The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor simvastatin reduces thrombolytic-induced intracerebral hemorrhage in embolized rabbits

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The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor simvastatin reduces thrombolytic-induced intracerebral hemorrhage in embolized rabbits Paul A. Lapchak a,⁎,1 , Moon Ku Han b,1 a

Department of Neuroscience, University of California San Diego, 9500 Gilman Drive MTF316, La Jolla, CA 92093-0624, USA Department of Neurology, Stroke Center, Neuroscience Center, Seoul National University Bundang Hospital, Seoul National University College of Medicine, 300 Gumidong, Bundang-gu, Seongnam-si, Gyeonggi-do 463-707, South Korea b

A R T I C LE I N FO

AB S T R A C T

Article history:

Statins were designed as cholesterol-lowering drugs for the prevention of coronary artery

Accepted 15 September 2009

disease. It is estimated that there are currently 33.5 million U.S. patients on chronic statin

Available online 23 September 2009

treatment regimens. Recently, statins have been shown to have pleiotropic including antiinflammatory and neuroprotective effects. In this study, we assessed the pharmacological

Keywords:

effects of simvastatin administered alone and in combination with tissue plasminogen

Ischemic stroke

activator (tPA) on measures of ischemia and hemorrhage in large clot embolized New

Neuroprotection

Zealand white rabbits. For these studies, simvastatin (20 mg/kg, SC in DMSO) was

Intracerebral hemorrhage

administered 24 and 4 h prior to large clot embolization in order to achieve a “loading

Translational science

dose” pretreatment with the drug. In combination studies, tPA (3.3 mg/kg, IV) was administered 1 h following embolization. Intravenous tPA administration significantly increased hemorrhage volume by 175% (p = 0.015) and hemorrhage incidence by 60% (p > 0.05) compared to control, but did not affect infarct incidence or volume. Simvastatin treatment significantly decreased tPA-induced hemorrhage incidence (p = 0.022) and volume (p = 0.0001) following embolization. The study suggests that simvastatin treatment blocks or attenuates mechanisms involved in tPA-induced hemorrhage. Based upon our results, patients on simvastatin treatment may have significantly reduced tPA-induced side effects should they require tPA administration following an embolic stroke. © 2009 Elsevier B.V. All rights reserved.

1.

Introduction

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoA reductase) inhibitors, more commonly known as statins, were initially designed as cholesterol-lowering drugs for the primary and secondary prevention of coronary artery disease (CAD) and acute ischemic stroke (AIS) (Delanty and Vaughan, 1997; Hebert et al., 1997; Kashyap, 1997), which is the third

leading cause of death and the leading cause of adult disability in the U.S. (Ingall, 2004). According to the current 2009 U.S. stroke statistics (Lloyd-Jones et al., 2009), each year, approximately 795,000 people suffer a stroke (one every 40 s with one mortality every 3 min). The World Health Organization (WHO) estimates that 15 million people suffer strokes worldwide. More than 5 million stroke victims die from the brain insult and approximately 5.5 million are permanently disabled

⁎ Corresponding author. Fax: +1 858 822 4106. E-mail address: [email protected] (P.A. Lapchak). 1 Both authors contributed equally to this translational science study. 0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2009.09.064

