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Lack of robust neurologic benefits with simvastatin or atorvastatin treatment after acute thoracic spinal cord contusion injury
Experimental Neurology 221 (2010) 285–295
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Experimental Neurology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x n r
Lack of robust neurologic benefits with simvastatin or atorvastatin treatment after acute thoracic spinal cord contusion injury Cody M. Mann a, Jae H.T. Lee a, Jessica Hillyer a, Anthea M.T. Stammers a, Wolfram Tetzlaff a,c, Brian K. Kwon a,b,⁎ a b c
International Collaboration on Repair Discoveries (ICORD), University of British Columbia, Vancouver, British Columbia, Canada Combined Neurosurgical and Orthopaedic Spine Program (CNOSP), Department of Orthopaedics, University of British Columbia, Vancouver, British Columbia, Canada Department of Zoology, University of British Columbia, Vancouver, British Columbia, Canada
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Article history: Received 20 September 2009 Revised 28 October 2009 Accepted 6 November 2009 Available online 18 November 2009 Keywords: Simvastatin Atorvastatin Neuroprotection Spinal cord injury
a b s t r a c t Although much progress has been made in the clinical care of patients with acute spinal cord injuries, there are no reliably effective treatments, which minimize secondary damage and improve neurologic outcome. The time and expense needed to establish de novo pharmacologic or biologic therapies for acute SCI has encouraged the development of neuroprotective treatments based on drugs that are already in clinical use and, therefore, have the advantage of a well-characterized safety and pharmacokinetic profile in humans. Statins are the most commonly prescribed class of lipid-lowering drugs, and recently, it has been recognized that statins also have powerful immunomodulatory and anti-inflammatory effects. This paper describes a series of experiments that were performed to evaluate the comparative neuroprotective effects of simvastatin and atorvastatin. We observed a promising signal of neurologic benefit with simvastatin in our first experiment, but in repeated attempts to replicate that effect in three subsequent experiments, we failed to reveal any behavioral or histologic improvements. We would conclude that simvastatin given orally or subcutaneously at doses previously reported by other investigators to be effective in different neurologic conditions does not confer a significant neurologic benefit in a thoracic contusion injury model (OSU Impactor) when administered with a 1-h delay in intervention. We contend that further preclinical investigation of atorvastatin and simvastatin is warranted before considering their translation into human SCI. © 2009 Elsevier Inc. All rights reserved.
Introduction Although much progress has been made in the clinical care of patients with acute spinal cord injuries, convincingly effective neuroprotective treatments to minimize secondary damage and ultimately improve neurologic outcome remain elusive. To date, intravenous methylprednisolone is the only neuroprotective drug to have achieved widespread adoption, although the efficacy and safety of this practice has been intensely questioned, leading many to abandon it (Hurlbert and Hamilton, 2008). Clearly there is a compelling need for better pharmacologic treatment options for this injury. The time and expense needed to establish de novo pharmacologic or biologic therapies for acute SCI has encouraged the development of neuroprotective treatments based on drugs that are already in clinical use, often for unrelated applications. Such drugs have the advantage ⁎ Corresponding author. D6 Heather Pavilion, Vancouver General Hospital, Department of Orthopaedics, University of British Columbia, 2733 Heather Street, Vancouver, BC, Canada V5Z 3J5. Fax: +1 604 875 5858 E-mail address: [email protected] (B.K. Kwon). 0014-4886/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2009.11.006
of a well-characterized safety and pharmacokinetic profile in humans. Examples of such pharmacologic agents include minocycline—a semisynthetic antibiotic used in the treatment of acne, erythropoietin—a recombinant hormone used in the treatment of anemia, riluzole—a sodium channel blocker used in the treatment of amyotrophic lateral sclerosis, and statins—cholesterol-lowering agents. All of these have demonstrated the ability to attenuate certain aspects of secondary damage procured after CNS injury (Bartesaghi et al., 2005; Domercq and Matute, 2004; Gorio et al., 2002; Kaptanoglu et al., 2004; Pannu et al., 2007; Schwartz and Fehlings, 2001; Stirling et al., 2004; Wells et al., 2003; Pannu et al., 2005). The statins are a group of drugs that inhibit hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase, the enzyme responsible for the conversion of HMG-CoA to mevalonate, the rate-limiting step in de novo cholesterol synthesis (Schachter, 2005). They are the most commonly prescribed class of lipid-lowering drugs and include lovastatin (Mevacor; Merck, Whitehouse Station, NJ), atorvastatin (Lipitor; Pfizer), pravastatin (Pravachol; Bristol-Myers Squibb, New York, NY), and simvastatin (Zocor; Merk). Recently it has been recognized that statins also have powerful immunomodulatory and anti-inflammatory effects (Stuve et al., 2003), a pleiotropic nature
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resulting from the downstream inhibition of isoprenoid intermediates (Liao and Laufs, 2005). Statins possess antioxidant properties and upregulate endothelial nitric oxide synthase (eNOS; Cimino et al., 2007). Furthermore, statins have been shown to inhibit the expression of several pro-inflammatory cytokines that play a role in immune mediated damage after SCI such as monocyte chemotactic protein (MCP)-1, IL-2, IL-12, and interferon gamma (Romano et al., 2000; Youssef et al., 2002). The potential for statins to promote neurologic recovery in acute SCI was first reported by Pannu et al., who revealed that the administration of atorvastatin for 1 week prior to spinal cord injury resulted in a significant reduction of inflammatory cytokines, neuronal cell death at the injury site, and a significant improvement in locomotor scores (Pannu et al., 2005). A subsequent study by these authors reported that post-injury treatment, starting 2, 4, or 6 h after SCI, had similar beneficial neuroprotective effects, with dramatic improvements in hindlimb locomotor function (Pannu et al., 2007). The efficacy of statins in protecting nervous tissue has also been demonstrated after stroke and traumatic brain injury (TBI; Cimino et al., 2005; Wang et al., 2007). The widespread clinical use of statins makes them an appealing choice for translation into human SCI. Recognizing that initiating a clinical trial in human SCI is an enormous endeavor and a huge commitment of time and resources, we undertook this series of preclinical studies in order to better characterize the neuroprotective effect. First, we investigated the relative neurologic efficacy of atorvastatin and simvastatin in a model of thoracic SCI. Because either drug could be utilized in a clinical SCI setting, we felt that it is important to pre-clinically assess whether one appeared more promising for human translation. Atorvastatin and simvastatin differ in their clinical pharmacology, as simvastatin is more lipophilic and therefore more capable of crossing the blood–brain barrier, increasing its potential to target injured CNS tissue (Igel et al., 2001; Igel et al., 2002). The relative efficacy of atorvastatin and simvastatin has been compared in two previous studies of traumatic brain injury. Wang et al. (2007) showed no difference in the neuroprotection afforded by these drugs, but Lu et al. (2007) concluded that simvastatin was superior at improving neurologic recovery. A comparison in acute SCI has not been previously reported. We then conducted a series of experiments to examine different dosing and drug formulations for simvastatin, in an attempt to optimize the neuroprotective properties of the therapy.
Manual bladder expression was performed 3 times per day until reflexive voiding returned. The animals received a subcutaneous injection of buprenorphrine (0. 02 mg/kg, Temgesic®, Reckitt Benkiser Healthcare, UK) just prior to their surgery and then again twice on post-operative days 1 and 2. All animal surgeries and care were conducted in accordance to UBC Animal Care guidelines. Behavioral outcome assessment Open-field locomotor testing was performed using the Basso, Bresnahan, and Beattie (BBB) locomotor score (Basso et al., 1995)and the later-developed subscore, which measures fine details of locomotion (Basso, 2004). Animals were tested pre-injury to establish a baseline and to acclimatize the animals to the testing apparatus, and then on post-injury days 2, 4, 7, 14, 28, and 42 or 45 when applicable. All open-field locomotor testings were performed under the same conditions (including time of day) on each scheduled testing date. At 45 days post-injury, the animals were video-taped walking over a horizontally laid ladder with unevenly and randomly spaced rungs. The number of hindlimb slips between the rungs (“footfalls”) was documented, as a measure of the animals' ability to sense and accommodate for changes in the walking surface. A CatWalk™ gait analysis system (Noldus, Ashville, NC) was acquired during the course of these series of experiments and was utilized in latter studies to specifically measure the following gait parameters: regularity index, average hindpaw base of support, and average hindpaw intensity (Hamers et al., 2006). The animals underwent hindpaw mechanical stimulation threshold testing at 42 days post-injury using a von Frey monofilament device (Semmes-Weinstein monofilament, Stoelting, Wood Dale, IL) to test for the development of below-level mechanical allodynia. The animal was placed on an elevated grid and observed for 5 to 10 min to ensure that it was calm. When both hindpaws were evenly resting upon the rungs of the grid, the monofilament was pressed into the center of the volar surface of the hindpaw. The mechanical force at which hindlimb withdrawal occurred and the time from stimulus application to hindlimb withdrawal were recorded. For all behavioral outcome assessments (hindlimb motor and sensory testing) baseline scores were first established by evaluating the animals prior to injury. All post-injury evaluations were performed by individuals that were blinded to the treatment group. Anatomic outcome assessment
Materials and methods Animal model and surgical procedures Male Sprague–Dawley rats weighing 320– 340 g were anesthetized with an intraperitoneal injection of ketamine hydrochloride ( 72 mg/kg; Bimeda-MTC, Cambridge, ON) and xylazine hydrochloride ( 9 mg/kg; Bayer, Etobicoke, ON) diluted in 20 mM Phosphate Buffered Solution (PBS) and a dorsal midline incision was made over the midthoracic spine to expose the posterior spinal elements at T9–10. A laminectomy was performed, and the bases of the adjacent spinous processes were secured with modified Allis clamps, which then held the animal secure within a custom frame. With half of the animal's weight suspended, the animal was positioned under the Ohio State University (OSU) Impactor, and the impactor tip was gently lowered to apply a pre-load force of 0.2 kdyn onto the dura. The impactor was then triggered to deliver a 1. 5-mm displacement injury at 300 m/s. Animals were immediately excluded if the peak force of the impact fell outside the range of 200– 260 kdyn. The dorsal wound was irrigated and then closed with clips. During the acute post-operative period, the animal temperature was monitored and maintained at 37 °C in an incubator to avoid hypothermia. Five milliliters of Ringer's lactate solution were administered subcutaneously for hydration.
