Spinal cord injury is a devastating condition affecting. Magnesium efficacy in a rat spinal cord injury model. Laboratory investigation

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1 J Neurosurg Spine 10: , 10: , 2009 Magnesium efficacy in a rat spinal cord injury model Laboratory investigation *Di a n a Ba r r e t t Wi s e m a n, M.D., 1 And r e w T. Da i l e y, M.D., 1,3 Dav i d Lu n d i n, M.D., 1 Ji e g a n g Zh o u, M.D., 1 Ad a m Li p s o n, M.D., 1 Al e x i s Fa l i c o v, M.D., Ph.D., 2 a n d Ch r i s t o p h e r I. Sh a f f r ey, M.D. 4 Departments of 1 Neurological Surgery and 2 Orthopaedics, University of Washington, Seattle, Washington; 3 Department of Neurosurgery, University of Utah, Salt Lake City, Utah; and 4 Department of Neurological Surgery, University of Virginia Health System, Charlottesville, Virginia Object. Magnesium has been shown to have neuroprotective properties in short-term spinal cord injury (SCI) studies. The authors evaluated the efficacy of magnesium, methylprednisolone, and magnesium plus methylprednisolone in a rat SCI model. Methods. A moderate-to-severe SCI was produced at T9 10 in rats, which then received saline, magnesium, methylprednisolone, or magnesium plus methylprednisolone within 10 minutes of injury. The Basso-Beattie-Bresnahan (BBB) motor score was evaluated weekly, beginning on postinjury Day 1. After 4 weeks, the rats spinal cords were evaluated histologically to determine myelin index and gross white matter sparing. A second experiment was conducted to evaluate the effect of delayed administration (8, 12, or 24 hours postinjury) of magnesium on recovery. Results. The mean BBB scores at 4 weeks showed that rats in which magnesium was administered (BBB Score 6.9 ± 3.9) recovered better than controls (4.2 ± 2.0, p < 0.01). Insufficient numbers of animals receiving methylprednisolone were available for analysis because of severe weight loss. The rats given magnesium within 8 hours of injury had better motor recovery at 4 weeks than control animals (13.8 ± 3.7 vs 8.6 ± 5.1, p < 0.01) or animals in which magnesium was administered at 12 or 24 hours after injury (p < 0.01). Steroids (30.2%), magnesium (32.3%), and a combination of these (42.3%) had a significant effect on white matter sparing (p < 0.05), but the effect was not synergistic (p > 0.8). Neither steroids nor magnesium had a significant effect on the myelin index (p > 0.1). Conclusions. The rats receiving magnesium had significantly better BBB motor scores and white matter sparing 4 weeks after moderate-to-severe SCI than control animals. In addition, the groups given steroids only or magnesium and steroids had improved white matter sparing, although the limited numbers of animals reaching the study end point makes it difficult to draw firm conclusions about the utility of steroids in this model. The optimal timing of magnesium administration appears to be within 8 hours of injury. Ke y Wo r d s magnesium methylprednisolone rat spinal cord injury Spinal cord injury is a devastating condition affecting ~ 200,000 people in the US, with ~ 10,000 new cases each year. 1,7,45 The total annual cost of caring for patients with SCI in the US is estimated to be $9.73 billion. 45 Clearly, SCI is one of the most devastating survivable injuries an individual can suffer, and it has an enormous social and economic impact on our communities. In cases of SCI, although the primary injury is well established before the patient presents to the hospital, modulation of secondary injury caused by apoptotic cell death at the periphery of traumatic contusions can limit Abbreviations used in this paper: ANOVA = analysis of variance; BBB = Basso-Beattie-Bresnahan; LFB = Luxol fast blue; NASCIS = National Acute Spinal Cord Injury Studies; NMDA = N-methyl-d-aspartate; SCI = spinal cord injury; TBI = traumatic brain injury. * Drs. Wiseman and Dailey contributed equally to this work. the size of the lesion and improve final outcome. Improving outcome after SCI would improve patient quality of life and lessen the social and economic impact of this condition. Pharmacological treatment after SCI is aimed at decreasing the severity of the injury and reducing the extent of permanent paralysis. Only 1 pharmacological therapy, high-dose methylprednisolone, has been approved for use in the treatment of SCI. The NASCIS II and III have shown that methylprednisolone administered within 8 hours of SCI improved motor and sensory outcome when given as a 30 mg/kg bolus loading dose followed by a 5.4 mg/kg/hr continuous intravenous infusion. 5,6 Methyl prednisolone use has not been without controversy, however, because the small beneficial effect may be outweighed by increased infection rates and generation of gastric and duodenal ulcers. 15,16,33,35 In addition, 308