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(www.strokecenter.org/patients/stats.htm). The recent JUPITER report indicated that more than 44.7 million Americans 50 years old and older might have an indication for statin therapy and that 33.5 million older Americans are currently taking a statin (Spatz et al., 2009). Nevertheless, some percentage of statin patients will still have an AIS, but the incidence of AIS in statin patients remains unknown. There has been some interest in using statins to treat AIS (Montaner, 2005) and prevent stroke (Amarenco et al., 2006) and there is also some interest in treating hemorrhagic stroke with statin therapy (Kramer et al., 2008; Meier et al., 2009; Naval et al., 2008; Uyttenboogaart et al., 2008). The SPARCL trial, which tested the effects of atorvastatin (80 mg per day) in patients with a history of recent stroke or transient ischemic attack, showed that the drug reduced the overall incidence of strokes and cardiovascular events, but caused a small increase in the incidence of hemorrhagic stroke (Amarenco et al., 2006). In a post-hoc analysis Goldstein et al. (2008) reported that hemorrhagic stroke was more frequent when patients were treated with atorvastatin and in patients with a hemorrhagic stroke as an entry event into the trial. The incidence of hemorrhagic stroke was also elevated in men, increased with age and was higher in patients with stage 2 hypertension (JNC-7). There were no relationships between hemorrhage risk and baseline low-density lipoprotein (LDL) cholesterol level or recent LDL cholesterol level in treated patients. Recently, Naval et al. (2008) completed a retrospective analysis of patients with intracerebral hemorrhage (ICH) treated with statins and found that there was a significant association of prior statin use with decreased mortality, thus suggesting that additional studies are warranted. Based upon the clinical information cited above, there is no clear indication for the use of statin to treat any form of stroke because of the confusing data. Of great interest to scientists are the pleiotropic effects of statins, which include protective effects on the vasculature and neurons (Bays, 2006; Endres, 2005; Halcox and Deanfield, 2004; Liao, 2002; Moonis and Fisher, 2004; Rutishauser, 2006; Schmeer et al., 2006; Vivancos-Mora and Gil-Nunez, 2005; Waldman and Kritharides, 2003; Werner et al., 2002). In animal models of hemorrhagic stroke, atorvastatin has been shown to improve neurological functional outcome, reduces cerebral cell loss, and promotes regional cellular plasticity when administered following hemorrhage in rats (Seyfried et al., 2004). In chronically atorvastatin-treated rats, there was a significant reduction in the severity of neurological deficits induced by hemorrhage, an effect that may involve increased neuronal plasticity. Jung et al. (2004) have also demonstrated that application of atorvastatin may reduce perihematomal cell death due to suppressed apoptosis and a reduced inflammatory response. Of importance to this study is a report which showed that simvastatin can reduce tPAinduced membrane or matrix metalloproteinase-9 (MMP-9) levels in cultured cortical astrocytes (Wang et al., 2006). This finding is important because we have previously shown that tPA-induced ICH in the rabbit embolic stroke model, the model used in the present study, can be regulated by treatment with the MMP-2/MMP-9 inhibitor BB-94 (Lapchak et al., 2000). Given the pleiotropic nature of simvastatin and the drug's ability to both reduce MMP activity and promote neuroprotec-

tion (Cimino et al., 2005; Hess et al., 2000; Montaner, 2005), we tested the hypothesis that administration of simvastatin prior to a large clot embolic stroke may attenuate stroke-induced hemorrhage and ischemia. This treatment regimen was used to parallel simvastatin use in approximately 40 million patients who are at risk for CAD and AIS, some of whom will have a stroke and be treated with tPA if they present to a clinic within 4.5 h of the event (Hacke et al., 2008). In addition, in parallel, we determined if prior statin administration would affect tPA-induced hemorrhage following large clot embolic strokes in rabbits as a follow-up to the Naval retrospective analysis described above (Naval et al., 2008). For these studies, we used the rabbit large clot embolic stroke model (RLCEM), a middle cerebral artery occlusion (MCAO) model that is sensitive to intravenously administered tPA, resulting in ICH (Lapchak et al., 2000; Lapchak, 2007).

2.

Results

2.1.

Stroke infarct incidence

Table 1 presents the infarct incidence resulting from quantitation of brain sections following embolization. Fig. 1 shows a representative brain section indicating the presence of an infarct. The infarct incidence was similar in all four treatment groups and there was no statistically significant difference between the groups. There was a trend for reduced infarct incidence in the simvastatin-treated group that did not reach statistical significance. There was also no significant effect of any treatment on infarct volume.