All animals were sacrificed with a lethal injection of Pentobarbital Sodium ( 107 mg/kg, Bimeda-MTC Animal Health, Cambridge, Ontario, Canada) and then perfused with phosphate buffered saline followed by fixation with cooled 4% paraformaldehyde. A 15-mm segment of spinal cord centered around the injury site was harvested, post-fixed overnight in 4% paraformaldehyde, cryoprotected in increasing concentrations of sucrose dissolved in PBS (18 and 24%), and then snap frozen over dry ice. The cords were cut in cross-section at 20-μm thickness for histological analysis of the injury site. For the analysis of spared white and gray matters through the injury site, sections 200 μm apart were stained with Eriochrome Cyanine (EC) as per described by Rabchevsky et al. (2001), counterstained with Neutral Red, then imaged on a Zeiss Axioskop microscope at 5× objective. Regions of spared white and gray matters were manually traced and then quantified using SigmaScan Pro version 5.0.0 (Systat Software). For the quantification of macrophages/microglia, a non-fluorescent DAB staining protocol was utilized, in which endogenous peroxidase activity was quenched with 0.3% hydrogen peroxide in methanol, and the sections incubated overnight with the monoclonal mouse ED1 antibody (1:500, Serotec) after appropriate blocking. After incubation for 1 h at room temperature with a biotin-conjugated secondary
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antibody, the ABC technique (Vectastain® ABC Kit, Vector Laboratories, Burlingame, CA) was applied in exactly the same manner for all slides. Images were captured with a Zeiss microscope (Axioplan 2) and Northern Eclipse software (Empix Imaging). A semi-quantitative analysis of ED1 staining was performed by first thresholding each section to remove non-specific background signal, and then capturing the extent of ED1 immunoreactivity. The proportion of ED1 positive area was calculated by sampling the total spared spinal cord area in the crosssection, as described previously (Popovich et al., 1997). Statistical analysis All statistical analyses were performed using SPSS 10.0 for Windows. Differences among the treatment groups were tested using either a Student's t-test or a one-way analysis of variance (ANOVA) with a least significant difference (LSD) multiple comparisons test when warranted. Differences with a p-value less than 0.05 were considered statistically significant. Experimental paradigms The following series of experiments and behavioral/anatomic outcome measurements was conducted. These experiments were carried out serially, with the results of one dictating the conduct of the next. Experiment 1. Comparison of atorvastatin and simvastatin dissolved in saline, administered via oral-gastric gavage
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were loaded into a syringe, and the syringe held by the mouth of the animal. The plunger was depressed slowly as the animal began to lap up the drug/Ensure mixture, allowing it to voluntarily swallow the drug. Animals received the treatment once a day, for 1 week. Experiment 4. Evaluation of purified simvastatin administered subcutaneously To overcome the issue of variability in ingestion and intestinal absorption, we obtained a purified form of simvastatin (via an MTA with Merck) for subcutaneous injection, a mode of administration that had been described by other numerous authors to be an effective method of drug delivery in rat models of stroke and traumatic brain injury (Balduini et al., 2001; Balduini et al., 2003; Endres et al., 1998; Lu et al., 2007; McGirt et al., 2002; Sironi et al., 2003; Wang et al., 2007). The drug was activated by alkaline hydrolysis prior to injection, as described previously (Endres et al., 1998; Kugi et al., 2002). One hour after thoracic contusion injury, animals were randomized to receive a subcutaneous injection of either simvastatin at 20 mg/kg/day (n = 16), or saline (n = 17), once per day. After 3 days, 5 animals from each group were sacrificed for evaluation of inflammatory cell invasion into the cord. Thereafter, in the remaining animals, the simvastatin dose was reduced to 5 mg/kg/day. In a previous pilot study, we observed weight loss and general lack of activity when the 20 mg/kg/day dose was administered systemically beyond 5 days (unpublished data). The lower 5 mg/kg/ day dose was delivered subcutaneously each day up to post-injury day 45 when the animals were sacrificed. Results
Oral tablets of atorvastatin (Lipitor®, Pfizer, New York, NY) and simvastatin (Zocor®, Merck, Whitehouse Station, NJ) were crushed and dissolved in saline. One hour after thoracic contusion injury, animals were randomized to receive either atorvastatin at 5 mg/kg (n = 4), simvastatin at 20 mg/kg (n = 5), or saline (n = 9) via oralgastric gavage using a 16-gauge feeding needle (Fine Science Tools, Cat#18061-10, North Vancouver, Canada). Animals received the treatment once a day, for 1 week, and were evaluated for behavioral recovery over 6 weeks. Histologic analysis of the spinal cord injury site was then conducted. Experiment 2. Comparison of atorvastatin and simvastatin dissolved in saline, administered via oral-gastric gavage (replication) The oral-gastric gavage technique that we utilized initially in Experiment 1 led to significant animal morbidity and mortality (hence the small numbers). It was therefore repeated here with a new gavage technique utilizing a different method for holding the animals. While in Experiment 1 the animals were wrapped in a cloth, in Experiment 2 we scruffed them by the hair on the back of their neck so their neck movements were more limited and they were more compliant. One hour after thoracic contusion injury, animals were randomized to receive either atorvastatin at 5 mg/kg (n = 8), simvastatin at 20 mg/kg (n = 8), or saline (n = 8) via oral-gastric gavage. Animals received the treatment once a day for 1 week and were evaluated for behavioral recovery over 6 weeks. Experiment 3. Comparison of atorvastatin and simvastatin dissolved in Ensure®, administered orally To obviate the need to gavage the animals at all, we explored the route of mixing the drugs in Ensure® (Abbott Nutrition, Columbus, Ohio) and feeding it orally to the animals. Oral tablets of atorvastatin and simvastatin were crushed and suspended in chocolate-flavored Ensure®. One hour after thoracic contusion injury, animals were randomized to receive either atorvastatin at 5 mg/kg (n = 8), simvastatin at 20 mg/kg (n = 8), or Ensure® (n = 8). All suspensions
Experiment 1: Comparison of atorvastatin and simvastatin dissolved in saline, administered via oral-gastric gavage Our first experiment with these drugs was complicated by difficulties with the oral-gastric gavage technique, such that nearly half of the animals died of respiratory complications within the first 5–7 days. We believe that this was related to the method by which we were holding the animals, which allowed them to move their necks while the gavage needle was inserted and thus resulting in upper airway and pharyngeal trauma. The animals also appeared to object to the taste of the atorvastatin and simvastatin and thus struggled a great deal more during the gavaging of these drugs than the animals gavaged with saline; hence the greater extent of local trauma (and subsequent death) in the atorvastatin- and simvastatin-treated groups. The final numbers of drug-treated animals was low (4 for atorvastatin, 5 for simvastatin) and was 9 for saline-treated animals. Open-field locomotor testing revealed a significant improvement in the simvastatin-treated animals as compared to the saline controls —an improvement that was apparent from day 28 to day 45 (Fig. 1). Simvastatin-treated animals reached an average score of 13.2 ± 1.02, which was significantly more than the controls' average score of 11.3 ± 0.23 (Fig. 1A). These results were mirrored in the BBB subscore analysis (Fig. 1B), with simvastatin-treated animals reaching an average score of 9.2 ± 1.07 and saline-treated animals remained at 4.44 ± 1.39. Despite this improvement, the percentage of hindlimb errors observed while crossing the horizontal ladder did not differ between simvastatin, atorvastatin, and saline controls (Fig. 2). No significant differences among all groups were seen in the paw withdrawal thresholds on sensory testing (data not shown). All histologic analyses were conducted at 6 weeks post-injury. Eriochrome C staining of serial cross-sections of the injury site (Fig. 3) revealed significantly greater white matter sparing in simvastatintreated animals (but not atorvastatin-treated) as compared to saline controls. The average white matter area at the injury epicenter in simvastatin, atorvastatin, and saline-treated groups was 0.78 ± 0. 04 mm2, 0.50 ± 0. 18 mm2, and 0.41 ± 0. 04 mm2, respectively (Fig. 3B).
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Fig. 1. In Experiment 1, open-field locomotor testing in control, simvastatin-, and atorvastatin-treated animals revealed significant improvements in both BBB (A) scores and (B) subscores in simvastatin-treated animals.
Summing the spared white matter through the central 400 μm of the injury epicenter injury revealed a significantly greater cumulatively white matter sparing in the simvastatin animals (2.40 ± 0. 11 mm2, 1.63 ± 0. 49 mm2, and 1.61 ± 0. 189 mm2 for simvastatin, atorvastatin, and saline groups, respectively; Fig. 3C). Identical analyses of gray matter sparing at 6 weeks post-injury revealed no significant sparing differences between treatment groups (Fig. 3D and E).
Experiment 2. Comparison of atorvastatin and simvastatin dissolved in saline, administered via oral-gastric gavage (replication) Obviously, the small numbers of animals in Experiment 1 made it impossible to make meaningful conclusions about the efficacy of atorvastatin and simvastatin, but we did believe that the simvastatin findings might be “real” and should be investigated further. Using an improved gavage technique, we replicated Experiment 1 with a larger series of animals (n = 8 per group). The significant animal morbidity/ mortality that impaired Experiment 1 was not observed. Hindlimb recovery was assessed with the BBB open-field locomotor score. Unlike in Experiment 1, however, there was no improvement in the simvastatin-treated animals. At 6 weeks post-injury, atorvastatin, simvastatin, and saline control-treated animals reached average BBB scores of 12.0 ± 0.33, 12.63 ± 1.08, and 12.0 ± 0.27, respectively (p = 0.398; Fig. 4A). These results were mirrored in the BBB subscore analysis, where, at 6 weeks post-injury, average scores were 8.375 ± 1.18, 7.375 ± 1.47, and 7.75 ± 1.29 (p = 0.558; Fig. 4B). No further histologic analysis was performed, given the lack of any locomotor improvements. Experiment 3. Comparison of atorvastatin and simvastatin mixed in Ensure®, administered orally
Fig. 2. In Experiment 1, the percentage of hindlimb errors 6 weeks post-injury while crossing a horizontal ladder did not differ between control, atorvastatin-, and simvastatin-treated animals.
While the new oral gavage technique was a vast improvement in Experiment 2 and animal morbidity/mortality was not observed, we felt that it was rational to attempt this comparative evaluation of atorvastatin and simvastatin without any gavage manipulation of the
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Fig. 3. In Experiment 1, Eriochrome cyanine staining of serial cross-sections at the injury site 6 weeks post-injury revealed increased white matter sparing in simvastatin-treated animals. (A) Representative cross-sections through the injury site. (B) Spared white matter from 3. 2 mm rostral to the injury epicenter through 3. 2 mm caudal to the injury epicenter. (C) Spared white matter summed through the central 400 μm of the injury epicenter. (D) Spared gray matter from 3. 2 mm rostral to the injury epicenter through 3. 2 mm caudal to the injury epicenter. (E) Spared gray matter summed through the central 400 μm of the injury epicenter.
animals; hence, the strategy of mixing the pulverized statin drugs in chocolate-flavored Ensure®. We then replicated Experiments 1 and 2, with 8 animals per group (saline, atorvastatin, and simvastatin). Again, we saw no improvements in either BBB score or BBB subscore in the atorvastatin- and simvastatin-treated animals as compared to saline controls. At 42 days post-injury, atorvastatin, simvastatin, and control-treated animals reached average BBB scores of 11.5 ± 0.18, 11.38 ± 0.17, and 11.75 ± 0.29, respectively (Fig. 5A). These results were mirrored in the BBB subscore analysis (Fig. 5B), where, on the
last day of testing, average scores were 6.5 ± 1.24, 6.25 ± 1.01, and 5.375 ± 1.4, p = 0.947. No further histologic analysis was performed, given the lack of any locomotor improvements. Experiment 4. Evaluation of purified simvastatin administered subcutaneously We were disappointed with the lack of any behavioral improvements in both the atorvastatin- and simvastatin-treated groups in
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Fig. 4. In Experiment 2, open-field locomotor testing revealed no improvements in BBB (A) scores or (B) subscores in either atorvastatin- or simvastatin-treated animals. As in Experiment 1, the drugs were administered via oral-gastric gavage.
