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Introduction of calpain inhibitors in traumatic brain injury: A novel approach?
Medical Hypotheses 79 (2012) 358–360
Contents lists available at SciVerse ScienceDirect
Medical Hypotheses journal homepage: www.elsevier.com/locate/mehy
Introduction of calpain inhibitors in traumatic brain injury: A novel approach? Marina Bralic a,⇑, Valter Stemberga b, Sanja Stifter c a
Department of Neurology, Clinical Hospital Centre Rijeka, Cambierieva 17 Rijeka, Croatia Department of Forensic Medicine and Criminalistics, School of Medicine, University of Rijeka, Croatia c Department of Pathology, School of Medicine, University of Rijeka, Croatia b
a r t i c l e
i n f o
Article history: Received 19 March 2012 Accepted 23 May 2012
a b s t r a c t Traumatic brain injury (TBI) is a major cause of death and disability throughout the world. In recent years, researchers focused on the pathological significance of calcium accumulation in the brain after TBI. Neuronal calcium homeostasis disturbances may result in the activation of calpain a ubiquitous calcium-sensitive protease. The calpain family has a well-established causal role in neuronal cell death following acute brain injury: their activation has been observed to progressively increase after either contusive or diffuse brain trauma in animals, suggesting calpain to be a mediator of early neuronal damage. We hypothesize that pretreatment with the calpain inhibitors in population at objective risk (military soldiers’ pre combat) in appropriate dose would open therapeutic time window expected to prevent and reduce extensive brain damage by providing optimal TBI neuroprotection. Additional therapeutic strategy for TBI, based on calpain modulating actions such as pretreatment with calpain inhibitors has been proposed. Since calpain overexpression has been well established in acute neuronal injury and further subsequent neurodegeneration, from a clinical viewpoint, we speculate that if this hypothesis proves correct pretreatment inhibitors introduction may become a therapeutic option for different brain pathologies to be approached and treated with. Ó 2012 Elsevier Ltd. All rights reserved.
Introduction Traumatic brain injury (TBI) is a major direct and indirect cause of death and disability throughout the world. It is a serious neuronal disorder commonly caused by car accidents, sports related events or violence [1]. Solely in the United States, approximately 52,000 people die and 80,000 people suffer permanent disability as the consequence of TBI. TBI accounts for 25% of all combat casualties as the leading cause of death (approaching 50% incidence) among wounded soldiers [2,3]. TBI represents a major public health problem both in military service and civil life. Preventive measures are highly recommended as stated by some authors in attempt to reduce the risk and number of TBI cases. The primary injury to the brain initiates a secondary injury process that spreads through multiple molecular mechanisms in the pathogenesis of TBI. TBI is also the leading cause of death and disability in young population less than 24 years old [4]. The Centers for Disease Control and Prevention (CDC) estimate that at least 5.3 million Americans currently have a long-term
⇑ Corresponding author. Tel.: +385 51 658 311; fax: +385 51 334 606. E-mail address: [email protected] (M. Bralic). 0306-9877/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mehy.2012.05.034
requirement for assistance with daily living activities as a result of TBI [5]. The events after TBI are generalized into four categories and we are focusing on primary injury that disrupts brain tissues while all discussed throughout the paper potentially impacts the secondary injury and neurodegeneration following to some extent TBI [1]. Despite its prevalence and severity effective treatment has not been developed yet for this form of injury. Many investigators have pointed out that the absence of reliable brain injury biochemical markers could have contributed to these failures [6]. There are currently no approved drug treatments for acute TBI injury although a large number of clinical trials are investigating potential therapies. In recent years, main stream research has focused on the pathological significance of calcium accumulation in the brain after TBI. Disturbances in neuronal calcium homeostasis may result in the activation of calpain a ubiquitous calcium-sensitive protease [7]. Calpain family is at the crossroad between physiology and pathology: they are essential for normal physiologic neuronal function and their transient activation triggers numerous cell signaling and remodeling events in brain parenchyma [8]. However, alterations in calcium homeostasis lead to persistent, pathologic activation of calpain in a number of neurodegenerative diseases. It has been confirmed that sustained calpain activation
M. Bralic et al. / Medical Hypotheses 79 (2012) 358–360