2.2. Effect of simvastatin on hemorrhage rate and volume: combination studies In these experiments, rabbis received two doses of simvastatin prior to embolization. In combination studies, tPA was given 1 h following embolization. We found that in simvastatintreated embolized rabbits, the hemorrhage rate was less than that measured in control rabbits, but the difference was not significant (Table 2). Fig. 1 shows representative brain section identifying the three types of hemorrhage that may be present

Table 1 – Infarct Incidence and volume. Treatment group Control Simvastatin tPA tPA + simvastatin

Infarct incidence

Infarct volume (number of faces)

11/16 (68.75%) 7/16 (43.75%) ⁎p = 0.28 10/15 (66.66%) ⁎p = 0.79 10/15 (66.66%) ⁎p = 0.79

5.46 ± 1.38 (11) 3.56 ± 1.38 (7) ⁎p = 0.338 7.86 ± 2.25 (10) ⁎p = 0.364 3.73 ± 0.99 (10) ⁎p = 0.322 vs. control #p = 0.16 vs. tPA

Table 1 presents infarct incidence for the four groups included in the study. Results are presented as percentage population. There were no statistically significant differences between the treatment groups and the control group (⁎p > 0.05). # indicates significance vs. the tPA group.

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When hemorrhage volume was measured, we found that there was no significant effect of simvastatin treatment, but tPA did significantly increase hemorrhage volume (p = 0.015) compared to control. Hemorrhage volume in the tPA plus simvastatin-treated rabbits was zero since simvastatin treatment prevented tPA-induced hemorrhage in embolized rabbits (Table 2).

3.

Fig. 1 – A rabbit brain exhibiting a PH (top panel), HPT (middle panel) and HIN (bottom panel). See all white arrows for hemorrhage location. The red arrows show the location of an infarct (white tissue) in the top and bottom panels.

in rabbit brain following large clot embolization. In tPA-treated rabbits, 6 of 15 or 40% of the group had hemorrhages compared to 4 of 16 (25%) in the control group. This difference was also not significant (p > 0.05) compared to control. In an additional group, we determined the effects of simvastatin in the presence of tPA administration on ICH incidence. We found that by treating with simvastatin, the incidence of hemorrhage associated with the administration of tPA following embolization was reduced to zero and that was statistically different from the tPA group (p = 0.022) (see Table 2).

Discussion

In the present study, we assessed the effects of simvastatin on measures of hemorrhage and ischemia following large clot embolic strokes in rabbits because of the usefulness of the model at predicting beneficial effects of pharmacological treatments (Lapchak et al., 2000; Lapchak, 2007). We found that simvastatin treatment significantly blocked tPA-induced hemorrhage. There have been a few preclinical reports documenting protective effects of statins, including atorvastatin and simvastatin, on various measures in rodent hemorrhage models. For example, Karki et al. (2009) showed that both simvastatin and atorvastatin significantly improved neurological recovery and decreased necrosis when administered for 1 week after primary ICH was induced in rats and Jung et al. (2004) showed that atorvastatin significantly reduced apoptosis and markers of inflammation in perihematomal tissue following experimental ICH, changes that promoted sensorimotor recovery. An interesting study by Trinkl et al. (2006) showed that pravastatin pretreatment resulted in a reduction of microvascular basal lamina damage (measured using collagen type IV) and hemoglobin extravasation following transient ischemia using a rat suture model. Interestingly, the authors concluded that pravastatin had protective effects on the cerebral microvascular system. Liu and colleagues (2006) showed that atorvastatin treatment was able to block the upregulation of cerebral endothelial genes (i.e., MMP-2 and MMP-9) that mediate thrombosis and BBB permeability following a stroke in rodents. Taken together, the results suggest that statins may have a variety of effects on neuronal and BBB integrity when applied before and after either ischemic or hemorrhagic insults. The therapeutic benefit of simvastatin therapy given using a two dose simvastatin pretreatment regimen followed by embolization with or without tPA treatment may be attributed to statin's beneficial effect(s) on cerebral vascular integrity, an effect unrelated to its primary effect of reducing serum lipids and inhibiting cholesterol production (Chmielewski et al., 2002; Corsini et al., 1999; Kolovou et al., 2008). The benefit may be associated with the action of simvastatin to reduce or attenuate one or more known molecular pathways involved in tPA-induced ICH. For example, we have identified MMPs, free radicals and TNF-α as possible mediators of ICH in the RLCEM (Lapchak et al., 2000, 2002; Lapchak, 2007). We have previously shown that MMP-2 or MMP-9 may be involved tPA-induced ICH because treatment with a broad spectrum MMP inhibitor significantly reduces ICH incidence following tPA administration (Lapchak et al., 2000; Lapchak, 2007). There is some evidence that treatment with tPA may upregulate MMP-9 expression in