Experiments 2 and 3, given the suggestion that simvastatin was beneficial in Experiment 1. We considered the possibility that the oral administration route was not sufficient (oral being a poor choice for a neuroprotective agent in any event) and chose to pursue a systemic approach. Others had published on the subcutaneous administration for simvastatin (Balduini et al., 2001; Balduini et al., 2003; Endres et al., 1998; Lu et al., 2007; McGirt et al., 2002; Sironi et al., 2003; Wang et al., 2007). Given the observations from Experiment 1, we obtained a purified version of simvastatin from Merck for subcutaneous injection (via an MTA) and tested this against saline, using the same initial dosage ( 20 mg/kg) that others had used with success. We were unable to obtain a similar version of purified atorvastatin from Pfizer.
Additionally, given that Pannu et al. had administered atorvastatin for the entire length of their experiment (Pannu et al., 2005, 2007) and Holmberg et al. had suggested a role for simvastatin in inducing sprouting (Holmberg et al., 2006, 2008) we also administered simvastatin over 42 days, hoping that this prolonged drug exposure might promote a neuroregenerative response over and above an acute neuroprotective function. As in Experiments 2 and 3, however, we observed no behavioral improvements in simvastatin-treated animals on either the BBB score or BBB subscore. At 42 days post injury, simvastatin and control groups had average BBB scores of 12.32 ± 0.79 and 12.04 ± 0.53, respectively (Fig. 6A). At no time point during the post-operative
Fig. 5. In Experiment 3, open-field locomotor revealed no improvements in BBB (A) scores or (B) subscores in either atorvastatin- or simvastatin-treated animals. Unlike Experiments 1 and 2, the drugs were mixed in Ensure® and fed orally to the animals.
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period was there any statistically significant difference between the groups (p N 0.05). Similarly, the BBB subscores revealed no significant differences between the groups (Fig. 6B). For gait testing on the horizontal ladder, all animals demonstrated increased numbers of stepping errors post-injury, but there were no significant differences between groups at 42 days post-injury (p N 0.05; Fig. 7. After injury, the percentage of hindlimb stepping errors was 25.6 ± 3.3 and 29.9 ± 5.9 for simvastatin and control groups, respectively. Between Experiments 3 and 4, we obtained a CatWalk system and utilized it here. The three parameters chosen from the CatWalk analysis system also showed no significant neurological improvements as a result of treatment (p N 0.05; Fig. 8). The regularity index, a relative measure of coordination, decreased after injury and, at 42 days post-injury, was 87.65 ± 2.01% and 92.01 ± 0.96% for control and simvastatin groups, respectively (Fig. 8A). The hindpaw base of support, which indicates gait stability, increased after injury and, at 42 days post injury, was 33.53 ± 1. 13 mm and 30.42 ± 1. 79 mm (Fig. 8B). Lastly, the hindpaw intensity, which estimates how much pressure or weight is supported on the hindpaws, decreased after injury and, at 42 days post injury, was 58.01 ± 5.86 and 58.88 ± 3.70 for control and simvastatin groups, respectively (Fig. 8C). In accordance with our behavioral results, histologic analysis revealed that simvastatin did not lead to more spared white or gray matter as compared to control treatment, either at the lesion epicenter or as the sum of sections through the injury epicenter (“cumulative” white matter sparing; Fig. 9). The extent of ED1 staining at 42 days post-
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Fig. 7. In Experiment 4, the percentage of hindlimb errors while crossing a horizontal ladder was not improved in simvastatin-treated animals.
injury also did not differ between simvastatin- and saline-treated animals (0.25 ± 0.017 and 0.24 ± 0.012, respectively, Fig. 10). Interestingly, however, in a pilot study of 10 animals euthanized at 3 days postinjury, there was a significant reduction in the ED1 staining in the simvastatin-treated animals, with average proportions of 0.0417 ± 0.006 and 0.023 ± 0.004 for control and simvastatin groups, respectively (data not shown). Clearly, this effect was not borne out to the 42-day post-injury time point.
Fig. 6. In Experiment 4, open-field locomotor revealed no improvements in BBB (A) scores or (B) subscores in simvastatin-treated animals (3 days of 20 mg/kg then 39 days of 5 mg/kg, via subcutaneous injection).
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Fig. 8. CatWalk analysis in control and simvastatin-treated animals. (A) Results of the regularity index, a relative measure of coordination. (B) Results of the hindpaw base of support, which indicates gait stability. (C) Results of the hindpaw intensity, which estimates how much pressure or weight is supported on the hindpaws. There were no improvements in the simvastatin-treated animals.
Discussion This paper describes a series of experiments that were performed to evaluate the comparative neuroprotective effects of simvastatin and atorvastatin. We observed what we thought was a promising “signal” of improved tissue sparing and locomotor recovery with simvastatin in our first experiment and were obviously compelled to replicate this with a larger series. Two subsequent attempts with orally administered simvastatin failed to reveal any behavioral benefit on open-field locomotor testing, and a final attempt with a subcutaneously administered dose of purified simvastatin given everyday for 42 days also failed to show any behavioral or histologic improvements. We would conclude that, in a thoracic contusion injury model (using the OSU Impactor) with a 1-h delay in intervention, simvastatin did not confer robust locomotor improvement as measured with the BBB scale or horizontal ladder. In our first three experiments, we did not observe any behavioral or histologic improvements with atorvastatin either. Our interest in studying statins for spinal cord injury was initially stimulated by the results of Pannu et al., who reported in 2005 of dramatic histologic and behavioral improvements in a rodent thoracic SCI model with atorvastatin at the same dose ( 5 mg/kg) for 1 week pre-injury followed by daily oral-gastric gavage administration for 42 days post-injury (Pannu et al., 2005). During the course of our series of experiments, the same authors reported even more impressive
histologic and behavioral improvements with atorvastatin begun at 2-, 4-, or even 6-h post-injury (Pannu et al., 2007). While we also utilized a 5 mg/kg dose of atorvastatin, there are important distinctions between our experimental designs that should be taken into consideration as potential reasons why we would not observe the same efficacy as Pannu et al. Our injury model differed from that of Pannu et al. in that they utilized a weight drop device while we utilized an electromechanical impactor (Ohio State University Impactor), and our injury site (T10) was more rostral than theirs (T12). The biomechanical severity of our contusion model is likely to be much greater than that of Pannu et al., judging by the published figures of the injury epicenter in their “control” animals (Pannu et al., 2005). While this may be a rationale explanation for the differences in experimental outcome, we recognize that human injuries occur with great biomechanical variety (and severity), and so treatments will be needed to be efficacious in similarly severe injuries. Additionally, we began our oral administration of atorvastatin 1-h post-injury, but maintained it for only 1 week post-injury and not 6 weeks post-injury. This may be an important difference, given the potential effect of statins in promoting axonal sprouting via the rho pathway (Pannu et al., 2007). Nonetheless, during these 7 days of atorvastatin treatment in our injury model, there was no improvement in BBB scores that would suggest that it was having an early neuroprotective effect. The application of simvastatin in an animal model of spinal cord injury has more recently been reported for the first time by Holmberg et al. (2008). Their initial in vitro work demonstrated that simvastatin promoted neurite outgrowth in the presence of growth-inhibitory molecules such as myelin associated glycoprotein, Nogo, and oligodendrocyte myelin glycoprotein (Holmberg et al., 2006). They went on to demonstrate that after a T9 thoracic contusion SCI, simvastatin reduced CSPG immunoreactivity in a dose-dependent fashion, particularly away from the injury site (Holmberg et al., 2008). However, similar to our observations, simvastatin by itself did not increase the extent of spared tissue, and administration either orally or intrathecally did not improve locomotor recovery as measured with BBB scores. The authors also point out the discrepancy between these negative behavioral results and those of Pannu et al. who reported near-normal BBB scores after atorvastatin treatment. Important distinctions such as the level of injury (T8/9 versus T12) and severity of injury were pointed out by Holmberg et al. Our experimental paradigm (T10 level) with a contusive injury that destroys over 90% of the cross-sectional area of the cord at the injury epicenter is seemingly more similar to that of Holmberg et al. (2008). In such in vivo pharmacologic neuroprotection studies, dose and mode of administration undoubtedly play critical roles. At the time that we initiated these studies, there was evidence that 20 mg/kg of simvastatin was neuroprotective in an animal model of cerebral ischemia (Cimino et al., 2005). Other authors have also utilized this simvastatin dose ( 20 mg/kg) and mode of administration (subcutaneous injection) with beneficial effects (Balduini et al., 2001, 2003; Endres et al., 1998; Lu et al., 2007; McGirt et al., 2002; Sironi et al., 2003; Wang et al., 2007). While these doses significantly exceeded the tolerable human dose of simvastatin, given the paucity of in vivo neuroprotection studies utilizing simvastatin, we felt that 20 mg/kg would be a justifiable initial dose to start our experiments with, particularly given that others had reported beneficial effects using the same dose. The in vivo work of Holmberg et al. utilized more clinically applicable oral simvastatin doses in their rodent SCI model (0. 57 mg/kg/day or 2. 3 mg/kg/day), and while they noted significant reductions in CSPG staining within the injured spinal cord, no behavioral recovery was noted. It is, of course, difficult to know exactly what the optimal drug concentration is within the injured spinal cord parenchyma and how to best get it there. We found in a pilot study that prolonging the subcutaneous injections of 20 mg/kg simvastatin beyond 3 days was quite toxic to the animals, and hence, in Experiment 4, we only maintained this 20 mg/kg dose for 3 days, before reducing it down to 5
C.M. Mann et al. / Experimental Neurology 221 (2010) 285–295 Fig. 9. In Experiment 4, Eriochrome C staining of serial cross-sections of the injury site revealed no significant improvements in either (A, B) white matter or (C, D) gray matter sparing in simvastatin-treated animals. The extent of spared white and gray matters was summed through the central 400 μm around the injury epicenter.
