Treatment with a histone deacetylase inhibitor, valproic acid, is associated with increased platelet activation in a large animal model of traumatic brain injury and hemorrhagic shock

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ScienceDirect journal homepage: www.JournalofSurgicalResearch.com

Treatment with a histone deacetylase inhibitor, valproic acid, is associated with increased platelet activation in a large animal model of traumatic brain injury and hemorrhagic shock Simone E. Dekker, BSc,a,b Martin Sillesen, MD,c,d Ted Bambakidis, MSc,a Anuska V. Andjelkovic, MD, PhD,e,f Guang Jin, MD, PhD,a Baoling Liu, MD,a Christa Boer, PhD,b Pa¨r I. Johansson, MD, Dmsc, MPA,g,h Durk Linzel, MD,a,i Ihab Halaweish, MD,a and Hasan B. Alam, MDa,* a

Department of Surgery, University of Michigan Hospital, Ann Arbor, Michigan Department of Anesthesiology, VU University Medical Center, Institute for Cardiovascular Research, Amsterdam, the Netherlands c Division of Trauma, Emergency Surgery And Surgical Critical Care, Department of Surgery, Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts d Department of Surgery, Copenhagen University Hospital, Hillerød, Denmark e Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan f Department of Neurosurgery, University of Michigan Medical School, Ann Arbor, Michigan g Capital Region Blood Bank, University of Copenhagen, Rigshospitalet, Denmark h Department of Surgery, University of Texas Medical School, Centre for Translational Injury Research (CeTIR), Houston, Texas i Department of Emergency Medicine, VU University Medical Center, Amsterdam, the Netherlands b

article info

abstract

Article history:

Background: We have previously shown that resuscitation with fresh frozen plasma (FFP) in

Received 15 January 2014

a large animal model of traumatic brain injury (TBI) and hemorrhagic shock (HS) decreases

Received in revised form

the size of the brain lesion, and that addition of a histone deacetylase inhibitor, valproic

15 January 2014

acid (VPA), provides synergistic benefits. In this study, we hypothesized that VPA admin-

Accepted 25 February 2014

istration would be associated with a conservation of platelet function as measured by

Available online xxx

increased platelet activation after resuscitation. Materials and methods: Ten swine (42-50 kg) were subjected to TBI and HS (40% blood loss).

Keywords:

Animals were left in shock for 2 h before resuscitation with either FFP or FFP þ VPA

Traumatic brain injury

(300 mg/kg). Serum levels of platelet activation markers transforming growth factor

Hemorrhagic shock

beta, CD40 L, P-selectin, and platelet endothelial cell adhesion molecule (PECAM) 1

Histone deacetylase inhibitor

were measured at baseline, postresuscitation, and after a 6-h observation period. Plate-

Fresh frozen plasma

let activation markers were also measured in the brain whole cell lysates and

Neuroprotection

immunohistochemistry.

This article was presented at the ninth Annual Academic Surgical Congress, San Diego, California, February 4e6, 2014. * Corresponding author. Department of Surgery, 2920 Taubman Center, University of Michigan Hospital, 1500 E. Medical Center Drive, Ann Arbor, MI 48109. Tel.: þ1 734 936 5823; fax: þ1 734 936 6927. E-mail address: [email protected] (H.B. Alam). 0022-4804/$ e see front matter ª 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2014.02.049

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Platelet activation

Results: Circulating P-selectin levels were significantly higher in the FFP þ VPA group

Resuscitation

compared with the FFP alone group (70.85  4.70 versus 48.44  7.28 ng/mL; P < 0.01).

