3

3

Experimental characteristics of asphyxial cardiac arrest and resuscitation

Baseline and post-ROSC characteristics are shown in Table. There was no significant difference among groups in body weight, baseline blood gas values, total ischemia time (induction time + cardiac arrest duration + CPR duration), baseline mean arterial pressure and heart rate, and lactate values.

Fig. 2 shows the Blood pressure and heart rate changes during the ex- perimental period. Mean arterial pressure was not different among groups, but heart rate was lower in the HT-treated groups (the NS/HT and VPA/HT groups) than in the NT-treated groups (the NS/NT and VPA/NT groups) during 10 to 90 minutes of the postresuscitation period.

Functional neurologic outcomes

Fig. 3 shows the NDS scores for 72 hours. Groups administered VPA had significantly higher NDS score than the NS-administered groups

Fig. 2. Body Temperature control and hemodynamic change during CPR and postresuscitation care.

Fig. 3. Neurologic deficit scale score.

(NS/NT vs VPA/NT, P b .05 at 48 hours and 72 hours; NS/HT vs VPA/ HT, P b .05 at 72 hours).

To assess sensorimotor function, a sticky tape removal test was per- formed (Fig. 4). At baseline, the latency and success rates for tape remov- al were not significantly different among groups. Although the administration of VPA did not result in significant differences between the NT-treated groups (NS/NT vs VPA/NT, P N .05 at all time points), the VPA/HT group had a higher success rate and lower latency than the HT-treated control group (VPA/HT vs NS/HT, P b .05 at 48 and 72 hours).

Histologic hippocampal injury

The hippocampi were examined for Ischemic injury using a light mi- croscope (Fig. 5). The rats administered VPA showed significantly lower histologic injury scores in sector CA1 than the NT-treated control group (NS/NT vs VPA/NT; P b .05). In the HT-treated groups, histologic injury score in the hippocampal CA1 sector showed a tendency to be lower in the VPA/HT group than the NS/HT group (NS/HT vs VPA/HT; P = .06). However, there were no significant differences between groups with re- spect to injury scores in other sectors of the hippocampus.

Discussion

This study demonstrated that the administration of VPA had a syner- gistic effect with therapeutic HT on neurologic outcomes and histologic cerebral injury in an asphyxial cardiac arrest rat model.

The neuroprotective effect of VPA has been reported in various ani- mal models of cerebral ischemia including cardiac arrest [3-5]. Although the precise mechanism responsible for this effect has not been identi- fied, epigenetic regulation including acetylation of histone and nonhis- tone proteins to activate transcription and gene expression, modification of target protein function, and decreased excitotoxicity have been suggested as possibilities [15,16]. Therapeutic HT is a treat- ment that has been recommended by clinical guidelines and adopted in clinical practice [8]. A new therapeutic modality needs to be evaluat- ed using current practices. The objective of this study was to investigate the synergistic effect of VPA with therapeutic HT on neurologic injury after cardiac arrest; this synergistic effect was found in an asphyxial car- diac arrest rat model. This finding might be consistent with an in vitro study published recently [10]. Jin et al [10] reported that VPA and ther- apeutic HT showed a synergistic neuroprotective effect in hypoxic hip- pocampal cells. In that study, increased phosphorylation of glycogen synthase kinase-3? and decreased expression of hypoxia-inducible

Fig. 4. Sticky tape removal test.

factor 1? and high mobility group box 1 protein were found to be asso- ciated with synergistic neuroprotective effect of VPA and HT. Although our study did not investigate the precise mechanisms responsible, this is the first in vivo study assessing the synergistic neuroprotective effects

of VPA and HT in a cardiac arrest rat model. Previously, we reported on the neuroprotective effects of VPA in an asphyxia cardiac arrest model [5]. In that study, VPA as a histone deacetylase inhibitor showed a ten- dency to increase the expression of acetylated histone H3 while

Fig. 5. Histologic hippocampal injury.

expression of cleaved caspase 3 was significantly decreased. Jin et al [10] reported that VPA significantly increased expression of acetylated his- tone H3, but HT did not. Thus, the combined neuroprotective effects might not be directly related with the enhanced effect of HT on histone hyperacetylation. Further study will be needed to identify the molecular mechanisms responsible for the synergistic effects of HT and VPA.