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Table 2 – Hemorrhage incidence, volume and types. Treatment group

n

Hemorrhage incidence (%)

Hemorrhage volume

Control

16

4/16 (25%)

1.62 ± 0.89

Statin

16

1/16 (6.25%) ⁎p = 0.33

2.25 ± 1.12 ⁎p = 0.663

tPA

15

6/15 (40%) ⁎p = 0.61

4.46 ± 0.62 ⁎p = 0.015

Statin + tPA

15

0/15 (0%) #p = 0.022 vs. tPA

0 ⁎p = 0.088 vs. control #p = 0.0001 vs. tPA

Hemorrhage types PH 1 HIN 3 HPT 1 PH 0 HIN 1 HPT 0 PH 3 HIN 3 HPT 0 PH 0 HIN 0 HPT 0

Table 2 presents hemorrhage volumes (number of faces) as mean ± SEM for only those rabbits exhibiting an ICH (n) and the types of hemorrhage found in the brain of embolized rabbits in each experimental group. PH, parenchymal hemorrhage; HIN, hemorrhagic infarction; HPT, punctuate hemorrhage. Hemorrhage volume was increased by tPA treatment (p = 0.015) and tPA-induced hemorrhage volume was decreased by simvastatin pretreatment (#p = 0.0001).

order to mediate blood–brain barrier damage resulting in ICH and hemorrhagic transformation (Lo et al., 2002; Pfefferkorn and Rosenberg, 2003; Rosenberg et al., 2001). Both atorvastatin and simvastatin have previously been shown to decrease MMP-9 levels in vitro and in vivo (Zhang et al., 2005). Thus, the existing data would suggest that the beneficial effect of statin treatment may be related to the ability of simvastatin to down-regulate MMP expression and/or activity, an action that may involve a variety of different pathways, possibly including the Rho kinase signaling pathway (Wang et al., 2006), PI3K/Akt pathway (Sugawara et al., 2008b) or JAK–STAT signaling pathway (Wu et al., 2006). Since we have established that prior simvastatin treatment can block tPAinduced hemorrhage, we are now elucidating the mechanisms and pathways involved in the simvastatin effect using the RLCEM. There are a number of caveats to the use of statins to treat tPA-induced ICH. Although simvastatin has been shown to be beneficial in hemorrhagic stroke patients (Naval et al., 2008), a small uncontrolled retrospective study (Uyttenboogaart et al., 2008) found that statin use had no significant influence on symptomatic ICH (sICH) or functional outcome in patients treated with tPA. Moreover, acute simvastatin treatment has no beneficial effect in patients with subarachnoid hemorrhage (Vergouwen et al., 2009), although Kerz et al. (2008) found that there was a trend for decreased mortality in patients given simvastatin, even though there was a trend for increased delayed cerebral ischemia in the same patients. Moreover, in a post-hoc analysis of the SPARCL atorvastatin trial, Goldstein et al. (2008) reported that there was a relationship between hemorrhagic stroke and patients with a hemorrhagic stroke as an entry event into the trial and there was also a connection with age, sex and other complicating factors such as hypertension. The available data related to the use of simvastatin to treat, prevent and/or treat stroke (including hemorrhagic stroke) are quite confusing. Because of the possible usefulness of statins to treat stroke, additional preclinical and clinical studies are necessary to determine whether the class of drugs should be used to treat hemorrhagic stroke or commonly known complications of tPA therapy. Future mechanistic

studies from translational studies may also provide additional insight into pharmacological regulation of thrombolyticinduced hemorrhage following embolic strokes. In conclusion, we have demonstrated that simvastatin administered before thrombolytic therapy can significantly reduce hemorrhage incidence and volume associated with thrombolytic therapy, the first line of therapy provided to AIS patients. Our study suggests that statins may protect the integrity of cerebral vessels, and reduce ICH following intravenous thrombolytic treatment in patients on a simvastatin regimen.

4.

Experimental procedures

4.1.