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Fig. 10. In Experiment 4, the 3-day treatment with 20 mg/kg simvastatin significantly reduced ED1 immunostaining at the injury epicenter. Prolonged treatment at this dose was toxic to the animals, necessitating a reduction to 5 mg/kg/day after the initial 3 days.
mg/kg/day. The simvastatin toxicity that we observed is quite contradictory to studies that also utilized a daily 20 mg/kg subcutaneous dosing regimen for at least a week, without noting any problems (Balduini et al., 2001, 2003; Endres et al., 1998; Lu et al., 2007; McGirt et al., 2002; Sironi et al., 2003; Wang et al., 2007). Our experiments were largely proof-of-concept studies, in which we hypothesized that there would be a neuroprotective effect with atorvastatin, but that it would be greater with simvastatin. Our interest in simvastatin was based on a number of factors, including the fact that the relative potency of HMG-CoA reductase inhibition is greater with simvastatin (Zacco et al., 2003), simvastatin was found to be more neuroprotective than atorvastatin in an in vitro assay of excitotoxicity (Zacco et al., 2003), and with its greater lipophilicity we believed that simvastatin might make it a better agent in the context of acute neurotrauma. After our initial histologic analysis of the injury site in Experiment 1, we did not pursue histologic assessments in Experiment 2 or 3, given that the behavioral outcomes were essentially identical among the saline, atorvastatin, and simvastatin groups. This reflects our own bias toward the importance of functional recovery above all other metrics, and in the absence of any behavioral improvements we did not feel that extensive histologic assessments of the cord would substantially influence our perspective on the efficacy of simvastatin (or lack thereof). In our recently conducted survey, we asked members of the SCI research community whether they were of the opinion that improvements in histologic, biochemical, or physiologic outcome measures without any behavioral improvements constituted “clinically meaningful efficacy” (Kwon et al., 2009). Of the 324 respondents, 59% disagreed that this represented clinically meaningful efficacy, while only 28% were of the opinion that it did. In fact, 31% “strongly disagreed” with improvements in non-behavioral outcomes in the absence of behavioral effects being considered a demonstration of “clinically meaningful efficacy”, while only 5% “strongly agreed” with this statement. While we would certainly not argue against the importance of mechanistic insights garnered from more extensive histological, biochemical, or physiological studies, from a translational perspective in deciding upon the “promising clinical potential” of an experiment treatment, we share the SCI community's emphasis on behavioral recovery and focused on this throughout the studies described in this paper. In Experiment 4, we did change the therapeutic regimen to include a prolonged, 42-day dosing schedule. Again, with the absence of any behavioral improvements on BBB score, BBB subscore, CatWalk, and horizontal ladder, the fact that ED1 immunostaining was reduced in our pilot study of simvastatin-treated animals at the 72-h post-injury mark is mechanistically interesting but of modest importance. This
significant reduction in ED1 immunostaining within the injury site with daily 20 mg/kg subcutaneous simvastatin injections at least indicated to us that the intervention was having some biological effect on the spinal cord, although this in itself was not particularly surprising, given the numerous studies that other authors have published with the use of this simvastatin dose ( 20 mg/kg) and mode of administration (subcutaneous injection; Balduini et al., 2001; Balduini et al., 2003; Endres et al., 1998; Lu et al., 2007; McGirt et al., 2002; Sironi et al., 2003; Wang et al., 2007). It is, of course, of interest to us that these other investigators observed substantive beneficial effects with this same dose and administration mode of simvastatin, albeit in different neurologic settings. The immunomodulatory effects of statins are now well recognized, and so it is certainly conceivable that macrophage/microglial activation may be somewhat attenuated. We did not undertake a semi-quantitative analysis of CSPG deposition at and around the spinal cord injury site as Holmberg et al. (2008) performed but would agree with their contention that while simvastatin may influence the properties of cells within the injury mileu (and possibly make for a more permissive environment), the drug by itself is not sufficient to promote functional recovery. Ultimately, these series of experiments were instructive to us, in that we admittedly were quite enthusiastic about the positive results from our first experiment where simvastatin appeared quite effective in both behavioral and histologic outcomes. The animal mortality in the first experiment was unacceptably high, and obviously, with only 4–5 animals, we were unable to make strong conclusions. It seemed, nonetheless, that there was some therapeutic potential there, and that we could more validly demonstrate that with a repetition of the experiment, albeit with better gavage technique. Having conducted 3 additional studies—all of which were resoundly negative—we would conclude that our initial positive findings with simvastatin represented an “alpha” error, related to the small numbers. In our recent survey of the SCI community, questions were included to garner the perspectives of our field on the issue of bias and how this might influence the interpretation of preclinical studies. A large proportion of respondents opined that scientists tend to repeat negative experiments in the attempt to generate positive findings (Kwon et al., 2009). Indeed, this paper illustrates our own repeated (and failed) attempts to demonstrate a positive neuroprotective response with atorvastatin and simvastatin, with a particular interest in confirming a fleeting positive response observed with the latter. Evidently, we too are not immune to this “bias” either. For us it is an interesting reflection on the difficulties in replicating positive experimental results, which is an issue that has become quite evident within the SCI community. The NIH has sponsored formal replication studies of a variety of therapeutic agents, including erythropoietin, minocycline, and NEP1-40 (Pinzon et al., 2008b; Pinzon et al., 2008a; Steward et al., 2008). To date, none of these formal replication attempts have successfully reproduced the efficacy reported in the original study. While differences in experimental design such as animal species, weight, age, gender, anesthetic agent, injury model, injury severity, and outcome assessors may be blamed for the lack of reproducibility in such studies, it is hard to imagine how a therapy could be successful at conferring improved neurologic function in the vastly variable setting of human SCI if such experimental differences are truly to blame for the lack of reproducibility in preclinical studies. In summary, in our hands, using a T10 OSU contusion SCI in Sprague–Dawley rats, we could not demonstrate a beneficial neurologic effect to either atorvastatin or simvastatin. The atorvastatin dose and mode of administration utilized was the same as that of Pannu et al. (2007), and although we did not extend the treatment for the entire post-injury period, we did not see any of the spectacular recovery that they reported. Recently, a study by Dery et al. reports a positive response to 5 mg/kg atorvastatin administered intraperitoneally after a T10 weight drop contusion SCI (Dery et al., 2009), so this does stand as some level of independent confirmation of Pannu et
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al.'s results. Further atorvastatin testing should arguably explore its efficacy in a cervical injury model and with doses more comparable to tolerable human doses. As for simvastatin, although we utilized a dose ( 20 mg/kg) and administration method (subcutaneous injection) also used successfully by others, we were unable to demonstrate a neuroprotective response in our SCI model, despite repeated efforts. Further dosing studies and investigation in a cervical injury model may be warranted, although we submit these negative findings to the research community so as to prevent others from attempting the exact same experimental approach that we have expended great time and effort on. Acknowledgments BKK holds a New Investigator Award from the Canadian Institutes for Health Research and WT is the Rick Hansen Man in Motion Chair of Spinal Cord Injury Research, UBC. This research was supported by the Wings for Life Spinal Cord Injury Foundation and Scoliosis Research Society. References Balduini, W., De V, A., Mazzoni, E., Cimino, M., 2001. Simvastatin protects against longlasting behavioral and morphological consequences of neonatal hypoxic/ischemic brain injury. Stroke 32, 2185–2191. Balduini, W., Mazzoni, E., Carloni, S., De Simoni, M.G., Perego, C., Sironi, L., Cimino, M., 2003. Prophylactic but not delayed administration of simvastatin protects against long-lasting cognitive and morphological consequences of neonatal hypoxicischemic brain injury, reduces interleukin-1beta and tumor necrosis factor-alpha mRNA induction, and does not affect endothelial nitric oxide synthase expression. Stroke 34, 2007–2012. Bartesaghi, S., Marinovich, M., Corsini, E., Galli, C.L., Viviani, B, 2005. Erythropoietin: a novel neuroprotective cytokine. Neurotoxicology 26 (5), 923–928. Basso, D.M., 2004. Behavioral testing after spinal cord injury: congruities, complexities, and controversies. J. Neurotrauma 21, 395–404. Basso, D.M., Beattie, M.S., Bresnahan, J.C., 1995. A sensitive and reliable locomotor rating scale for open field testing in rats. J. Neurotrauma 12, 1–21. Cimino, M., Gelosa, P., Gianella, A., Nobili, E., Tremoli, E., Sironi, L., 2007. Statins: multiple mechanisms of action in the ischemic brain. Neuroscientist 13 (3), 208–213 (Review). Cimino, M., Balduini, W., Carloni, S., Gelosa, P., Guerrini, U., Tremoli, E., Sironi, L, 2005. Neuroprotective effect of simvastatin in stroke: a comparison between adult and neonatal rat models of cerebral ischemia. Neurotoxicology 26 (5), 929–933. Dery, M.A., Rousseau, G., Benderdour, M., Beaumont, E., 2009. Atorvastatin prevents early apoptosis after thoracic spinal cord contusion injury and promotes locomotion recovery. Neurosci. Lett. 453, 73–76. Domercq, M., Matute, C., 2004. Neuroprotection by tetracyclines. Trends Pharmacol. Sci. 25, 609–612. Endres, M., Laufs, U., Huang, Z., Nakamura, T., Huang, P., Moskowitz, M.A., Liao, J.K., 1998. Stroke protection by 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors mediated by endothelial nitric oxide synthase. Proc. Natl. Acad. Sci. U. S. A. 95, 8880–8885. Gorio, A., Gokmen, N., Erbayraktar, S., Yilmaz, O., Madaschi, L., Cichetti, C., Di Giulio, A.M., Vardar, E., Cerami, A., Brines, M., 2002. Recombinant human erythropoietin counteracts secondary injury and markedly enhances neurological recovery from experimental spinal cord trauma. Proc. Natl. Acad. Sci. U.S.A. 99, 9450–9455. Hamers, F.P., Koopmans, G.C., Joosten, E.A., 2006. CatWalk-assisted gait analysis in the assessment of spinal cord injury. J. Neurotrauma 23, 537–548. Holmberg, E., Nordstrom, T., Gross, M., Kluge, B., Zhang, S.X., Doolen, S., 2006. Simvastatin promotes neurite outgrowth in the presence of inhibitory molecules found in central nervous system injury. J. Neurotrauma 23, 1366–1378. Holmberg, E., Zhang, S.X., Sarmiere, P.D., Kluge, B.R., White, J.T., Doolen, S., 2008. Statins decrease chondroitin sulfate proteoglycan expression and acute astrocyte activation in central nervous system injury. Exp. Neurol. Hurlbert, R.J., Hamilton, M.G., 2008. Methylprednisolone for acute spinal cord injury: 5-year practice reversal. Can. J. Neurol. Sci. 35, 41–45. Igel, M., Sudhop, T., von, B.K., 2001. Metabolism and drug interactions of 3-hydroxy-3methylglutaryl coenzyme A-reductase inhibitors (statins). Eur. J. Clin. Pharmacol. 57, 357–364.
