BASIC INVESTIGATIONS. Neuroprotective Effects of Nicotinamide after Experimental Spinal Cord Injury. Kori L. Brewer, PhD, J. Shane Hardin, MD, PhD

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1 ACAD EMERG MED d February 2004, Vol. 11, No. 2 d BASIC INVESTIGATIONS Neuroprotective Effects of Nicotinamide after Experimental Spinal Cord Injury Abstract Objective: To investigate the ability of nicotinamide to protect against secondary damage in spinal cord tissue after an experimental injury. Trauma to the spinal cord induces a cascade of cellular events that lead to progressive tissue injury over time. Nicotinamide has been shown to affect many elements of this cascade, including excitatory amino acid release, inflammation, apoptosis, and cellular energy balance. Methods: Male Long Evans (n = 12) rats received an excitotoxic spinal cord injury by intraspinal injection of quisqualic acid (QUIS), a glutamate receptor agonist. A second set of rats (n = 4) received intraspinal saline as a sham injury. Thirty minutes after injury, animals that had QUIS injections received an intraperitoneal injection of either saline (control, n = 4) or nicotinamide (500 mg/kg, n = 8). Seven days postinjury, the spinal cords were removed, and serial sections were cut, mounted on slides, Kori L. Brewer, PhD, J. Shane Hardin, MD, PhD and stained. By using light microscopy, the extent of tissue damage was assessed at the epicenter of injury as well as sections up to 450-mm rostral and 450-mm caudal to the epicenter. Results: Only those animals receiving QUIS injections showed damaged tissue. There was no significant difference in the amount of damage at the epicenter of injury between the saline- and nicotinamide-treated animals. However, when comparing the total amounts of damage over the 975-mm length of cord examined, the rostro-caudal extent of injury was significantly reduced in the nicotinamide-treated animals compared with the saline-treated animals. Conclusions: Systemic nicotinamide serves to limit the rostro-caudal extent of cell death after experimental spinal cord injury. Key words: neurotrauma; neuroprotection; excitotoxicity; vitamin B3. ACADEMIC EMERGENCY MEDICINE 2004; 11: Spinal cord injuries (SCIs) are a major cause of morbidity in the United States and other developed nations. 1,2 In addition to paralysis, SCIs can result in spasticity, autonomic dysfunction, chronic pain syndromes, pressure ulcers, and other debilitating sequelae. 3 5 Because most of these injuries occur in young people, the financial costs of SCI can be enormous due to the number of years of lost wages and extensive medical care. An ischemic or traumatic insult to the spinal cord triggers a sequence of cellular events that leads to a selfperpetuating cascade of neuronal death. This cascade of events includes anatomical, neurochemical, excitotoxic, and inflammatory changes that ultimately impact the function of spinal neurons, including survival pathways. 6 The primary trauma of the injury induces secondary effects such as activation of excitatory amino acids, second messengers, and cytokines From the Department of Emergency Medicine, Brody School of Medicine at East Carolina University (KLB, JSH), Greenville, NC. Received July 14, 2003; revision received September 3, 2003; accepted September 23, Presented at the SAEM annual meeting, Boston, MA, May Address for correspondence and reprints: Kori L. Brewer, PhD, Department of Emergency Medicine, Division of Research, Brody School of Medicine at East Carolina University, Physician s Quadrangle, Building M, Greenville, NC Fax: ; brewerk@mail.ecu.edu. doi: /j.aem at the level of injury. Secondary effects also include free radical formation, activation of N-methyl-D-aspartate (NMDA) and 2-amino-3-propionic acid (AMPA) receptors, nuclear factor kappa-b (NFkB) activation, release of tumor necrosis factor-alpha (TNFa), interleukin 1-beta (IL-1b), expression of inducible nitric oxide synthase (inos), and poly-adp-ribose polymerase (PARP) activation. 7 9 Initiation of this secondary central cascade of injury will lead to necrosis, apoptosis, gliosis, and cavitation that extend beyond the site of the spinal injury. Although the primary injury depends on the biomechanical forces associated with the injury and cannot be altered, it is possible to interrupt the secondary events that are triggered by those forces. If the cascade of destructive cellular events is stopped, the neural tissue surrounding the primary injury site can be spared, potentially sparing function. This is the basis for the idea of neuroprotection after central nervous system (CNS) injury. Currently, the only option for emergency treatment of patients with spinal injuries is administration of methylprednisolone, which serves to reduce the inflammatory reaction to the injury. However, the effectiveness of this intervention remains controversial and is not without risks In addition, the therapeutic window is not well defined, and the guidelines for its use are often difficult to meet. 13 Research has identified other substances that provide some degree of neuroprotection after experimental SCI, including anti-inflammatory agents, 15