2 Magnesium in spinal cord injury the therapeutic window for methylprednisolone is small in rodent models of SCI, while the potential side effects of methylprednisolone administration in these models include potentiation of inflammatory and ischemic neuronal injury. 20,35,37 Because of these limitations, alternative pharmacological agents that may improve outcome are under investigation and may ultimately supplement or replace methylprednisolone therapy. 2,10 Magnesium is a functioning component of ~ 300 key enzymes involved in protein synthesis, energy transformation, adenosine triphosphate consumption and production, and lipid and nucleic acid metabolism. 44 Magnesium sulfate has been shown to have neuroprotective properties in short-term studies of animal TBI and ischemia as well as SCI models. 3,12,14,21,22,26,27,39,40,42,47 The primary mechanism of action appears to be limiting levels of intracellular calcium by inhibition of excitotoxicity and blockage of voltage-gated calcium channels. Interestingly, magnesium has had positive effects on outcome in rodent cerebral ischemia, even when given up to 24 hours after injury. 14 In this study, we evaluated the effect of magnesium on a rodent SCI model when given within 10 minutes of injury and at various other time points, up to 24 hours after injury. We also evaluated how magnesium affects outcome in comparison with methylprednisolone and when used in combination with methylprednisolone. Injury Procedure Methods Adult female Sprague-Dawley rats weighing between 250 and 300 g were used. The animals received enrofloxacin (Baytril, Bayer Corp.) as antibiotic prophylaxis immediately before the procedure and each day for 7 days afterward. The animals were anesthetized with 29 mg/ kg of intraperitoneal pentobarbital. After each animal was adequately anesthetized, the region over the lower thoracic spine was shaved and then prepared in a sterile fashion. A midline incision was made from T-8 to T-11. Laminectomies were performed at T9 10, and the NYU impactor model was used to generate all injuries. For Experiment 1, a moderate-to-severe injury was created when a 10-g weight was dropped 25 mm. A less severe injury caused by 10 g dropped from 12.5 mm was used for animals in Experiment 2. The overlying muscle was then closed with interrupted absorbable sutures (Ethicon), and the skin was closed with skin clips. The animals body temperature was maintained by use of a heating blanket postoperatively, and the animals were closely observed until they demonstrated the ability to drink and eat independently. Postoperative signs of pain were assessed and treated with buprenorphine hydrochloride (0.3 mg/kg). All experimental protocols were approved by the University of Washington animal care committee. Treatment Groups In Experiment 1, 70 animals were separated into 4 groups. Each group underwent the SCI procedure as described above and then received by random selection either 1 ml of normal saline (control, 13 rats); 600 mg/kg magnesium, in similar volume (20 rats); 30 mg/kg methylprednisolone, in similar volume (17 rats); or magnesium plus methylprednisolone at the aforementioned doses (20 rats). All injections were intraperitoneal and occurred within 10 minutes of injury. For Experiment 2, in which 52 rats were used, the animals were again separated into 4 groups, with animals receiving 1 ml of saline (control, 13 rats) within 10 minutes of injury or 600 mg/kg magnesium, in 1-ml volume, at 8, 12, or 24 hours postinjury (13 animals per time point). All animals underwent motor scoring by the BBB method. This was performed on postoperative Day 1 and then again each week for 4 weeks by 2 observers who were blinded to the treatment. Bladder expression was performed twice daily. Animals were monitored for signs of autophagia, urinary tract infections, and weight loss. Animals that demonstrated signs of significant autophagia, urinary tract infections recalcitrant to antibiotic treatment, or weight loss > 20% of body weight were killed. Animals that lost > 10% but 20% of body weight were started on aggressive twice-daily highcalorie oral supplementation. Histological Evaluation Animals from Experiment 1 that completed the 4-week observation period were given 50 mg/kg pentobarbital intraperitoneally, and then intracardiac perfusion was performed with 4% paraformaldehyde (300 ml/ animal). The spinal cord from T-6 to L-1 was removed and placed in paraformaldehyde. An 8-mm longitudinal section was harvested around the epicenter of the lesion to include the entire spinal cord lesion. The cords were embedded in paraffin, and 20-µm-thick longitudinal sections of spinal cord that were cut through the lesion epicenter were collected and stained with LFB/cresyl violet. Because of the attrition in the methylprednisolone-treated groups, histological analysis was performed in only 4 animals from each group in Experiment 1. White matter sparing was calculated for spinal cords within each treatment group, because other investigators have shown this to be an excellent measure by which to characterize the degree of injury. 