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Lu, D., Qu, C., Goussev, A., Jiang, H., Lu, C., Schallert, T., Mahmood, A., Chen, J., Li, Y., Chopp, M., 2007. Statins increase neurogenesis in the dentate gyrus, reduce delayed neuronal death in the hippocampal CA3 region, and improve spatial learning in rat after traumatic brain injury. J. Neurotrauma 24, 1132–1146. McGirt, M.J., Lynch, J.R., Parra, A., Sheng, H., Pearlstein, R.D., Laskowitz, D.T., Pelligrino, D.A., Warner, D.S., 2002. Simvastatin increases endothelial nitric oxide synthase and ameliorates cerebral vasospasm resulting from subarachnoid hemorrhage. Stroke 33, 2950–2956. Pannu, R., Barbosa, E., Singh, A.K., Singh, I., 2005. Attenuation of acute inflammatory response by atorvastatin after spinal cord injury in rats. J. Neurosci. Res. 79, 340–350. Pannu, R., Christie, D.K., Barbosa, E., Singh, I., Singh, A.K., 2007. Post-trauma Lipitor treatment prevents endothelial dysfunction, facilitates neuroprotection, and promotes locomotor recovery following spinal cord injury. 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Romano, M., Diomede, L., Sironi, M., Massimiliano, L., Sottocorno, M., Polentarutti, N., Guglielmotti, A., Albani, D., Bruno, A., Fruscella, P., Salmona, M., Vecchi, A., Pinza, M., Mantovani, A., 2000. Inhibition of monocyte chemotactic protein-1 synthesis by statins. Lab. Invest. 80, 1095–1100. Schachter, M., 2005. Chemical, pharmacokinetic and pharmacodynamic properties of statins: an update. Fundam. Clin. Pharmacol. 19, 117–125. Schwartz, G., Fehlings, M.G., 2001. Evaluation of the neuroprotective effects of sodium channel blockers after spinal cord injury: improved behavioral and neuroanatomical recovery with riluzole. J. Neurosurg. 94, 245–256. Sironi, L., Cimino, M., Guerrini, U., Calvio, A.M., Lodetti, B., Asdente, M., Balduini, W., Paoletti, R., Tremoli, E., 2003. Treatment with statins after induction of focal ischemia in rats reduces the extent of brain damage. Arterioscler. Thromb. Vasc. Biol. 23, 322–327. Steward, O., Sharp, K., Yee, K.M., Hofstadter, M., 2008. 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Contents lists available at ScienceDirect
Experimental Neurology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x n r
Lack of robust neurologic benefits with simvastatin or atorvastatin treatment after acute thoracic spinal cord contusion injury Cody M. Mann a, Jae H.T. Lee a, Jessica Hillyer a, Anthea M.T. Stammers a, Wolfram Tetzlaff a,c, Brian K. Kwon a,b,⁎ a b c
International Collaboration on Repair Discoveries (ICORD), University of British Columbia, Vancouver, British Columbia, Canada Combined Neurosurgical and Orthopaedic Spine Program (CNOSP), Department of Orthopaedics, University of British Columbia, Vancouver, British Columbia, Canada Department of Zoology, University of British Columbia, Vancouver, British Columbia, Canada
a r t i c l e
i n f o
Article history: Received 20 September 2009 Revised 28 October 2009 Accepted 6 November 2009 Available online 18 November 2009 Keywords: Simvastatin Atorvastatin Neuroprotection Spinal cord injury
a b s t r a c t Although much progress has been made in the clinical care of patients with acute spinal cord injuries, there are no reliably effective treatments, which minimize secondary damage and improve neurologic outcome. The time and expense needed to establish de novo pharmacologic or biologic therapies for acute SCI has encouraged the development of neuroprotective treatments based on drugs that are already in clinical use and, therefore, have the advantage of a well-characterized safety and pharmacokinetic profile in humans. Statins are the most commonly prescribed class of lipid-lowering drugs, and recently, it has been recognized that statins also have powerful immunomodulatory and anti-inflammatory effects. This paper describes a series of experiments that were performed to evaluate the comparative neuroprotective effects of simvastatin and atorvastatin. We observed a promising signal of neurologic benefit with simvastatin in our first experiment, but in repeated attempts to replicate that effect in three subsequent experiments, we failed to reveal any behavioral or histologic improvements. We would conclude that simvastatin given orally or subcutaneously at doses previously reported by other investigators to be effective in different neurologic conditions does not confer a significant neurologic benefit in a thoracic contusion injury model (OSU Impactor) when administered with a 1-h delay in intervention. We contend that further preclinical investigation of atorvastatin and simvastatin is warranted before considering their translation into human SCI. © 2009 Elsevier Inc. All rights reserved.
Introduction Although much progress has been made in the clinical care of patients with acute spinal cord injuries, convincingly effective neuroprotective treatments to minimize secondary damage and ultimately improve neurologic outcome remain elusive. To date, intravenous methylprednisolone is the only neuroprotective drug to have achieved widespread adoption, although the efficacy and safety of this practice has been intensely questioned, leading many to abandon it (Hurlbert and Hamilton, 2008). Clearly there is a compelling need for better pharmacologic treatment options for this injury. The time and expense needed to establish de novo pharmacologic or biologic therapies for acute SCI has encouraged the development of neuroprotective treatments based on drugs that are already in clinical use, often for unrelated applications. Such drugs have the advantage ⁎ Corresponding author. D6 Heather Pavilion, Vancouver General Hospital, Department of Orthopaedics, University of British Columbia, 2733 Heather Street, Vancouver, BC, Canada V5Z 3J5. Fax: +1 604 875 5858 E-mail address: [email protected] (B.K. Kwon). 0014-4886/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2009.11.006
of a well-characterized safety and pharmacokinetic profile in humans. Examples of such pharmacologic agents include minocycline—a semisynthetic antibiotic used in the treatment of acne, erythropoietin—a recombinant hormone used in the treatment of anemia, riluzole—a sodium channel blocker used in the treatment of amyotrophic lateral sclerosis, and statins—cholesterol-lowering agents. All of these have demonstrated the ability to attenuate certain aspects of secondary damage procured after CNS injury (Bartesaghi et al., 2005; Domercq and Matute, 2004; Gorio et al., 2002; Kaptanoglu et al., 2004; Pannu et al., 2007; Schwartz and Fehlings, 2001; Stirling et al., 2004; Wells et al., 2003; Pannu et al., 2005). The statins are a group of drugs that inhibit hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase, the enzyme responsible for the conversion of HMG-CoA to mevalonate, the rate-limiting step in de novo cholesterol synthesis (Schachter, 2005). They are the most commonly prescribed class of lipid-lowering drugs and include lovastatin (Mevacor; Merck, Whitehouse Station, NJ), atorvastatin (Lipitor; Pfizer), pravastatin (Pravachol; Bristol-Myers Squibb, New York, NY), and simvastatin (Zocor; Merk). Recently it has been recognized that statins also have powerful immunomodulatory and anti-inflammatory effects (Stuve et al., 2003), a pleiotropic nature
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resulting from the downstream inhibition of isoprenoid intermediates (Liao and Laufs, 2005). Statins possess antioxidant properties and upregulate endothelial nitric oxide synthase (eNOS; Cimino et al., 2007). Furthermore, statins have been shown to inhibit the expression of several pro-inflammatory cytokines that play a role in immune mediated damage after SCI such as monocyte chemotactic protein (MCP)-1, IL-2, IL-12, and interferon gamma (Romano et al., 2000; Youssef et al., 2002). The potential for statins to promote neurologic recovery in acute SCI was first reported by Pannu et al., who revealed that the administration of atorvastatin for 1 week prior to spinal cord injury resulted in a significant reduction of inflammatory cytokines, neuronal cell death at the injury site, and a significant improvement in locomotor scores (Pannu et al., 2005). A subsequent study by these authors reported that post-injury treatment, starting 2, 4, or 6 h after SCI, had similar beneficial neuroprotective effects, with dramatic improvements in hindlimb locomotor function (Pannu et al., 2007). The efficacy of statins in protecting nervous tissue has also been demonstrated after stroke and traumatic brain injury (TBI; Cimino et al., 2005; Wang et al., 2007). The widespread clinical use of statins makes them an appealing choice for translation into human SCI. Recognizing that initiating a clinical trial in human SCI is an enormous endeavor and a huge commitment of time and resources, we undertook this series of preclinical studies in order to better characterize the neuroprotective effect. First, we investigated the relative neurologic efficacy of atorvastatin and simvastatin in a model of thoracic SCI. Because either drug could be utilized in a clinical SCI setting, we felt that it is important to pre-clinically assess whether one appeared more promising for human translation. Atorvastatin and simvastatin differ in their clinical pharmacology, as simvastatin is more lipophilic and therefore more capable of crossing the blood–brain barrier, increasing its potential to target injured CNS tissue (Igel et al., 2001; Igel et al., 2002). The relative efficacy of atorvastatin and simvastatin has been compared in two previous studies of traumatic brain injury. Wang et al. (2007) showed no difference in the neuroprotection afforded by these drugs, but Lu et al. (2007) concluded that simvastatin was superior at improving neurologic recovery. A comparison in acute SCI has not been previously reported. We then conducted a series of experiments to examine different dosing and drug formulations for simvastatin, in an attempt to optimize the neuroprotective properties of the therapy.
Manual bladder expression was performed 3 times per day until reflexive voiding returned. The animals received a subcutaneous injection of buprenorphrine (0. 02 mg/kg, Temgesic®, Reckitt Benkiser Healthcare, UK) just prior to their surgery and then again twice on post-operative days 1 and 2. All animal surgeries and care were conducted in accordance to UBC Animal Care guidelines. Behavioral outcome assessment Open-field locomotor testing was performed using the Basso, Bresnahan, and Beattie (BBB) locomotor score (Basso et al., 1995)and the later-developed subscore, which measures fine details of locomotion (Basso, 2004). Animals were tested pre-injury to establish a baseline and to acclimatize the animals to the testing apparatus, and then on post-injury days 2, 4, 7, 14, 28, and 42 or 45 when applicable. All open-field locomotor testings were performed under the same conditions (including time of day) on each scheduled testing date. At 45 days post-injury, the animals were video-taped walking over a horizontally laid ladder with unevenly and randomly spaced rungs. The number of hindlimb slips between the rungs (“footfalls”) was documented, as a measure of the animals' ability to sense and accommodate for changes in the walking surface. A CatWalk™ gait analysis system (Noldus, Ashville, NC) was acquired during the course of these series of experiments and was utilized in latter studies to specifically measure the following gait parameters: regularity index, average hindpaw base of support, and average hindpaw intensity (Hamers et al., 2006). The animals underwent hindpaw mechanical stimulation threshold testing at 42 days post-injury using a von Frey monofilament device (Semmes-Weinstein monofilament, Stoelting, Wood Dale, IL) to test for the development of below-level mechanical allodynia. The animal was placed on an elevated grid and observed for 5 to 10 min to ensure that it was calm. When both hindpaws were evenly resting upon the rungs of the grid, the monofilament was pressed into the center of the volar surface of the hindpaw. The mechanical force at which hindlimb withdrawal occurred and the time from stimulus application to hindlimb withdrawal were recorded. For all behavioral outcome assessments (hindlimb motor and sensory testing) baseline scores were first established by evaluating the animals prior to injury. All post-injury evaluations were performed by individuals that were blinded to the treatment group. Anatomic outcome assessment
Materials and methods Animal model and surgical procedures Male Sprague–Dawley rats weighing 320– 340 g were anesthetized with an intraperitoneal injection of ketamine hydrochloride ( 72 mg/kg; Bimeda-MTC, Cambridge, ON) and xylazine hydrochloride ( 9 mg/kg; Bayer, Etobicoke, ON) diluted in 20 mM Phosphate Buffered Solution (PBS) and a dorsal midline incision was made over the midthoracic spine to expose the posterior spinal elements at T9–10. A laminectomy was performed, and the bases of the adjacent spinous processes were secured with modified Allis clamps, which then held the animal secure within a custom frame. With half of the animal's weight suspended, the animal was positioned under the Ohio State University (OSU) Impactor, and the impactor tip was gently lowered to apply a pre-load force of 0.2 kdyn onto the dura. The impactor was then triggered to deliver a 1. 5-mm displacement injury at 300 m/s. Animals were immediately excluded if the peak force of the impact fell outside the range of 200– 260 kdyn. The dorsal wound was irrigated and then closed with clips. During the acute post-operative period, the animal temperature was monitored and maintained at 37 °C in an incubator to avoid hypothermia. Five milliliters of Ringer's lactate solution were administered subcutaneously for hydration.