2 126 Brewer and Hardin d NICOTINAMIDE NEUROPROTECTION IN SCI glutamate receptor antagonists, 16,17 and free radical scavengers. 18 Each of these agents targets a specific point on the injury cascade. Nicotinamide is a naturally occurring B vitamin (B3) which has been shown to be protective in models of ischemic and traumatic brain injury This compound would potentially target multiple points of the injury cascade by limiting the effects of nitric oxide synthase (NOS) activation, preventing cellular energy depletion through the activation of PARP, 22,23 blocking pathways of cell death leading to apoptosis, 24 and reducing free radical formation secondary to membrane peroxidation. 19 In addition, nicotinamide is inexpensive, and it is clinically well tolerated. 25 In the current study, we examined the ability of nicotinamide to act as a neuroprotective agent in an animal model of acute SCI. The excitotoxic model used previously produced pathological changes similar to those seen with traumatic injury. 26 METHODS Study Design. This was a laboratory investigation of the neuroprotective effects of nicotinamide in a rat model of SCI. All experimental procedures were approved by the Institutional Animal Care and Use Committee. Animal Use and Preparation. Intraspinal Injection. The technique of intraspinal injection was similar to that described in previous reports. 15,26 Male Long Evans rats ( g) were anesthetized with inhaled isoflurane (1.5 2%). Animals were shaved and scrubbed with Betadine. A midline incision was made, and the muscle layers were bluntly dissected to expose the vertebral column. After the vertebral column was exposed, the spinous process and vertebral lamina were removed from one spinal level, and the dura was incised longitudinally and reflected unilaterally. Unilateral injections were made in a single segment at spinal levels ranging from T12 to L2. Glass micropipettes (tip diameter 5 10 mm), attached to a Hamilton syringe (volume 10 ml; Sigma, St. Louis, MO), were used for injections. The syringe was mounted on a microinjector (Kopf 5000, Tujunga, CA) attached to a micromanipulator. Injections were made between the dorsal vein and dorsal root entry zone at a depth of 1,000 mm below the surface of the cord. A stock solution of 125 mm quisqualic acid (QUIS; Sigma, St. Louis, MO) was made by using phosphate-buffered saline (PBS). Each animal received 1.2 ml of PBS (SHAM, n = 4) or QUIS (n = 12), injected over a 60-second time interval (three tracks of 0.4 ml separated by 0.3 mm parallel to the long axis of the cord). Following injections, muscles were closed in layers, the skin was closed with wound clips, and animals were returned to their home cages. Study Protocol. Thirty minutes after quisqualic acid administration, the animals were randomized to receive intraperitoneal injections of either 500 mg/kg of nicotinamide (experimental, n = 8) or an equal volume of saline (control, n = 4). Equal numbers of syringes were filled with saline or nicotinamide solution and coded prior to surgery. An investigator who was blinded as to which solutions were in each syringe randomly selected a syringe, administered injections, and recorded the code from the syringe used. Tissue Collection and Processing. Seven days after injury, animals were deeply anesthetized with sodium pentobarbital (100 mg/kg intraperitoneal). Spinal cords were removed and placed in 10% formalin for 24 hours and then transferred to 30% sucrose for 48 hours. By using a sledge microtome (Leica 2400; Leica Microsystems, Wetzler, Germany), 75-mm sections were then cut through the length of the spinal cord, collected in PBS, and mounted onto gelatincoated slides. Serial sections were collected at the level of injection as well as rostral and caudal to the epicenter to determine the extent of tissue damage. Sections were then stained with cresyl violet. Lesion Analysis. Quantification of QUIS-induced neuronal loss was carried out by using a technique described previously. 