Swine

Likewise, immunohistochemistry data showed elevated P-selectin in the VPA treatment group (22.30  10.39% versus 8.125  3.94%, P < 0.01). Serum sCD40L levels were also higher in the FFP þ VPA group (3.21  0.124 versus 2.38  0.124 ng/mL; P < 0.01), as was brain sCD40L levels (1.41  0.15 versus 1.22  0.12 ng/mL; P ¼ 0.05). Circulating transforming growth factor beta levels were elevated in the FFP þ VPA group, but this did not reach statistical significance (11.20  1.46 versus 8.09  1.41 ng/mL; P ¼ 0.17). Brain platelet endothelial cell adhesion molecule 1 levels were significantly lower in the FFP þ VPA group compared with the FFP group (5.22  2.00 pg/mL versus 7.99  1.13 pg/mL; P ¼ 0.03). Conclusions: In this clinically relevant large animal model of combined TBI þ HS, the addition of VPA to FFP resuscitation results in an early upregulation of platelet activation in the circulation and the brain. The previously observed neuroprotective effects of VPA may be due to a conservation of platelet function as measured by a higher platelet activation response after resuscitation. ª 2014 Elsevier Inc. All rights reserved.

1.

Introduction

Trauma is the leading cause of death among young people, accounting for nearly 6 million deaths worldwide each year [1]. Hemorrhagic shock (HS) and traumatic brain injury (TBI) frequently co-occur and account for most trauma-related mortalities [2]. Approximately one-third of TBI patients [3] and one-quarter of general trauma patients [4] exhibit abnormal coagulation tests on hospital admission. Although the frequency and severity of coagulation disturbances is well documented [4,5], the role of platelets in these disturbances is poorly understood. Platelet count has been shown to predict lesion progression and mortality in TBI patients [6]. In addition, we recently found that platelet function decreases after TBI þ HS [7], and dysfunction is associated with increased mortality [8,9] and bleeding complications [10] in general trauma and TBI patients. However, relatively little is known about platelet activation and function after the initial TBI insult as well as the importance of these processes in the initiation or attenuation of secondary brain injury. The method of resuscitation following TBI merits particular attention, as the choice of fluid may play a critical role in the development of platelet dysfunction. Data from our lab have indicated that fresh frozen plasma (FFP) resuscitation increases platelet function in the first hours after resuscitation [11]. We have also shown that valproic acid (VPA), a commonly prescribed anti-epileptic drug, can improve platelet functions. VPA when given in large doses acts as a histone deacetylation inhibitor to improve outcomes in large animal models of lethal insults [12]. Addition of VPA to hetastarch resuscitation was found to be neuroprotective in a large animal model of TBI þ HS [13]. Similarly, early administration of VPA to the FFP-resuscitated animals was associated with smaller lesion size and decreased brain swelling when compared with FFP infusion alone [14]. In the present study, we investigated whether these differences in lesion size and brain swelling are associated with differential effects of FFP and FFP þ VPA on in vivo platelet activation. We hypothesized that resuscitation with FFP þ VPA would preserve platelet functions, as measured by increased platelet activation markers in brain tissue and serum, compared with FFP alone.

2.

Materials and methods

All experiments were conducted in accordance with the Animal Welfare Act and other federal statutes and regulations related to animal research. The study complied with the Guide for the Care and Use of Laboratory Animals, Institute for Laboratory Animal Research (1996) and was approved by the institutional animal care and use committee. All experiments were performed under the supervision of a veterinarian.

2.1.

Animal preparation and monitoring

Ten female Yorkshire swine (42e50 kg; Tufts Veterinary School, Grafton, MA) were allowed to acclimate for 3 d and examined by a veterinarian to ensure good health. Animals were anesthetized and prepared as previously described [14]. To summarize, animals were sedated, intubated, and supported on a mechanical ventilator with inhaled isoflurane maintained at 1%e3% for the duration of the experiment. Invasive hemodynamic monitoring was accomplished by cannulating the left femoral artery, left femoral vein, right femoral artery, and right external jugular vein. The animal was moved to a sternal position, and the head was fixed in a custom-made stereotactic frame to prevent movement. A craniotomy was performed to provide access for brain oxygenation and intracranial pressure monitoring, and also for the TBI insult.

2.2.