Animals and animal welfare

Male New Zealand white rabbits weighing 2–2.5 kg were purchased from Rabbit Source, Ramona, CA. Rabbits were supplied food (alfalfa cubes) and water ad libitum while under quarantine in an enriched environment for at least 5 days prior to experimental use. Surgery was done in a sterile controlled environment with a room temperature between 22.8 and 23.2 °C. All surgical, embolization and histological procedures were done as described previously (Lapchak et al., 2000; Lapchak, 2007; Lyden et al., 1989). The Department of Veterans Affairs and Institutional Animal Care and Use Committee (IACUC) approved the surgical and treatment procedures used in this study. Veterinary assistance and care were used throughout the study to minimize pain and discomfort. Per the IACUC-approved protocol, rabbits were euthanized if they were in pain, showed extreme discomfort or if they were unable to reach food or water.

4.2.

Surgery and embolization

Rabbits were anesthetized with isoflurane via face mask, 5% in 3 l/min at induction, and 3% in 3 l/min as a maintenance dose. The right internal carotid artery was exposed, and the external carotid artery and the common carotid artery were

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ligated. If any other branches were seen originating from the internal carotid artery, these were also ligated. A Becton, Dickinson and Company (B-D) plastic catheter oriented toward the brain was inserted into the common carotid and secured with ligatures. The incision was closed around the catheter so that the distal end was accessible outside. The catheter was filled with 0.2 ml of heparinized saline (33 U/ml) and plugged with injection caps. The animals were allowed to recover from anesthesia for at least 2 h before embolization (Lapchak et al., 2008). Emboli were prepared by withdrawing 2–4 ml of arterial blood from a single donor rabbit. Because the studies had a 24-h endpoint, there was no concern of immunogenicity of blood clots prepared from a single donor rabbit that were subsequently used in experiment for a group of rabbits on a particular experiment day. The blood was mixed with a trace quantity of 57Co labeled plastic NEN-Trac microspheres (New England Nuclear, Inc.), and allowed to clot for at least 3 h at 39 °C. The clot was cut into small cubes weighing approximately 2.5–3.5 mg and they were suspended in phosphatebuffered saline containing 0.1% bovine serum albumin. The amount of radiolabel present in each blood clot was measured using a mobile gamma counter. Prior to embolization, each animal was placed in a Plexiglas restrainer and the injection cap of the catheter was removed to allow the rabbit's blood to fill the catheter and wash out the heparinized saline. The line was then filled with heparinfree normal saline. One of the clot cubes was placed inside the injection port of the catheter and rapidly injected with 3 ml of saline flush, followed immediately by a 5 ml flush. Care was taken during both flushes to be sure that no air bubbles were present in the catheter or syringe. If the animal did not have a behavioral stroke reaction, which could include nystagmus, kicking, rolling, loss of balance, hemiparesis, seizure or a coma, then another blood clot was injected in the same way 3 min after the first embolization. If there was no behavioral reaction to either embolization, no further blood clots were administered. After the embolization process was completed, the catheter was ligated close to the neck and the rest of the catheter and injection port were heat-sealed. Animals that died prior to sacrifice, but received treatment(s), were included in this study; the brains were fixed and sectioned as below. The surviving animals were euthanized 24 h post-embolization with 1–1.5 ml of Beuthanasia-D via the marginal ear vein. The brains were removed and immersion fixed in 4% paraformaldehyde for at least a week, and then examined by an observer naïve to the treatment groups.

(2) hemorrhagic infarction (HIN) consists of red speckling of an area, usually surrounded by soft infarcted tissue; and (3) punctate hemorrhages (HPT) which are small isolated red marks within tissue that do not extend through the tissue like a blood vessel would. Fig. 1 presents representative brain sections with the different types of hemorrhage observed in the study. The panel also shows an infarct in the cortex after standard fixation. For infarct analysis, we noted the presence of infarct damage which is grossly visible as pale, softer tissue surrounded by yellow-tinted normal brain tissue on the brain sections (due to formaldehyde fixation). After evaluation of the brain tissue, the total radioactivity in the brain was measured by placing the slices into a portable gamma counter and the amount of radiolabel present in the brain was compared to that contained in the labeled blood clot prior to embolization. Table 1 presents the fraction of counts received in brain for the four treatment groups included in this study. If fewer than 10% of the total counts were found in the brain, it was assumed that the labeled blood clot had not reached the brain (Lapchak et al., 2000; Lapchak, 2007) and the animal was excluded from the study. Fig. 2 shows a rabbit brain with a clot lodged in the middle cerebral artery after embolization.