31,32 Every sixth section was sampled throughout the spinal cord, so that 7 9 sections were evaluated from each animal. Spared white matter volumes were assessed using stained longitudinal tissue sections cut through the lesion and the surrounding tissues, by creating a binary image of myelin-stained compared with unstained or scarred tissues. 25,36 Images were collected using a Nikon TE2000-S inverted microscope attached to a motorized stage (Prior Scientific, Inc.) and a Coolsnap HQ camera (Photometrics). Image analysis was performed using Metamorph Image Analysis (Universal Imaging Corp.). Tissue sections were imaged and analyzed by an observer blinded to treatment groups. An 8-mm length of each spinal cord was centered on the lesion epicenter, a grid was overlaid, and sequential 20 images were collected and stitched together to form a single color image of the lesion epicenter and the penumbra. A binary image was created on a 309

3 D. B. Wiseman et al. computer workstation showing myelinated tissues (those stained with LFB) and scarred or unstained tissues. As a result, a binary histogram could be created to calculate the areas of myelinated tissue in relation to the entire area of tissue in the spinal cord. 31 A 1-in-6 series of serial 20- µm sections was analyzed in this manner, so that between 7 and 9 sections were analyzed per specimen, and a mean area of myelinated tissue per specimen was calculated. Areas of preserved white matter are expressed as the percentage of preserved myelin-stained tissues per total area of tissue for the 8-mm longitudinal section of spinal cord surrounding the lesion epicenter. Finally, lesion length was estimated by measuring the length of the scarred tissue on a single midline image. Statistical Analysis Statistical analysis was accomplished using StatView software (Abacus Concepts, Inc.). Two-way ANOVA was used to determine the effect of each treatment at the 1-week time points (group week). The final week s (4th week) median BBB data were compared using an unpaired t-test. One-way ANOVA was used to compare differences between the experimental groups for white matter sparing and myelin index. A probability value < 0.05 was considered significant. Analysis of BBB Scores Results In Experiment 1, the mean BBB scores were significantly better for the magnesium group (BBB Score 6.9 ± 3.9) than for the control group (4.2 ± 2.0) after 4 weeks (p < 0.01, 2-sample unpaired t-test) (Fig. 1), but there were no significant differences at earlier time points among any of the groups (p > 0.1, ANOVA). From an initial group of 13 animals in each treatment category, the majority of the animals receiving methylprednisolone alone as well as most of the animals receiving methylprednisolone plus magnesium suffered weight loss > 20% of initial body weight. University of Washington animal regulations require any animal that exceeds 20% weight loss to be killed. Even the addition of up to 7 rats per group did not yield a sufficient number of animals in the 2 steroidtreated groups to perform statistical analysis of their BBB outcomes at 4 weeks postinjury (Table 1). In Experiment 2, the mean BBB data at 4 weeks revealed that the animals in which magnesium was administered within 8 hours (BBB Score 13.8 ± 3.7) had significantly better function than animals in the control (8.6 ± 5.1), 12-hour (8.1 ± 3.1), or 24-hour (7.9 ± 2.8) groups (p < 0.01, unpaired t-tests). The difference in motor improvement started at 2 weeks for the animals that received magnesium within 8 hours of injury, compared with the late-administration or control groups (p < 0.01, ANOVA; Fig. 2). Histological Findings Fig. 1. Graph showing the mean BBB scores for animals administered magnesium or saline (control) immediately after SCI. In Experiment 1, the LFB/cresyl violet stained sections were used to determine white matter sparing (Fig. 3). Measurements of spared white matter and total crosssectional area were made of the lesion epicenter. All treatment groups had a significantly greater volume of white matter spared after the injury (magnesium 32.3%, methylprednisolone 30.3%, and