TBI, hemorrhage, and resuscitation protocol

TBI þ HS insults and resuscitation were conducted as previously published [14]. Briefly, a computer-controlled cortical impact device was used to deliver a precise and reproducible TBI [15]. Volume-controlled hemorrhage commenced concurrent with TBI. The total blood volume was estimated, and 40%e45% was withdrawn through the femoral artery at a rate of 3.15% total blood volume per minute. Animals were left in shock (Mean arterial pressure maintained between 30e35 mmHg) for 120 min after hemorrhage. After 2 h of shock, animals were randomly resuscitated with either (i) FFP at 50 mL/min or (ii) FFP at 50 mL/min plus VPA 300 mg/kg (EMD

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Biosciences Inc, La Jolla, CA). The volume of FFP matched the volume of shed blood.

2.3.

Observation and collection of samples

All animals were monitored under anesthesia for 6 h after resuscitation [14] and then killed by an intravenous injection of Euthasol (sodium pentobarbital [100 mg/kg]). Brains were harvested, and samples were flash-frozen at 80 C for storage and subjected later to whole cell lysis [15]. Blood samples were collected at baseline, postresuscitation, and after 6 h of observation, and processed as previously described [7].

2.4.

Enzyme-linked immunosorbent assay

In-vivo platelet activation was assessed by measuring markers of platelet activation in the circulation (serum) and in brain tissue. Approximately 50 mg of brain tissue was homogenized in 1 mL of cell lysis buffer (Whole Cell Extraction Kit; Millipore, Temecula, CA) and sonicated with an ultrasonic cell disruptor (Series 36,810; Cole Palmer, Vernon Hill, IL). Optical densitometry was used to measure protein content, which was subsequently equalized among samples. Enzyme-linked immunosorbent assays were performed according to manufacturer’s instructions using serum and brain lysates for the following markers of platelet activation: soluble P-selectin (Glory Science Co, Ltd, Del Rio, TX), transforming growth factor b1 (TGF-b1; R&D systems, Minneapolis, MN), and soluble CD40 ligand (CD40L; BlueGene Biotech, Shanghai, China). Platelet endothelial cell adhesion molecule (PECAM-1, CD31; Novateinbio Biosciences, Cambridge, MA) was used as a marker for negative platelet activation. Protein concentrations were measured by optical densitometry on a SpectramaxPlus 384 microplate reader (Molecular Devices, Sunnyvale, CA) at 450 nm.

2.5.

3

deparaffinized with xylene, rehydrated through a descending ethanol series (100%, 95%, 70%, 50%, 3 min each) and washed in Tris-buffered saline for 2e5 min. Antigen retrieval was achieved by immersing slides in 0.01 M citrate buffer (pH 6) and microwaving at 800 W for 6 min, followed by a 20 min rest period. After that, samples were preincubated in blocking solution (5% bovine serum albumin, 5% normal goat serum, 0.05% Triton100X, and phosphate buffered saline) and then incubated overnight with primary antibody mouse anti-CD62P antibody (P-selectin; LifeSpan BioSciences Inc, Seattle, WA) and mouse anti-CD61 antibody (Integrin Beta-3; Millipore, Billerica, MA) at 4 C. Reaction was visualized by Fluorescein isothiocyanate-conjugated anti-mouse antibody (Vector Laboratory, Burlingame, CA). For double labeling immunohistochemistry, the brain sections were further incubated with rabbit anti-ZO-1 antibody (Zonula Occludens-1; Abcam, Cambridge, MA) followed by incubation with Texas Red conjugated anti-rabbit antibody (Vector Laboratory). The sections were mounted in VECTASHIELD Mounting Medium (Vector Laboratory) and viewed on a confocal laser scanning microscope (LSM 510, objective 40 1.3 NA; Zeiss, Germany). For quantification of CD61 and CD62P expression, three brain slices per each group were analyzed. Microscope data were acquired with a 20 objective numerical aperture with constant laser power (45% of laser power), pinhole, zoom, focus, gain, and duration of image capturing. A total of five images were randomly selected and captured per slide. Brain sections were evaluated by counting the number of capillaries with positive staining for CD61 and CD62P and ZO-1 in selected area and compared with number of only ZO1 þ blood vessels. A mean of the percentage of positive CD61 or CD62P blood vessels  standard deviation was computed by dividing the number of vessels that expressed CD61 or CD62P by the total number of ZO-1 positive blood vessels multiplied by 100. Slides were coded, so that the counter was blind to the identity of the slides being counted.