4.4.

Drug treatment

The RLCEM was primarily used to determine whether simvastatin treatment in the absence or presence of tPA administration would alter hemorrhage or ischemia incidence (rate) or volume following embolic strokes in rabbits.

4.5.

Simvastatin therapy

Pharmaceutical grade (United States Pharmacopeia) simvastatin was obtained from Chong Kun Dang Pharmaceutical Co. Ltd. (Seoul, South Korea). Rabbits were treated with simvastatin (20 mg/kg (Sugawara et al., 2008a)) using a subcutaneous

4.3. Intracerebral hemorrhage and radioactivity quantitation The brain was cut into 10 coronal slices, each having two faces. We noted the presence and type of each hemorrhage and the number of section faces showing hemorrhage(Lapchak et al., 2008). Three major types of ICH can be identified:

(1) parenchymal hemorrhages (PH) are large homogenous masses of blood within tissue;

Fig. 2 – A rabbit brain with a large clot lodged (white arrow) in the middle cerebral artery following embolization.

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(SC) injection at the nape of the neck, with one injection given 24 h prior to embolization and a second injection given 4 h before large clot embolization. We used this pretreatment regimen with simvastatin at pharmacological doses to parallel a “loading” effect with the drug. Simvastatin was dissolved in 100% HPLC grade DMSO purchased from Fisher Scientific.

4.6.

Power analysis and statistical analysis

Power calculations for ICH rate were based on a two-sided chisquare test for detecting a difference between two proportions assuming a type 1 error of 0.05. With a sample size of 15–20 rabbits in each group and assuming a baseline ICH rate of approximately 20% in the control embolized group, we have 80% power to detect a hemorrhage rate of 70% in the treated group. Power analysis for ICH volume indicates that, assuming α = .05 and β = .90, a coefficient of variation of 15% and a difference between means of 20% for ICH volume, we would require a sample size of 14 animals per group. Data were analyzed using MedCalc version 9.4.1.0. for comparison of proportions for hemorrhage and infarct incidence rates as described previously by Lapchak et al. (2008) Analysis of variance (ANOVA) followed by the t-test was used for the analysis of differences for hemorrhage and infarct volumes using SigmaStat 3.5 (Lapchak et al., 2008).

4.8.

(4) simvastatin + intravenous thrombolytic group (as above under 2 and 3).

Acknowledgments This work was supported by a VA Merit Review grant and gift funds to P.A.L. at the University of California San Diego.

Thrombolytic therapy

For tPA administration, tPA purchased from Genentech, Inc. was infused 1 h following embolization using the marginal ear vein as described previously (Lapchak et al., 2008) at a dose of 3.3 mg/kg tPA, 20% as a bolus injection given over 1 min, followed by the remainder infused over 30 min (Lapchak et al., 2000; Lapchak, 2007; Lyden et al., 1989). This standard dose and timing of tPA administration has previously been shown to affect hemorrhage rate in rabbits following large clot embolization (Lapchak et al., 2000; Lapchak, 2007). In this study, all groups of embolized rabbits, excluding the simvastatin-treated groups, received equivalent volumes of DMSO according to the dosing schedule described above.

4.7.

149

Treatment groups

In the present study, a full control group was run in parallel to the three different treatment groups and all groups were rated simultaneously in order to preclude any experimental or investigator bias. For all experiments in this study, rabbits were randomly allocated into treatment groups before the embolization procedure, with concealment of the randomization guaranteed by using an independent party (PAL). The randomization sequence was not revealed until all postmortem analyses were complete. The four treatment groups studied were the following: (1) control (DMSO-treated) embolization group, (2) simvastatin embolization group (24 and 4 h prior to embolization), (3) intravenous tPA (embolization group) given 1 h postembolization and

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