magnesium plus methylprednisolone 42.3%) than did the control group (20.2%) (p < 0.05, ANOVA; Table 2). No differences in mean lesion length (2.82 mm magnesium, 3.30 mm control) could be found between the groups; however, the small number of specimens and the variance in the control group ( mm) were potential limitations. Discussion Evidence presented in both the basic science and the clinical literature has supported the suggestion that mag- TABLE 1: Number of rats in Experiment 1 exhibiting various conditions during the SCI study Treatment Group No. at Day 1 Survival at 4th Wk Weight Loss Signs of Autophagia Died in Immediate Postop Period control magnesium methylprednisolone methylprednisolone + magnesium

4 Magnesium in spinal cord injury TABLE 2: Histological analysis examining white matter preservation in rats in Experiment 1 Treatment Group No. of Rats % Myelin Preservation p Value control magnesium methylprednisolone methylprednisolone + magnesium Fig. 2. Graph showing the mean BBB scores for animals administered magnesium treatment at various time points after SCI. nesium acts as a potential therapeutic agent after SCI as well as TBI and ischemia. The loss of neurons and glia in traumatic CNS injury occurs not only by direct injury but also by apoptotic cell death at the periphery of traumatic contusions. 4,23 Apoptotic cell loss may account for a secondary zone of damage in regions distant from as well as adjacent to the initial injury site. Use of magnesium sulfate has been found to decrease apoptotic cell death in rodent models of short-term cerebral ischemia 26,40 as well as in TBI and SCI models. 14,22,42 The authors of most studies, however, have examined the efficacy of magnesium administered immediately after injury and have not focused on behavioral outcomes. Within the cerebral ischemia and TBI literature, magnesium therapy has shown promising results. 27,28 Magnesium serves as an NMDA receptor antagonist by competitive inhibition of the receptor ion channel. Activation of the NMDA receptor by glutamate in cases of trauma or ischemia, with a subsequent influx of calcium, is thought to be one of the components leading to cell death. 38 In addition, magnesium not only serves to stabilize the NMDA receptor but also is involved in many other key enzymatic reactions within the cell involved in adenosine triphosphate production. Maintained homeostasis of magnesium can theoretically play a role benefiting many reactions within the CNS. Optimal dosing of magnesium has been determined in rodent models of CNS neurotoxic injury. Wolf et al. 47 demonstrated the protective effects of magnesium against quinolinate-induced NMDA activation, noting that subcutaneous injection of either 600 or 300 mg/kg of magnesium sulfate was neuroprotective when given simultaneously with the quinolinate. Complete neuroprotection was noted when 600 mg/kg was administered within 1 hour of injury, whereas incomplete protection was noted for the animals treated after 3 hours. In a similar model of kainite-induced injury, magnesium at the higher dose of 600 mg/kg injected subcutaneously was the most effective dose for neuroprotection, compared with doses of 300 and 150 mg/kg. 46 Peak levels of 4.8 meq/l were reached at 1 hour. In a rat TBI model, Heath 14 and Vink and Cernak 44 found a neuroprotective effect for magnesium (750 µmol/ kg) even when it was administered intramuscularly up to 24 hours postinjury. Significant improvement in motor outcomes was noted at all time points in animals that received their infusion at 30 minutes, 8 hours, or 12 hours after injury. Animals that received magnesium 24 hours after injury demonstrated a more rapid recovery of function that was noted to be significantly better than recovery of the control animals by the end of the assessment week. Investigation of magnesium levels in animals that had received an intramuscular bolus of magnesium showed a maximum blood level at 2.5 hours after administration, which fell to preinjury levels 12 hours after trauma. However, repeated administration of magnesium at 12-hour intervals for 1 week did not yield significant motor improvement over single-dose administration. 14 In fact, repeated administration of magnesium sulfate to achieve serum levels > 1.25 mmol/l for 5 days was found to have a detrimental effect in the treatment of TBI in human subjects. 