In this study, we evaluated neurologic outcomes using validated NDS score and the sticky tape removal test. In addition, histologic injury was assessed in hippocampal regions. There may be discrepancies be- tween the clinical outcomes and the assessed histologic injuries. The NDS score and sticky tape removal test assessed general neurologic sta- tus and sensorimotor functions, but examination of histologic hippo- campal injuries was not consistent with these outcomes. However, cardiac arrest causes global cerebral ischemia, and CA1 pyramidal neu- rons in the hippocampus, medium-sized neurons in the striatum, and the Purkinje cells in the cerebellum have been shown to be susceptible to global cerebral ischemia [17]. Of those, the neurons in the CA1 zone are the most sensitive to ischemia. Thus, histologic injury in the hippo- campus might reflect the degree of global cerebral injury. Although the clinical outcomes evaluated in this study might be inconsistent with the histologic injury assessment, therapeutic HT and VPA improved clinical outcomes and resulted in decreased histologic injury in the CA1 zone of the hippocampus.

The dose of VPA used in this study was based on previous reports that investigated the neuroprotective effect of VPA in ischemic brain in- jury model of animals [18-21]. This may not be clinically relevant and could result in toxicity. However, it has been reported that a 300 mg/kg VPA dosage did not increase liver and kidney toxicity in a study investigating the effect of VPA in a polytrauma swine model [22]. In addition, a maximum-tolerated dose of 140 mg/kg per day over 3 days was reported in a phase I clinical trial testing VPA as an ad- junct anticancer treatment [23]. However, there have been no clinical reports on the effective and tolerated doses of VPA for neuroprotection. Thus, further study regarding the optimal neuroprotective dose of VPA will be required.

This study has several limitations. First, during the observation peri- od after weaning from mechanical ventilation, body temperature was not controlled. Some rats, which are assumed to have had severe inju- ries, had decreased spontaneous activity, which might influence body temperature and neurologic outcomes. However, if spontaneous HT had a protective effect, rats with severe injuries would have better out- comes than expected. Second, the molecular mechanism responsible for the synergistic effect of HT and VPA was not investigated in this study. Further study will be required. Third, the effective dose and dosing in- terval of VPA were not investigated in this study. Valproic acid was ad- ministered just after ROSC, and no additional administration was performed during the 72-hour observation period. Considering previ- ous reports of the neuroprotective effects of VPA in other experimental settings [4,24], multiple doses of VPA may have beneficial effects in car- diac arrest-induced cerebral injury. Further study is warranted. Fourth, cardiac arrest model used in this study is not clinically relevant. The du- ration of cardiac arrest and resuscitation was very short compared with real clinical situations. Thus, the beneficial effect of VPA may not be ex- pected in humans with longer durations of cardiac arrest and resuscita- tion. However, considering the pathophysiology of hypoxic cerebral injury from cardiac arrest, the beneficial effect of VPA combined with HT in a small animal cardiac arrest model can be expected in a large an- imal or human model. Further studies that reflect real clinical situations are required. Fifth, the duration of HT treatment was 4 hours, which may be contrasted with clinical use of HT. The duration of HT treatment used in this study was similar to previous studies investigating the ben- eficial effect of HT in a cardiac arrest rat model [25,26]. Four hours of HT may not be enough to improve neurologic outcomes in cases of clinical HT, and more prolonged HT could result in diminishing beneficial ef- fects of VPA. However, this may be a problem of balance. If the cardiac arrest duration was increased, the beneficial effect of therapeutic HT

might not have been prominent. The beneficial effect of VPA combined with HT needs to be confirmed in an adequate model of cardiac arrest that reflects the clinical situation.

Conclusion

Administration of VPA just after ROSC improved neurologic out- comes, and it added the neuroprotective effect of therapeutic HT in an asphyxial cardiac arrest rat model.

Acknowledgment

All authors have no conflict of interest.

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