15,16,26 Sections were examined under light microscopy (4 3 objective) and pictures were taken of the epicenter of damage as well as six sections rostral and six sections caudal to the epicenter. Analysis of the spinal cord sections was performed by using the National Institutes of Health (NIH) Image 1.60 software package (Bethesda, MD). This analysis was done by an individual blinded to treatment protocols. The criteria used in determining the boundary between intact and damaged tissue were based on the absence of neurons and the presence of degenerating neuron profiles and inflammatory cells. The total area of intact gray matter in sections at the epicenter of QUIS-induced lesions was compared with sections from normal (naïve) animals matched for spinal level. The area of normal gray matter in each injured animal was divided by the area of normal gray expected at that spinal level (as determined from normal animals) and expressed as the percentage of normal gray matter that remains in the injured animal. This value was then subtracted from 100 to obtain the percentage of damage at the lesion epicenter. The determination of total lesion volume was carried out by examining six sections rostral and six sections caudal to the epicenter of the lesion (0.975 mm total length) as described for the epicenter, adding the areas of normal gray matter obtained at each section and multiplying by 75, the known thickness of each individual section. This value was then divided by the expected total gray matter as obtained from an equal length of cord in

3 ACAD EMERG MED d February 2004, Vol. 11, No. 2 d normal animals matched for spinal level to determine the percent of normal gray matter remaining in the injured animal. Subtracting this value from 100 provided the total lesion volume for each animal. Data Analysis. After completion of the lesion analysis, the treatment groups were decoded and analysis was completed. Independent Student s t-test was used to determine differences in percent of tissue damage between the epicenters of control and nicotinamidetreated animals as well as the total amount of gray matter damaged over a 975-mm length of cord surrounding the epicenter. Comparisons between the amount of damage at each 75-mm interval were made by using a repeated-measures analysis of variance (RMANOVA), with the treatment group and distance from the epicenter as independent variables. The RMANOVA was followed by Fisher s least significant difference post hoc test; p-values and 95% confidence intervals (CIs) are reported. RESULTS Because injections were placed in the intermediate gray matter, no animals displayed motor complications from the injury. Animals that received PBS injections showed no signs of histological damage, indicating that any damage seen in the QUIS-injected animals was a result of the QUIS itself and not the surgical process (Figure 1A). Eight QUIS-injected animals received nicotinamide treatment and four received saline (control) treatment. Representative histological sections from the nicotinamide treatment group compared with control group are shown in Figures 1B and C. Although the nicotinamide-treated cord does not show the same amount of neuronal loss, a strong inflammatory reaction is still evident. When the mean percentages of gray matter damage at the epicenter of injury were compared, there was significant difference between the saline- and nicotinamidetreated animals (63.84% damage vs % damage, respectively; p = 0.088; 95% CI = ÿ3.04 to 36.31) (Figure 2A). Also, when the total volumes of gray matter damage along the length of 975 mm were compared, there was a significant difference between the saline- and nicotinamide-treated animals (53.74% vs %, respectively; p = 0.026; 95% CI = 3.4 to 42.6) (Table 1, Figure 2B). Figure 2A shows that the area of damage at five of the six sections measured rostrally from the epicenter and one of the six sections measured caudally from the epicenter was significantly decreased by nicotinamide treatment. Rostral to the epicenter, there was a significant effect of distance from the epicenter on lesion volume in the nicotinamide group, because sections beyond 150 mm from the epicenter had significantly decreased damage compared with the epicenter (p = 0.02). No effect of distance from the epicenter was apparent caudally in the nicotinamide group or in either direction in the saline group. DISCUSSION In the present study, we used an excitotoxic model of SCI to examine the neuroprotective effects of nicotinamide. A transient increase of excitatory amino acids following SCI is viewed as contributing to a central cascade of secondary pathological changes following Figure 1. Representative sections (4X magnification) taken from the epicenter of injury of a sham-injected animal (A), a QUISinjected animal treated with saline (control, B), and one treated with nicotinamide (C). By using a cresyl violet stain, no evidence of neuronal damage is seen in the cord of the animal injected with phosphate-buffered saline (PBS). Although the nicotinamidetreated animal does not show cavitation due to neuronal loss as seen in the control animal, a narrowing of the intermediate gray matter and a robust inflammatory response are still evident (arrows).