Immunohistochemistry 2.6.

Brain tissue samples, obtained from areas adjacent to the primary TBI lesions, were fixed in 10% buffered formalin and embedded in paraffin wax by conventional techniques. The paraffin blocks were then cut into 20 mm thick sections that were subsequently mounted on to slides. The slides were

Statistical analysis

Values at each time point are represented as mean  standard deviation. Independent sample t-tests were used to compare different brain markers in the FFP versus FFP þ VPA groups. Correlated data (serum samples postresuscitation and

Fig. 1 e Concentrations of serum CD40 ligand (CD40L) panel (A), P-selectin panel (B), and TGF-b1; panel (C) in FFP, and FFP D VPA groups. Data presented as group mean ± standard deviation. Asterisk (*) indicates P £ 0.05. P value represents differences in estimated marginal means based on the time points postresuscitation (PR) and after 6 h of observation (6HOB) using a linear mixed model with Bonferroni post hoc testing.

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Table e Plasma and brain markers of platelet activation and inhibition at different time points. Time point CD40 L (ng/mL) Circulation

Brain P-selectin (ng/mL) Circulation

Brain TGF-b (ng/mL) Circulation

Brain PECAM-1 (pg/mL) Circulation

Brain

FFP (n ¼ 5)

FFP þ VPA (n ¼ 5)

Baseline Postresuscitation 6HOB postresuscitation 6HOB postresuscitation

3.12  2.64  2.12  1.22 

0.91 0.60 0.16 0.12

2.80 3.50 2.92 1.41

   

0.46 0.42 0.31 0.15

Baseline Postresuscitation 6HOB postresuscitation 6HOB postresuscitation

50.43  45.96  51.56  3.48 

5.07 12.38 9.31 0.31

70.57 66.39 78.40 3.81

   

17.56 13.13 20.70 0.15

Baseline Postresuscitation 6HOB postresuscitation 6 HOB postresuscitation

10.47  7.80  8.38  0.22 

5.91 2.80 4.45 0.04

14.46 11.31 11.32 0.22

   

6.11 5.66 2.58 0.04

Baseline Postresuscitation 6HOB postresuscitation 6HOB postresuscitation

2.16  2.80  2.42  7.99 

0.50 0.61 0.23 1.13

1.73 2.07 2.00 5.22

   

0.45 0.80 0.67 2.00

P 0.50 <0.01* 0.05 0.18 <0.01* 0.06 0.36 0.17 0.98 0.19 0.26 0.03*

6HOB ¼ 6 h of observation; PR ¼ postresuscitation. Data presented as group means  standard deviation. Asterisk (*) indicates P  0.05. P value of the time points postresuscitation and 6 h of observation represents differences in serum estimated marginal means of both time points using a linear mixed model with Bonferroni post hoc testing.

following the 6-h observation period) were compared between groups by using a linear mixed model with Bonferroni post hoc testing, yielding one compound estimated mean  standard error of the mean. All statistical analyses were performed using IBM SPSS Statistics version 20.0 (IBM, NY).

3.

Results

3.1.

Circulating markers of platelet activation

Serum sCD40L was significantly greater in FFP þ VPA relative to FFP (3.21  0.12 ng/mL versus 2.38  0.12 ng/mL; P < 0.01). Serum P-selectin levels were significantly higher in the FFP þ VPA group compared with FFP alone (70.85  4.70 ng/mL versus 48.44  7.28 ng/mL; P < 0.01). TGF-b was elevated in FFP þ VPA animals, but the difference was not significant (11.20  1.46 ng/mL versus 8.09  1.41 ng/mL; P ¼ 0.17; Fig. 1, Table).

3.2.

Markers of platelet activation in brain tissue

Brain sCD40L levels were significantly higher in FFP þ VPA compared with FFP alone (1.41  0.15 ng/mL versus 1.22  0.12 ng/mL; P ¼ 0.05). No significant differences were found in brain levels of P-selectin (3.81  0.15 ng/mL versus 3.48  0.31 ng/mL; P ¼ 0.06) and TGF-b (0.22  0.04 ng/mL versus 0.22  0.04 ng/mL; P ¼ 0.98) between the treatment groups (Fig. 2, Table).