All animals were sacrificed with a lethal injection of Pentobarbital Sodium ( 107 mg/kg, Bimeda-MTC Animal Health, Cambridge, Ontario, Canada) and then perfused with phosphate buffered saline followed by fixation with cooled 4% paraformaldehyde. A 15-mm segment of spinal cord centered around the injury site was harvested, post-fixed overnight in 4% paraformaldehyde, cryoprotected in increasing concentrations of sucrose dissolved in PBS (18 and 24%), and then snap frozen over dry ice. The cords were cut in cross-section at 20-μm thickness for histological analysis of the injury site. For the analysis of spared white and gray matters through the injury site, sections 200 μm apart were stained with Eriochrome Cyanine (EC) as per described by Rabchevsky et al. (2001), counterstained with Neutral Red, then imaged on a Zeiss Axioskop microscope at 5× objective. Regions of spared white and gray matters were manually traced and then quantified using SigmaScan Pro version 5.0.0 (Systat Software). For the quantification of macrophages/microglia, a non-fluorescent DAB staining protocol was utilized, in which endogenous peroxidase activity was quenched with 0.3% hydrogen peroxide in methanol, and the sections incubated overnight with the monoclonal mouse ED1 antibody (1:500, Serotec) after appropriate blocking. After incubation for 1 h at room temperature with a biotin-conjugated secondary
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antibody, the ABC technique (Vectastain® ABC Kit, Vector Laboratories, Burlingame, CA) was applied in exactly the same manner for all slides. Images were captured with a Zeiss microscope (Axioplan 2) and Northern Eclipse software (Empix Imaging). A semi-quantitative analysis of ED1 staining was performed by first thresholding each section to remove non-specific background signal, and then capturing the extent of ED1 immunoreactivity. The proportion of ED1 positive area was calculated by sampling the total spared spinal cord area in the crosssection, as described previously (Popovich et al., 1997). Statistical analysis All statistical analyses were performed using SPSS 10.0 for Windows. Differences among the treatment groups were tested using either a Student's t-test or a one-way analysis of variance (ANOVA) with a least significant difference (LSD) multiple comparisons test when warranted. Differences with a p-value less than 0.05 were considered statistically significant. Experimental paradigms The following series of experiments and behavioral/anatomic outcome measurements was conducted. These experiments were carried out serially, with the results of one dictating the conduct of the next. Experiment 1. Comparison of atorvastatin and simvastatin dissolved in saline, administered via oral-gastric gavage
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were loaded into a syringe, and the syringe held by the mouth of the animal. The plunger was depressed slowly as the animal began to lap up the drug/Ensure mixture, allowing it to voluntarily swallow the drug. Animals received the treatment once a day, for 1 week. Experiment 4. Evaluation of purified simvastatin administered subcutaneously To overcome the issue of variability in ingestion and intestinal absorption, we obtained a purified form of simvastatin (via an MTA with Merck) for subcutaneous injection, a mode of administration that had been described by other numerous authors to be an effective method of drug delivery in rat models of stroke and traumatic brain injury (Balduini et al., 2001; Balduini et al., 2003; Endres et al., 1998; Lu et al., 2007; McGirt et al., 2002; Sironi et al., 2003; Wang et al., 2007). The drug was activated by alkaline hydrolysis prior to injection, as described previously (Endres et al., 1998; Kugi et al., 2002). One hour after thoracic contusion injury, animals were randomized to receive a subcutaneous injection of either simvastatin at 20 mg/kg/day (n = 16), or saline (n = 17), once per day. After 3 days, 5 animals from each group were sacrificed for evaluation of inflammatory cell invasion into the cord. Thereafter, in the remaining animals, the simvastatin dose was reduced to 5 mg/kg/day. In a previous pilot study, we observed weight loss and general lack of activity when the 20 mg/kg/day dose was administered systemically beyond 5 days (unpublished data). The lower 5 mg/kg/ day dose was delivered subcutaneously each day up to post-injury day 45 when the animals were sacrificed. Results
Oral tablets of atorvastatin (Lipitor®, Pfizer, New York, NY) and simvastatin (Zocor®, Merck, Whitehouse Station, NJ) were crushed and dissolved in saline. One hour after thoracic contusion injury, animals were randomized to receive either atorvastatin at 5 mg/kg (n = 4), simvastatin at 20 mg/kg (n = 5), or saline (n = 9) via oralgastric gavage using a 16-gauge feeding needle (Fine Science Tools, Cat#18061-10, North Vancouver, Canada). Animals received the treatment once a day, for 1 week, and were evaluated for behavioral recovery over 6 weeks. Histologic analysis of the spinal cord injury site was then conducted. Experiment 2. Comparison of atorvastatin and simvastatin dissolved in saline, administered via oral-gastric gavage (replication) The oral-gastric gavage technique that we utilized initially in Experiment 1 led to significant animal morbidity and mortality (hence the small numbers). It was therefore repeated here with a new gavage technique utilizing a different method for holding the animals. While in Experiment 1 the animals were wrapped in a cloth, in Experiment 2 we scruffed them by the hair on the back of their neck so their neck movements were more limited and they were more compliant. One hour after thoracic contusion injury, animals were randomized to receive either atorvastatin at 5 mg/kg (n = 8), simvastatin at 20 mg/kg (n = 8), or saline (n = 8) via oral-gastric gavage. Animals received the treatment once a day for 1 week and were evaluated for behavioral recovery over 6 weeks. Experiment 3. Comparison of atorvastatin and simvastatin dissolved in Ensure®, administered orally To obviate the need to gavage the animals at all, we explored the route of mixing the drugs in Ensure® (Abbott Nutrition, Columbus, Ohio) and feeding it orally to the animals. Oral tablets of atorvastatin and simvastatin were crushed and suspended in chocolate-flavored Ensure®. One hour after thoracic contusion injury, animals were randomized to receive either atorvastatin at 5 mg/kg (n = 8), simvastatin at 20 mg/kg (n = 8), or Ensure® (n = 8). All suspensions
Experiment 1: Comparison of atorvastatin and simvastatin dissolved in saline, administered via oral-gastric gavage Our first experiment with these drugs was complicated by difficulties with the oral-gastric gavage technique, such that nearly half of the animals died of respiratory complications within the first 5–7 days. We believe that this was related to the method by which we were holding the animals, which allowed them to move their necks while the gavage needle was inserted and thus resulting in upper airway and pharyngeal trauma. The animals also appeared to object to the taste of the atorvastatin and simvastatin and thus struggled a great deal more during the gavaging of these drugs than the animals gavaged with saline; hence the greater extent of local trauma (and subsequent death) in the atorvastatin- and simvastatin-treated groups. The final numbers of drug-treated animals was low (4 for atorvastatin, 5 for simvastatin) and was 9 for saline-treated animals. Open-field locomotor testing revealed a significant improvement in the simvastatin-treated animals as compared to the saline controls —an improvement that was apparent from day 28 to day 45 (Fig. 1). Simvastatin-treated animals reached an average score of 13.2 ± 1.02, which was significantly more than the controls' average score of 11.3 ± 0.23 (Fig. 1A). These results were mirrored in the BBB subscore analysis (Fig. 1B), with simvastatin-treated animals reaching an average score of 9.2 ± 1.07 and saline-treated animals remained at 4.44 ± 1.39. Despite this improvement, the percentage of hindlimb errors observed while crossing the horizontal ladder did not differ between simvastatin, atorvastatin, and saline controls (Fig. 2). No significant differences among all groups were seen in the paw withdrawal thresholds on sensory testing (data not shown). All histologic analyses were conducted at 6 weeks post-injury. Eriochrome C staining of serial cross-sections of the injury site (Fig. 3) revealed significantly greater white matter sparing in simvastatintreated animals (but not atorvastatin-treated) as compared to saline controls. The average white matter area at the injury epicenter in simvastatin, atorvastatin, and saline-treated groups was 0.78 ± 0. 04 mm2, 0.50 ± 0. 18 mm2, and 0.41 ± 0. 04 mm2, respectively (Fig. 3B).
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Fig. 1. In Experiment 1, open-field locomotor testing in control, simvastatin-, and atorvastatin-treated animals revealed significant improvements in both BBB (A) scores and (B) subscores in simvastatin-treated animals.
Summing the spared white matter through the central 400 μm of the injury epicenter injury revealed a significantly greater cumulatively white matter sparing in the simvastatin animals (2.40 ± 0. 11 mm2, 1.63 ± 0. 49 mm2, and 1.61 ± 0. 189 mm2 for simvastatin, atorvastatin, and saline groups, respectively; Fig. 3C). Identical analyses of gray matter sparing at 6 weeks post-injury revealed no significant sparing differences between treatment groups (Fig. 3D and E).
Experiment 2. Comparison of atorvastatin and simvastatin dissolved in saline, administered via oral-gastric gavage (replication) Obviously, the small numbers of animals in Experiment 1 made it impossible to make meaningful conclusions about the efficacy of atorvastatin and simvastatin, but we did believe that the simvastatin findings might be “real” and should be investigated further. Using an improved gavage technique, we replicated Experiment 1 with a larger series of animals (n = 8 per group). The significant animal morbidity/ mortality that impaired Experiment 1 was not observed. Hindlimb recovery was assessed with the BBB open-field locomotor score. Unlike in Experiment 1, however, there was no improvement in the simvastatin-treated animals. At 6 weeks post-injury, atorvastatin, simvastatin, and saline control-treated animals reached average BBB scores of 12.0 ± 0.33, 12.63 ± 1.08, and 12.0 ± 0.27, respectively (p = 0.398; Fig. 4A). These results were mirrored in the BBB subscore analysis, where, at 6 weeks post-injury, average scores were 8.375 ± 1.18, 7.375 ± 1.47, and 7.75 ± 1.29 (p = 0.558; Fig. 4B). No further histologic analysis was performed, given the lack of any locomotor improvements. Experiment 3. Comparison of atorvastatin and simvastatin mixed in Ensure®, administered orally
Fig. 2. In Experiment 1, the percentage of hindlimb errors 6 weeks post-injury while crossing a horizontal ladder did not differ between control, atorvastatin-, and simvastatin-treated animals.