4 128 Brewer and Hardin d NICOTINAMIDE NEUROPROTECTION IN SCI TABLE 1. Percentage of Gray Matter that Was Damaged along the 975-mm Length of Cord Examined in Each of the Nicotinamide-treated versus Saline-Treated Animals Nicotinamidetreated Saline-treated Mean 6 SD Figure 2. (A) Comparison of percentages of total gray matter damage at epicenter and adjacent sections between salineand nicotinamide-treated animals. The data are shown at 75- mm intervals for a total of 975 mm (*p \ 0.05). (B) Comparison of total lesion volumes along a 975-mm length of spinal cord between saline- and nicotinamide-treated animals. Treatment with nicotinamide reduced the total lesion volume by 23% (*p = 0.026; 95% CI = 3.40 to 42.6). In both cases, error bars represent the standard error of the mean. SCI 27,28 that ultimately result in neuronal loss, demyelination, and cavitation of the spinal cord. Previous studies have shown that intraspinal injection of the AMPA/metabotropic receptor agonist, QUIS, simulates this increase in excitatory amino acid and produces excitotoxic injury with pathological characteristics similar to those associated with ischemic and traumatic SCI. 26,29 Although a traumatic injury will likely produce a more rapidly progressing injury with the potential for more complex cellular events, the excitotoxic model will induce the same blood flow and neurochemical, inflammatory, electrophysiological, demyelinating, and necrotic changes as seen in traumatic models of SCI. 26,29 One consequence of this excitotoxicity is an increase in activity of NOS. 7,30,31 The resulting free radical cascades involving nitric oxide and peroxynitrite can lead to membrane and deoxyribonucleic acid (DNA) damage. 18,32,33 In addition, NO-induced insults have been shown to activate the nuclear enzyme PARP. Poly-ADP-ribose polymerase is a nuclear enzyme that serves to promote DNA repair pathways. 34 However, the intense polymer synthesis that results from its activation requires an excessive amount of energy so that excessive activation of PARP may result in subsequent adenosine triphosphate (ATP) depletion. 34 This loss of cellular energy, in the face of an existing ischemia (as seen after SCI) would be devastating to any neurons that may have survived the initial injury. In addition, PARP may serve to amplify acute excitotoxicity by impairing reuptake of excitatory neurotransmitters. 35 The increased release of excitatory amino acids after SCI, coupled with decreased reuptake secondary to PARP activation, may lead to additional neuronal damage. Studies have shown that inhibitors of PARP can successfully reduce the functional deficits that occur following experimental traumatic brain injury or stroke. 36 However, despite the similarity in the pathogenesis of injury between traumatic brain injury and spinal cord injury, little research has been done using PARP inhibitors as neuroprotective agents in acute SCI. Nicotinamide is a naturally occurring B-vitamin (B3) that possesses both NO and PARP inhibitory activity 22,23 and has been proven to be effective in providing neuroprotection against ischemic or traumatic injury of the brain Nicotinamide may work to correct the initial energy imbalance that is created by ischemia. In addition, evidence suggests that it can prevent both necrotic and apoptotic cell death after CNS injury 24 and work to attenuate lipid peroxidation. 19 When administered systemically, nicotinamide reduced the damage caused by a seven-day excitotoxic spinal cord injury as indicated by a 23% decrease in overall lesion volume in the length of cord measured. In contrast, treatment with nicotinamide had no significant effect on the epicenter of injury (Figure 2). Although the amount of neuronal loss appeared to be lessened by nicotinamide, a robust inflammatory response was still present at the epicenter. Although no quantification of specific cellular reactions at the lesion epicenter was done, the fact that nicotinamide was ineffective in this region suggests that the