3.3.

Negative regulation of platelet activation

Brain PECAM-1 levels were significantly lower in the FFP þ VPA group compared with the FFP group (5.22  2.00 pg/mL versus 7.99  1.13 pg/mL; P ¼ 0.03). There was no significant difference in serum levels of PECAM-1 between treatment groups (2.15  0.27 pg/mL versus 2.62  0.27 pg/mL; P ¼ 0.26; Fig. 3, Table).

Fig. 2 e Concentrations of CD40 ligand (CD40L) panel (A), P-selectin panel (B), and TGF-b1; panel (C) in brain tissue between FFP and FFP D VPA. Data presented as group mean ± standard deviation. Asterisk (*) indicates P £ 0.05.

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Fig. 3 e Concentrations of PECAM-1 in serum panel (A) and brain tissue panel (B) between FFP and FFP D VPA. Data presented as group mean ± standard deviation. Asterisk (*) indicates P £ 0.05.

3.4.

Histologic quantification of platelet activation

Immunohistochemistry revealed differences in platelet activation between FFP and FFP þ VPA groups (Fig. 4, panel A). Platelet activation, as measured by CD62P (P-selectin), was significantly higher in the FFP þ VPA group compared with FFP (22.30  10.39% versus 8.125  3.94%, P < 0.01; Fig. 4, panel B). The FFP þ VPA group showed greater platelet density, as measured by CD61, relative to the FFP group (25.70  11.29% versus 12.88  5.44%; P ¼ 0.06), whereas this difference was not significant (Fig. 4, panel B).

4.

Discussion

This study investigated whether the addition of VPA to FFP resuscitation would be associated with increased platelet activation. Our data show that FFP þ VPA treatment resulted in elevated platelet activation in both the brain and serum compared with FFP alone. Brain immunohistochemistry and serum P-selectin levels were significantly higher in the FFP þ VPA group compared with the FFP group. The addition of VPA also resulted in significantly greater serum and brain

Fig. 4 e Representative images of double labeling immunohistochemical staining for CD61 (Integrin beta-3) or CD62P (Pselectin) and ZO-1 protein (Zonula Occludens-1) in brain tissue adjacent to the primary TBI lesion in FFP and FFP D VPA panel (A). Scale bar represents 50 mm. Semiquantitative analysis of CD61 and CD62P immunostaining in FFP and FFP D VPA panel (B). Data represent mean ± standard deviation of five chosen microscopic fields per section at 3200 magnification. Asterisk (*) indicates P £ 0.01. (Color version of figure is available online).