While the new oral gavage technique was a vast improvement in Experiment 2 and animal morbidity/mortality was not observed, we felt that it was rational to attempt this comparative evaluation of atorvastatin and simvastatin without any gavage manipulation of the
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Fig. 3. In Experiment 1, Eriochrome cyanine staining of serial cross-sections at the injury site 6 weeks post-injury revealed increased white matter sparing in simvastatin-treated animals. (A) Representative cross-sections through the injury site. (B) Spared white matter from 3. 2 mm rostral to the injury epicenter through 3. 2 mm caudal to the injury epicenter. (C) Spared white matter summed through the central 400 μm of the injury epicenter. (D) Spared gray matter from 3. 2 mm rostral to the injury epicenter through 3. 2 mm caudal to the injury epicenter. (E) Spared gray matter summed through the central 400 μm of the injury epicenter.
animals; hence, the strategy of mixing the pulverized statin drugs in chocolate-flavored Ensure®. We then replicated Experiments 1 and 2, with 8 animals per group (saline, atorvastatin, and simvastatin). Again, we saw no improvements in either BBB score or BBB subscore in the atorvastatin- and simvastatin-treated animals as compared to saline controls. At 42 days post-injury, atorvastatin, simvastatin, and control-treated animals reached average BBB scores of 11.5 ± 0.18, 11.38 ± 0.17, and 11.75 ± 0.29, respectively (Fig. 5A). These results were mirrored in the BBB subscore analysis (Fig. 5B), where, on the
last day of testing, average scores were 6.5 ± 1.24, 6.25 ± 1.01, and 5.375 ± 1.4, p = 0.947. No further histologic analysis was performed, given the lack of any locomotor improvements. Experiment 4. Evaluation of purified simvastatin administered subcutaneously We were disappointed with the lack of any behavioral improvements in both the atorvastatin- and simvastatin-treated groups in
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Fig. 4. In Experiment 2, open-field locomotor testing revealed no improvements in BBB (A) scores or (B) subscores in either atorvastatin- or simvastatin-treated animals. As in Experiment 1, the drugs were administered via oral-gastric gavage.
Experiments 2 and 3, given the suggestion that simvastatin was beneficial in Experiment 1. We considered the possibility that the oral administration route was not sufficient (oral being a poor choice for a neuroprotective agent in any event) and chose to pursue a systemic approach. Others had published on the subcutaneous administration for simvastatin (Balduini et al., 2001; Balduini et al., 2003; Endres et al., 1998; Lu et al., 2007; McGirt et al., 2002; Sironi et al., 2003; Wang et al., 2007). Given the observations from Experiment 1, we obtained a purified version of simvastatin from Merck for subcutaneous injection (via an MTA) and tested this against saline, using the same initial dosage ( 20 mg/kg) that others had used with success. We were unable to obtain a similar version of purified atorvastatin from Pfizer.
Additionally, given that Pannu et al. had administered atorvastatin for the entire length of their experiment (Pannu et al., 2005, 2007) and Holmberg et al. had suggested a role for simvastatin in inducing sprouting (Holmberg et al., 2006, 2008) we also administered simvastatin over 42 days, hoping that this prolonged drug exposure might promote a neuroregenerative response over and above an acute neuroprotective function. As in Experiments 2 and 3, however, we observed no behavioral improvements in simvastatin-treated animals on either the BBB score or BBB subscore. At 42 days post injury, simvastatin and control groups had average BBB scores of 12.32 ± 0.79 and 12.04 ± 0.53, respectively (Fig. 6A). At no time point during the post-operative
Fig. 5. In Experiment 3, open-field locomotor revealed no improvements in BBB (A) scores or (B) subscores in either atorvastatin- or simvastatin-treated animals. Unlike Experiments 1 and 2, the drugs were mixed in Ensure® and fed orally to the animals.
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period was there any statistically significant difference between the groups (p N 0.05). Similarly, the BBB subscores revealed no significant differences between the groups (Fig. 6B). For gait testing on the horizontal ladder, all animals demonstrated increased numbers of stepping errors post-injury, but there were no significant differences between groups at 42 days post-injury (p N 0.05; Fig. 7. After injury, the percentage of hindlimb stepping errors was 25.6 ± 3.3 and 29.9 ± 5.9 for simvastatin and control groups, respectively. Between Experiments 3 and 4, we obtained a CatWalk system and utilized it here. The three parameters chosen from the CatWalk analysis system also showed no significant neurological improvements as a result of treatment (p N 0.05; Fig. 8). The regularity index, a relative measure of coordination, decreased after injury and, at 42 days post-injury, was 87.65 ± 2.01% and 92.01 ± 0.96% for control and simvastatin groups, respectively (Fig. 8A). The hindpaw base of support, which indicates gait stability, increased after injury and, at 42 days post injury, was 33.53 ± 1. 13 mm and 30.42 ± 1. 79 mm (Fig. 8B). Lastly, the hindpaw intensity, which estimates how much pressure or weight is supported on the hindpaws, decreased after injury and, at 42 days post injury, was 58.01 ± 5.86 and 58.88 ± 3.70 for control and simvastatin groups, respectively (Fig. 8C). In accordance with our behavioral results, histologic analysis revealed that simvastatin did not lead to more spared white or gray matter as compared to control treatment, either at the lesion epicenter or as the sum of sections through the injury epicenter (“cumulative” white matter sparing; Fig. 9). The extent of ED1 staining at 42 days post-
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Fig. 7. In Experiment 4, the percentage of hindlimb errors while crossing a horizontal ladder was not improved in simvastatin-treated animals.
injury also did not differ between simvastatin- and saline-treated animals (0.25 ± 0.017 and 0.24 ± 0.012, respectively, Fig. 10). Interestingly, however, in a pilot study of 10 animals euthanized at 3 days postinjury, there was a significant reduction in the ED1 staining in the simvastatin-treated animals, with average proportions of 0.0417 ± 0.006 and 0.023 ± 0.004 for control and simvastatin groups, respectively (data not shown). Clearly, this effect was not borne out to the 42-day post-injury time point.
Fig. 6. In Experiment 4, open-field locomotor revealed no improvements in BBB (A) scores or (B) subscores in simvastatin-treated animals (3 days of 20 mg/kg then 39 days of 5 mg/kg, via subcutaneous injection).
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Fig. 8. CatWalk analysis in control and simvastatin-treated animals. (A) Results of the regularity index, a relative measure of coordination. (B) Results of the hindpaw base of support, which indicates gait stability. (C) Results of the hindpaw intensity, which estimates how much pressure or weight is supported on the hindpaws. There were no improvements in the simvastatin-treated animals.
Discussion This paper describes a series of experiments that were performed to evaluate the comparative neuroprotective effects of simvastatin and atorvastatin. We observed what we thought was a promising “signal” of improved tissue sparing and locomotor recovery with simvastatin in our first experiment and were obviously compelled to replicate this with a larger series. Two subsequent attempts with orally administered simvastatin failed to reveal any behavioral benefit on open-field locomotor testing, and a final attempt with a subcutaneously administered dose of purified simvastatin given everyday for 42 days also failed to show any behavioral or histologic improvements. We would conclude that, in a thoracic contusion injury model (using the OSU Impactor) with a 1-h delay in intervention, simvastatin did not confer robust locomotor improvement as measured with the BBB scale or horizontal ladder. In our first three experiments, we did not observe any behavioral or histologic improvements with atorvastatin either. Our interest in studying statins for spinal cord injury was initially stimulated by the results of Pannu et al., who reported in 2005 of dramatic histologic and behavioral improvements in a rodent thoracic SCI model with atorvastatin at the same dose ( 5 mg/kg) for 1 week pre-injury followed by daily oral-gastric gavage administration for 42 days post-injury (Pannu et al., 2005). During the course of our series of experiments, the same authors reported even more impressive
histologic and behavioral improvements with atorvastatin begun at 2-, 4-, or even 6-h post-injury (Pannu et al., 2007). While we also utilized a 5 mg/kg dose of atorvastatin, there are important distinctions between our experimental designs that should be taken into consideration as potential reasons why we would not observe the same efficacy as Pannu et al. Our injury model differed from that of Pannu et al. in that they utilized a weight drop device while we utilized an electromechanical impactor (Ohio State University Impactor), and our injury site (T10) was more rostral than theirs (T12). The biomechanical severity of our contusion model is likely to be much greater than that of Pannu et al., judging by the published figures of the injury epicenter in their “control” animals (Pannu et al., 2005). While this may be a rationale explanation for the differences in experimental outcome, we recognize that human injuries occur with great biomechanical variety (and severity), and so treatments will be needed to be efficacious in similarly severe injuries. Additionally, we began our oral administration of atorvastatin 1-h post-injury, but maintained it for only 1 week post-injury and not 6 weeks post-injury. This may be an important difference, given the potential effect of statins in promoting axonal sprouting via the rho pathway (Pannu et al., 2007). Nonetheless, during these 7 days of atorvastatin treatment in our injury model, there was no improvement in BBB scores that would suggest that it was having an early neuroprotective effect. The application of simvastatin in an animal model of spinal cord injury has more recently been reported for the first time by Holmberg et al. (2008). Their initial in vitro work demonstrated that simvastatin promoted neurite outgrowth in the presence of growth-inhibitory molecules such as myelin associated glycoprotein, Nogo, and oligodendrocyte myelin glycoprotein (Holmberg et al., 2006). They went on to demonstrate that after a T9 thoracic contusion SCI, simvastatin reduced CSPG immunoreactivity in a dose-dependent fashion, particularly away from the injury site (Holmberg et al., 2008). However, similar to our observations, simvastatin by itself did not increase the extent of spared tissue, and administration either orally or intrathecally did not improve locomotor recovery as measured with BBB scores. The authors also point out the discrepancy between these negative behavioral results and those of Pannu et al. who reported near-normal BBB scores after atorvastatin treatment. Important distinctions such as the level of injury (T8/9 versus T12) and severity of injury were pointed out by Holmberg et al. Our experimental paradigm (T10 level) with a contusive injury that destroys over 90% of the cross-sectional area of the cord at the injury epicenter is seemingly more similar to that of Holmberg et al. (2008). In such in vivo pharmacologic neuroprotection studies, dose and mode of administration undoubtedly play critical roles. At the time that we initiated these studies, there was evidence that 20 mg/kg of simvastatin was neuroprotective in an animal model of cerebral ischemia (Cimino et al., 2005). Other authors have also utilized this simvastatin dose ( 20 mg/kg) and mode of administration (subcutaneous injection) with beneficial effects (Balduini et al., 2001, 2003; Endres et al., 1998; Lu et al., 2007; McGirt et al., 2002; Sironi et al., 2003; Wang et al., 2007). While these doses significantly exceeded the tolerable human dose of simvastatin, given the paucity of in vivo neuroprotection studies utilizing simvastatin, we felt that 20 mg/kg would be a justifiable initial dose to start our experiments with, particularly given that others had reported beneficial effects using the same dose. The in vivo work of Holmberg et al. utilized more clinically applicable oral simvastatin doses in their rodent SCI model (0. 57 mg/kg/day or 2. 3 mg/kg/day), and while they noted significant reductions in CSPG staining within the injured spinal cord, no behavioral recovery was noted. It is, of course, difficult to know exactly what the optimal drug concentration is within the injured spinal cord parenchyma and how to best get it there. We found in a pilot study that prolonging the subcutaneous injections of 20 mg/kg simvastatin beyond 3 days was quite toxic to the animals, and hence, in Experiment 4, we only maintained this 20 mg/kg dose for 3 days, before reducing it down to 5
C.M. Mann et al. / Experimental Neurology 221 (2010) 285–295 Fig. 9. In Experiment 4, Eriochrome C staining of serial cross-sections of the injury site revealed no significant improvements in either (A, B) white matter or (C, D) gray matter sparing in simvastatin-treated animals. The extent of spared white and gray matters was summed through the central 400 μm around the injury epicenter.