5 ACAD EMERG MED d February 2004, Vol. 11, No. 2 d necessary substrate for action was not present (i.e., the delay in the time to treatment allowed for the development of a response that nicotinamide could not combat). If the nicotinamide had been given earlier, there might have been a difference seen at the epicenter. However, in a clinical setting, there would be a delay between the time of injury and the administration of any other neuroprotective agent. Because no drug has proved to be completely effective at protecting neurons against secondary cell death after SCI, it is likely that a cocktail approach of combining agents will be required to achieve optimal neuroprotection. On the basis of the histology of the current study, it appears that the addition of an anti-inflammatory agent to the nicotinamide would have had better success at the epicenter of injury. The greatest promise for the successful treatment of spinal cord injuries lies in the ability to interrupt the secondary injury that follows the initial insult, preventing the functional deficits that result from the death of spinal neurons. Major focused research efforts are needed to expedite the development of highly effective agents that will give the emergency physician the tools to provide protection to those who will suffer SCI in the future. CLINICAL RELEVANCE The results of the current study suggest that nicotinamide may have a place in the ultimate neuroprotective cocktail that will prevent secondary cell death after SCI. The ideal neuroprotective agent would be something that can be given empirically in the out-of-hospital setting. It must be easily administered and without deleterious side effects. Studies on nicotinamide have shown that it can be given systemically in high doses with few complications. 25 Therefore, nicotinamide could potentially be administered by first responders to a victim in whom spinal injury is suspected, especially when a lengthy extrication or prolonged transport time is involved. LIMITATIONS The current study used a single dose of nicotinamide to obtain some degree of neuroprotection. Future studies should examine the effects of multiple dosing on lesion volume. In addition, the mechanism of action of the nicotinamide was not established. It should be noted that there is no clinical evidence that the dose of 500 mg/kg would be tolerated in humans, because data suggest doses over 3 grams may be toxic. 25 Studies examining the effects of nicotinamide administration on PARP activation, NOS levels, excitatory amino acid release, etc., should be done to establish exactly what aspect of the secondary cascade is being affected by the treatment. The pharmacodynamics of nicotinamide protection of QUIS-induced injury should be examined by developing a dose response curve for nicotinamide. It also should be determined if the route of administration is related to its effectiveness and if there is a therapeutic window for its administration. Although no statistically significant difference was found in the damage at the epicenter of injury between the two groups, this result is likely due to the small number of animals in each group. The group size was selected on the basis of previous neuroprotective studies using similar groups. 17 A power analysis reveals that the current number of animals provides 50 80% power to detect a difference in the values obtained. Increasing the control group to eight animals would increase the power to 90 95%. This finding suggests that the nicotinamide may be effective in reducing the epicenter of the lesion as well. Larger studies should be performed to address this possibility. Finally, although a specific length of cord was examined for this study, significant gray matter damage appears to extend beyond that measured length. If this is the case, the effects of the nicotinamide may, in fact, be underestimated by this restriction. Although it is expected that the amount of damage would decrease as the distance from the epicenter increases, this trend was not seen statistically in the saline group as it was in the nicotinamide group. CONCLUSIONS Systemic nicotinamide provides some degree of neuroprotection in an animal model of spinal cord injury by limiting the rostro-caudal extent of the damage. Potentially, nicotinamide could be used alone or along with other pharmacological agents to help preserve the integrity of the neural tissue and neurological function in patients who have suffered spinal cord injury. References 1. Burke DA, Linden RD, Zhang YP, Maiste AC, Shields CB. Incidence rates and populations at risk for spinal cord injury: a regional study. 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