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sCD40L levels. Brain PECAM-1 levels were significantly lower in the FFP þ VPA group compared with the FFP group. We previously showed that the addition of VPA to FFP reduces brain lesion size following TBI [14]. Our results from the present study suggest that this neuroprotective effect is associated with an increase in platelet activation, both in circulation and in the brain. Shock and tissue injury are independently associated with platelet dysfunction. Our recent work has shown that TBI and hemorrhage induce platelet hypofunction within minutes after injury, and this response is associated with circulating markers of platelet activation [7]. These findings support Jacoby et al. [16], who found that TBI patients experience increased platelet activation but decreased function compared with non-TBI trauma patients. The precise mechanisms of this platelet dysfunction remain unclear, but it may be mediated by the so-called “exhausted platelet syndrome.” This phenomenon is characterized by initial platelet hyperactivation and concomitant depletion of intracellular mediators, ultimately resulting in unresponsiveness to stimulation [17]. However, in this study, we still found active platelets 8 h after the TBI insult in the VPA-treated group as shown by elevated sCD40L and P-selectin levels compared with FFP alone. Furthermore, we measured CD62P (P-selectin) levels in the brain using immunohistochemistry and found that these were significantly higher in the FFP þ VPA group compared with FFP resuscitation alone. Moreover, we found a higher level of CD61 (Integrin Beta-3), a marker for platelet density in the brains of the VPA-treated animals, although this value did not reach significance (P ¼ 0.06). However, this difference in platelet activation in the VPA group may be a function of increased platelet density. It is important to note that the total systemic platelet count was similar between the groups (M. Sillesen, unpublished data). Thus, one explanation for the increase in both platelet activation and density in the brain may be due to platelet recruitment from systemic circulation to the brain in response to TBI. In addition to platelet activation, inhibition may also occur via several different mechanisms. For example, PECAM-1, a cell surface glycoprotein receptor expressed on platelets, inhibits platelet aggregation [18,19]. In this study, we found significantly lower brain PECAM-1 levels in the FFP þ VPA group compared with the FFP group. Previous studies have reported that PECAM-1 may limit the growth of platelet thrombi on collagen surfaces [20,21]. This is in accordance with our findings, which show that FFP þ VPA-resuscitated animals exhibited increased platelet activation compared with FFP-resuscitated animals. Platelet activation is responsible for not only forming clots on damaged endothelium but also expressing and releasing substances that promote tissue repair. Functional platelets also act as key players in angiogenesis, coagulation, inflammation, and the immune response [22]. For these reasons, elevated platelet activation may play a role in attenuating secondary brain injury. One possible explanation for the increase in platelet activation in the FFP þ VPA group may be that VPA treatment attenuates the initial platelet hyperactivation. Unpublished data from our research group showed that VPA treatment reduces platelet activation 60 min after administration in the same TBI þ HS model. This initial

attenuation may mitigate the depletion of intracellular mediators and subsequent exhaustion, thereby preserving platelet function that may be critically important in attenuating secondary brain injury in the first 8 h after the TBI insult. In the FFP group, there may be more initial hyperactivation, dysfunction, and exhaustion compared with the FFP þ VPA group. Exhausted platelets can be removed from the bloodstream, which may explain why we see less platelet activation and fewer platelets in the brains of FFP-treated animals. Previous studies have described antiplatelet activity related to chronic administration of VPA, such as thrombocytopenia and platelet dysfunction [23e27], as well as acquired von Willebrand’s disease [28,29]. However, those symptoms rarely become symptomatic [30,31]. Chronic VPA use has been suggested to inhibit the platelet arachidonate pathway [25]. However, other studies have reported a limited effect of VPA on coagulation. Anderson et al. [32] previously described that VPA did not alter the clot strength contributed by platelets, and Ward et al. [33] showed that VPA treatment did not affect blood loss during surgery. To our knowledge, ours is the first study to demonstrate that VPA has a protective effect on platelet activation. We, however, only administered a single large dose of VPA, which most likely has very different effects on platelets compared with chronic exposure to a much lower dose. This clinically realistic model has several limitations. This nonsurvival study ended 6 h after resuscitation, which limited our ability to measure long-term outcomes of platelet activation. Our group is addressing this limitation by currently performing long-term survival experiments where we will further study platelet dysfunction. Furthermore, we measured platelet activation and not platelet function. Functional tests such as whole-blood impedance aggregometry may provide more insight into the role of VPA on platelet function.

5.

Conclusions

Combined treatment with FFP þ VPA increases platelet activation in both serum and brain tissue, which may attenuate lesion size and be protective after TBI and HS. Elevated platelet activation may play a role in attenuating secondary brain injury. For this reason, the role of platelets in TBI and HS should be further investigated.

Acknowledgment This study was funded by a grant from the US Army Medical Research Material Command GRANTT00521959 (H.B.A.) and a grant from the Lundbeck Foundation (M.S. and P.I.J.). Authors’ contributions: S.E.D., M.S., and H.B.A. contributed to conception and design; S.E.D., M.S., T.B., A.V.A., G.J., and I.H. for analysis and interpretation; S.E.D., M.S., T.B., A.V.A., G.J., B.L., D.L., and I.H. for data collection; S.E.D., and T.B. for writing the article; and M.S., C.B., P.I.J., I.H., and H.B.A. for critical revision of the article; and H.B.A. for obtaining funding.

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Disclosure [16]

The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in this article.

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