43 In our study, rats with moderate SCI that were given a single subcutaneous injection of 600 mg/kg of magnesium after injury showed significant improvement in motor scores as well as white matter sparing compared with control animals receiving saline injection. These animals (the ones receiving magnesium), however, experienced higher complication rates of autophagia compared with controls or animals in the group receiving steroid injections. The occurrence of autophagia usually began 7 10 days postoperatively but was noted to occur as early as 5 days. This increased incidence may be due to a return of sensation to the rats hind limbs that they find unpleasant. This dysesthetic sensation may stimulate the autophagia of toes and, ultimately, the feet if the animal is not controlled. 9 Tylenol use was instituted immediately when animals showed signs of autophagia, but this was often ineffective in stopping the activity, and thus the animals had to be killed. With a less severe injury, the animals in Experiment 2 experienced lower rates of autophagia and presumably experienced less dysesthesia. Previous studies investigating the protective effect of magnesium in SCI have shown that single-dose administration can reduce lipid peroxidation, 18,42 improve somatosensory evoked potentials, 19,42 and reduce caspase-3 311

5 D. B. Wiseman et al. Fig. 3. Photomicrographs showing longitudinal sections of spinal cord stained with LFB/PAS. All spinal cords were harvested 4 weeks postimpact and sectioned through the center of the injury. The control section (A) shows a larger area of tissue necrosis and myelin loss than the sections treated with magnesium (B), steroids (C), and magnesium and steroids (D). Original magnification 40. activity and thus diminish apoptosis.41 Studies evaluating the longer-term behavioral effects of magnesium have recently shown that 600 mg/kg administered almost immediately after the injury and then followed by a second dose 6 hours later could improve locomotor recovery in a rat clip compression model. The improvement in function correlated with a reduction in spinal cord lesion volume, although the reduction in volume was not statistically significant in comparison with saline-treated controls.9 Similar findings of preserved histological structure have been found in rabbit models of spinal cord ischemia22 and rat models of contusion,42 suggesting that magnesium sulfate does have neuroprotective effects when administered early after SCI. Although the current study suggests that the effective window for administration of magnesium is within 8 hours of the injury, further work regarding timing and dosing schedules needs to be completed and correlated with behavioral and histological preservation. Since the publication of the NASCIS studies, methylprednisolone has been widely used for the treatment of acute SCIs.5,6 Many centers have reported a number of side effects, especially in older patients, and the widespread use of methylprednisolone has been condemned by some clinicians.16,17,24 Because of the continued use of methylprednisolone by some clinicians, we opted to include animals treated with this drug in our comparisons. An unexpected finding was the severe weight loss noted among study animals that received methylprednisolone. Weight loss was rapid and noticeable within 48 hours of receiving the dose. Any animal that was noted to have weight loss between 10 and 20% of body weight was immediately started on high-calorie oral supplements that were hand-administered twice daily. Despite aggressive 312 attempts to maintain animal weights, most rats that received steroids lost > 20% of their body weight within the first 2 weeks, and the killing of these animals was required per the University of Washington animal care protocol. Other investigators have reported similar weight loss after administration of methylprednisolone. Constantini and Young8 found that all rats treated with methylprednisolone (30 mg/kg in a 1-time dose) lost 7 8% of body weight within 24 hours of injury. This weight loss was significantly greater than that in control animals, and it occurred irrespective of SCI severity. In a very revealing study, Nava et al.30 demonstrated that animals lost up to 19% of their body weight after 5 days of methylprednisolone treatment (80 mg/kg/day). Nutritional and water intake was statistically less than in control animals. Muscle histological investigations revealed loss of Type IIb muscle fibers within the gastrocnemius. Given these findings, methylprednisolone use in rats appears to be detrimental to the animals overall health. Further study would be necessary to evaluate whether methylprednisolone causes significant weight loss in humans when other causes of weight loss in the trauma patient are controlled. The use of high-dose methylprednisolone has been demonstrated to result in acute steroid myopathy based on electromyography and histological data in a limited number of patients with SCI,34 although no evidence to confirm or refute the widespread appearance of myopathy was presented in the NASCIS trials. Methylprednisolone has been found to have equivocal or even detrimental effects in several CNS injuries. After focal forebrain ischemia, administration of glucocorticoids has been shown to potentiate ischemic neuronal