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Fig. 10. In Experiment 4, the 3-day treatment with 20 mg/kg simvastatin significantly reduced ED1 immunostaining at the injury epicenter. Prolonged treatment at this dose was toxic to the animals, necessitating a reduction to 5 mg/kg/day after the initial 3 days.
mg/kg/day. The simvastatin toxicity that we observed is quite contradictory to studies that also utilized a daily 20 mg/kg subcutaneous dosing regimen for at least a week, without noting any problems (Balduini et al., 2001, 2003; Endres et al., 1998; Lu et al., 2007; McGirt et al., 2002; Sironi et al., 2003; Wang et al., 2007). Our experiments were largely proof-of-concept studies, in which we hypothesized that there would be a neuroprotective effect with atorvastatin, but that it would be greater with simvastatin. Our interest in simvastatin was based on a number of factors, including the fact that the relative potency of HMG-CoA reductase inhibition is greater with simvastatin (Zacco et al., 2003), simvastatin was found to be more neuroprotective than atorvastatin in an in vitro assay of excitotoxicity (Zacco et al., 2003), and with its greater lipophilicity we believed that simvastatin might make it a better agent in the context of acute neurotrauma. After our initial histologic analysis of the injury site in Experiment 1, we did not pursue histologic assessments in Experiment 2 or 3, given that the behavioral outcomes were essentially identical among the saline, atorvastatin, and simvastatin groups. This reflects our own bias toward the importance of functional recovery above all other metrics, and in the absence of any behavioral improvements we did not feel that extensive histologic assessments of the cord would substantially influence our perspective on the efficacy of simvastatin (or lack thereof). In our recently conducted survey, we asked members of the SCI research community whether they were of the opinion that improvements in histologic, biochemical, or physiologic outcome measures without any behavioral improvements constituted “clinically meaningful efficacy” (Kwon et al., 2009). Of the 324 respondents, 59% disagreed that this represented clinically meaningful efficacy, while only 28% were of the opinion that it did. In fact, 31% “strongly disagreed” with improvements in non-behavioral outcomes in the absence of behavioral effects being considered a demonstration of “clinically meaningful efficacy”, while only 5% “strongly agreed” with this statement. While we would certainly not argue against the importance of mechanistic insights garnered from more extensive histological, biochemical, or physiological studies, from a translational perspective in deciding upon the “promising clinical potential” of an experiment treatment, we share the SCI community's emphasis on behavioral recovery and focused on this throughout the studies described in this paper. In Experiment 4, we did change the therapeutic regimen to include a prolonged, 42-day dosing schedule. Again, with the absence of any behavioral improvements on BBB score, BBB subscore, CatWalk, and horizontal ladder, the fact that ED1 immunostaining was reduced in our pilot study of simvastatin-treated animals at the 72-h post-injury mark is mechanistically interesting but of modest importance. This
significant reduction in ED1 immunostaining within the injury site with daily 20 mg/kg subcutaneous simvastatin injections at least indicated to us that the intervention was having some biological effect on the spinal cord, although this in itself was not particularly surprising, given the numerous studies that other authors have published with the use of this simvastatin dose ( 20 mg/kg) and mode of administration (subcutaneous injection; Balduini et al., 2001; Balduini et al., 2003; Endres et al., 1998; Lu et al., 2007; McGirt et al., 2002; Sironi et al., 2003; Wang et al., 2007). It is, of course, of interest to us that these other investigators observed substantive beneficial effects with this same dose and administration mode of simvastatin, albeit in different neurologic settings. The immunomodulatory effects of statins are now well recognized, and so it is certainly conceivable that macrophage/microglial activation may be somewhat attenuated. We did not undertake a semi-quantitative analysis of CSPG deposition at and around the spinal cord injury site as Holmberg et al. (2008) performed but would agree with their contention that while simvastatin may influence the properties of cells within the injury mileu (and possibly make for a more permissive environment), the drug by itself is not sufficient to promote functional recovery. Ultimately, these series of experiments were instructive to us, in that we admittedly were quite enthusiastic about the positive results from our first experiment where simvastatin appeared quite effective in both behavioral and histologic outcomes. The animal mortality in the first experiment was unacceptably high, and obviously, with only 4–5 animals, we were unable to make strong conclusions. It seemed, nonetheless, that there was some therapeutic potential there, and that we could more validly demonstrate that with a repetition of the experiment, albeit with better gavage technique. Having conducted 3 additional studies—all of which were resoundly negative—we would conclude that our initial positive findings with simvastatin represented an “alpha” error, related to the small numbers. In our recent survey of the SCI community, questions were included to garner the perspectives of our field on the issue of bias and how this might influence the interpretation of preclinical studies. A large proportion of respondents opined that scientists tend to repeat negative experiments in the attempt to generate positive findings (Kwon et al., 2009). Indeed, this paper illustrates our own repeated (and failed) attempts to demonstrate a positive neuroprotective response with atorvastatin and simvastatin, with a particular interest in confirming a fleeting positive response observed with the latter. Evidently, we too are not immune to this “bias” either. For us it is an interesting reflection on the difficulties in replicating positive experimental results, which is an issue that has become quite evident within the SCI community. The NIH has sponsored formal replication studies of a variety of therapeutic agents, including erythropoietin, minocycline, and NEP1-40 (Pinzon et al., 2008b; Pinzon et al., 2008a; Steward et al., 2008). To date, none of these formal replication attempts have successfully reproduced the efficacy reported in the original study. While differences in experimental design such as animal species, weight, age, gender, anesthetic agent, injury model, injury severity, and outcome assessors may be blamed for the lack of reproducibility in such studies, it is hard to imagine how a therapy could be successful at conferring improved neurologic function in the vastly variable setting of human SCI if such experimental differences are truly to blame for the lack of reproducibility in preclinical studies. In summary, in our hands, using a T10 OSU contusion SCI in Sprague–Dawley rats, we could not demonstrate a beneficial neurologic effect to either atorvastatin or simvastatin. The atorvastatin dose and mode of administration utilized was the same as that of Pannu et al. (2007), and although we did not extend the treatment for the entire post-injury period, we did not see any of the spectacular recovery that they reported. Recently, a study by Dery et al. reports a positive response to 5 mg/kg atorvastatin administered intraperitoneally after a T10 weight drop contusion SCI (Dery et al., 2009), so this does stand as some level of independent confirmation of Pannu et
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al.'s results. Further atorvastatin testing should arguably explore its efficacy in a cervical injury model and with doses more comparable to tolerable human doses. As for simvastatin, although we utilized a dose ( 20 mg/kg) and administration method (subcutaneous injection) also used successfully by others, we were unable to demonstrate a neuroprotective response in our SCI model, despite repeated efforts. Further dosing studies and investigation in a cervical injury model may be warranted, although we submit these negative findings to the research community so as to prevent others from attempting the exact same experimental approach that we have expended great time and effort on. Acknowledgments BKK holds a New Investigator Award from the Canadian Institutes for Health Research and WT is the Rick Hansen Man in Motion Chair of Spinal Cord Injury Research, UBC. This research was supported by the Wings for Life Spinal Cord Injury Foundation and Scoliosis Research Society. References Balduini, W., De V, A., Mazzoni, E., Cimino, M., 2001. Simvastatin protects against longlasting behavioral and morphological consequences of neonatal hypoxic/ischemic brain injury. Stroke 32, 2185–2191. Balduini, W., Mazzoni, E., Carloni, S., De Simoni, M.G., Perego, C., Sironi, L., Cimino, M., 2003. Prophylactic but not delayed administration of simvastatin protects against long-lasting cognitive and morphological consequences of neonatal hypoxicischemic brain injury, reduces interleukin-1beta and tumor necrosis factor-alpha mRNA induction, and does not affect endothelial nitric oxide synthase expression. Stroke 34, 2007–2012. Bartesaghi, S., Marinovich, M., Corsini, E., Galli, C.L., Viviani, B, 2005. Erythropoietin: a novel neuroprotective cytokine. Neurotoxicology 26 (5), 923–928. Basso, D.M., 2004. Behavioral testing after spinal cord injury: congruities, complexities, and controversies. J. Neurotrauma 21, 395–404. Basso, D.M., Beattie, M.S., Bresnahan, J.C., 1995. A sensitive and reliable locomotor rating scale for open field testing in rats. J. Neurotrauma 12, 1–21. Cimino, M., Gelosa, P., Gianella, A., Nobili, E., Tremoli, E., Sironi, L., 2007. Statins: multiple mechanisms of action in the ischemic brain. Neuroscientist 13 (3), 208–213 (Review). Cimino, M., Balduini, W., Carloni, S., Gelosa, P., Guerrini, U., Tremoli, E., Sironi, L, 2005. Neuroprotective effect of simvastatin in stroke: a comparison between adult and neonatal rat models of cerebral ischemia. Neurotoxicology 26 (5), 929–933. Dery, M.A., Rousseau, G., Benderdour, M., Beaumont, E., 2009. Atorvastatin prevents early apoptosis after thoracic spinal cord contusion injury and promotes locomotion recovery. Neurosci. Lett. 453, 73–76. Domercq, M., Matute, C., 2004. Neuroprotection by tetracyclines. Trends Pharmacol. Sci. 25, 609–612. Endres, M., Laufs, U., Huang, Z., Nakamura, T., Huang, P., Moskowitz, M.A., Liao, J.K., 1998. Stroke protection by 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors mediated by endothelial nitric oxide synthase. Proc. Natl. Acad. Sci. U. S. A. 95, 8880–8885. Gorio, A., Gokmen, N., Erbayraktar, S., Yilmaz, O., Madaschi, L., Cichetti, C., Di Giulio, A.M., Vardar, E., Cerami, A., Brines, M., 2002. Recombinant human erythropoietin counteracts secondary injury and markedly enhances neurological recovery from experimental spinal cord trauma. Proc. Natl. Acad. Sci. U.S.A. 99, 9450–9455. Hamers, F.P., Koopmans, G.C., Joosten, E.A., 2006. CatWalk-assisted gait analysis in the assessment of spinal cord injury. J. Neurotrauma 23, 537–548. Holmberg, E., Nordstrom, T., Gross, M., Kluge, B., Zhang, S.X., Doolen, S., 2006. Simvastatin promotes neurite outgrowth in the presence of inhibitory molecules found in central nervous system injury. J. Neurotrauma 23, 1366–1378. Holmberg, E., Zhang, S.X., Sarmiere, P.D., Kluge, B.R., White, J.T., Doolen, S., 2008. Statins decrease chondroitin sulfate proteoglycan expression and acute astrocyte activation in central nervous system injury. Exp. Neurol. Hurlbert, R.J., Hamilton, M.G., 2008. Methylprednisolone for acute spinal cord injury: 5-year practice reversal. Can. J. Neurol. Sci. 35, 41–45. Igel, M., Sudhop, T., von, B.K., 2001. Metabolism and drug interactions of 3-hydroxy-3methylglutaryl coenzyme A-reductase inhibitors (statins). Eur. J. Clin. Pharmacol. 57, 357–364.
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