6 Magnesium in spinal cord injury injury, and the use of steroids after stroke has proven to be detrimental. 37 In SCI models specifically, although steroids have been shown to reduce lipid peroxidation immediately after injury, 13 methylprednisolone has not reliably demonstrated a positive effect on either behavioral outcomes or white matter sparing after spinal cord contusion. 35 When used in conjunction with other medications that are showing promise for neurological improvement after SCI, methylprednisolone can attenuate the positive effects of these experimental treatments. For example, methylprednisolone eliminates erythropoietin-mediated improvement after a spinal cord contusion through mechanisms independent of the antiinflammatory effects of the steroids. 11 Although modest clinical improvements were shown in the NASCIS trials, more recent evidence from basic science studies further clouds the positive role methylprednisolone may play in treating SCI. Unfortunately, because several rats that had received steroids (65% of those treated with methylprednisolone only and 50% of those receiving methylprednisolone plus magnesium) were eliminated from the study because of excessive weight loss, we were left with insufficient numbers to complete an accurate statistical evaluation of the BBB scores in Experiment 1. In addition, because of the limited number of animals in the steroid groups in Experiment 1, we were limited in the number of spinal cords that could be evaluated histologically. As a result, only 4 specimens from each group in Experiment 1 were examined histologically to match the number available from the steroid-treated group. Thus, any differences that exist between the treatment arms in Experiment 1 may be due to sampling error. Although white matter preservation has been used to predict motor recovery and the extent of the injury, 32 only a limited number of cords were available for examination, and the loss of spinal cords from the second series of experiments eliminated our ability to review the utility of early magnesium administration on myelin preservation. Further work to correlate white matter preservation, a qualitative myelin index, and lesion volume with behavioral improvement is needed to determine the benefits of early magnesium administration. The histological analysis was limited to a quantification of white matter preservation. A subjective, qualitative analysis of the morphological features of the myelinated tissues performed using a pathoanatomical index that examined necrosis, hyperemia, and morphological features of the nerve substance did not reveal any differences among the groups, 29 although the magnesium group showed a trend toward less necrosis and better preservation of myelin structures than the control group (see Fig. 3). In addition, because we cut longitudinal sections of the spinal cord through the lesion epicenter, it was difficult to determine the extent of injury to the gray matter, because in many of the parasagittal sections analyzed, only minimal portions of the dorsal and ventral horns were represented. Despite the limitations of the histological data, the behavioral data showing improvement with early magnesium administration and the suggestion that magnesium has a function similar to steroids in the preservation of myelinated tracts reveal the promise of magnesium as a potential modulator of secondary injury after SCI. Conclusions Administration of magnesium immediately after SCI has resulted in promising longer-term neurological as well as histological outcomes in treated animals compared with controls. Methylprednisolone use was associated with severe weight loss in our rat model. Given the controversy around methylprednisolone use and the relative safety of magnesium use, magnesium may ultimately prove to be a more effective pharmacological treatment option overall for patients with SCI. A future direction for study would be to evaluate whether magnesium is effective when given at different time intervals up to 24 hours after injury. 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Neurosci Lett 117: , 1990 Manuscript submitted July 22, Accepted January 7, Address correspondence to: Andrew Dailey, M.D., Department of Neurosurgery, University of Utah, 175 North Medical Drive, Salt Lake City, Utah andrew.dailey@hsc.utah.edu. 314