produced by trauma is associated with neuron death and axonal degeneration in multiple models of TBI. Further, the calpain family has a well-established causal role in neuronal cell death following acute brain injury: their activation has been observed within minutes to hours after either contusive or diffuse brain trauma in animals, suggesting calpain as an early mediator of neuronal damage [9,10]. Hypothesis We propose additional therapeutic strategy for TBI, based on calpain modulating actions such as pretreatment with calpain inhibitors. Our hypothesize is that the pretreatment with calpain inhibitors in population at risk (combat military) in appropriate dose and therapeutic time window (pre combat) may be expected to prevent extensive brain damage and provide optimal neuroprotection of TBI when it is expected to occur significantly. Discussion Current state of art recognizes the complexity and significant contribution of calpain induced molecular pathways in the settings of brain injury, though clinical appreciation and therapeutic impact is still limited. Calpain inhibition has emerged as an attractive prophylactic therapeutic approach since the correlation between prolonged calpain activation and trauma-induced neurodegeneration is well established and documented [11]. Additionally, observed relatively low basal levels of calpain activity in unaffected brain are suggestive that short-term inhibition of calpain secretion when there is an objectively higher risk for brain injury to be expected in near future may yield with fewer collateral since their effects antagonize with other common targets such as glutamate receptors. So far, the majority of studies have been conducted on animal models, aimed to reveal the role of calpain in the treatment of TBI. They confirmed that inhibition of calpain after trauma may attenuate cell death and improve functional outcome [12]. But short pretraumatic calpain inhibition as we suggest might be justify in cases of expected trauma to proceed. The cell death mainly induced by intrinsic apoptotic pathways has been recognized as dominant. This is the major cause of prolonged and by nature chronic, sub chronic as well as sub lethal neuronal damage leading to the progressive neurodegeneration [1]. Consequently, it has been shown that calpain inhibition is associated with functional decrease and behavioral deficits of manifested axonal pathology due to cell death in animal models of TBI. Post-traumatically delivered calpain inhibition reduces degradation of multiple neuronal or non neuronal substrate proteins such as cytoskeletal proteins and signaling molecules, reducing neurodegeneration after brain injury but the extent of neuronal deficit reduction significantly correlates with real time period of calpain inhibitor introduction [11]. This is actually well functional in animal models but our opinion is that human population in significant risk of brain injury should be premedicated as prophylaxis directly using specific calpain inhibitors or indirectly via other targets that modulate intracellular free calcium levels. Short term effects and fast half life degradation point of calpain inhibitors predispose them to be optimal precondition in population where we expect brain injury to occur. The effectiveness of pretreatment therapy has not been explored yet, may be due to assumption that the physiological roles
359
of calpain might be in jeopardy. But current vivid development in this field gives us impression that the time to come forward and step out with novel approaches should not be dismissed. We are aware that use of drugs calpain inhibitors is a source of concern because inhibition might affect its physiological function, causing the adverse side effects. According to studies conducted on genetically modified mice overexpressing the endogenous inhibitor calpastatin this seems unlikely to happen. Namely, the mice showed normal cytoskeletal components and synaptic markers as well as undisturbed development, fertility, morphology, motility, and life span when in normal conditions [13]. Thus, it is likely that calpain has a regulatory or signaling function in cells rather than a digestive function such as the lysosomal proteases, and therefore, undesired side effects by calpain inhibitors might be of limited potential. Furthermore, the calpain inhibitors E64 and BDA-410 showed no effects on synaptic function, cognitive behavior, and locomotion in mice used in model experiments of Alzheimer’s disease. In fact, calpain is likely to cleave polypeptides at a limited number of sites, leaving large, often catalytically active fragments [14]. Previous studies have demonstrated neuroprotective effects of postischemic calpain inhibitor therapy in animal models [15]. These points out cumulative effect of apoptotic cell death in progressive neurodegenerative milieu. Calpain inhibitor AK 295, tested in a murine model has confirmed efficacy on spinal cord structure, neurological function, and apoptosis after spinal cord injury [16]. Frederick and colleagues have demonstrated the effectiveness of delayed calpain inhibition after transient forebrain ischemia on rat model [17]. A study conducted by Kawamura et al. showed that calpain inhibitor MDL 28170 protects hypoxic-ischemic brain injury in neonatal rats by inhibition of both apoptosis and necrosis [18]. A recent study has demonstrated that pretreatment with the M1-specific muscarinic cholinergic receptor antagonist dicyclomine attenuated 145 kDa spectrin BDP levels in the CSF at 24 h after moderate lateral fluid percussion in rats [19]. Clinical implications and future directions We suggest novel potential use of calpain inhibitors, but the mechanism of brain protection remains to be elucidated, and several key questions are to be addressed such as whether a calpain inhibitor therapy results in sustained neuroprotection and its optimal duration, time point of application or solely dosage determination necessary for perquisite result. Since calpain overexpression has been well established in acute neuronal injury and further subsequent neurodegeneration, from a clinical viewpoint, we speculate that if this hypothesis proves to be correct pretreatment therapy may become a therapeutic option for different brain pathologies in acute and chronic progressive stage to be treated with. Conflicts of interest statement None declared. References [1] Ray SK, Dixon CE, Banik NL. Molecular mechanisms in the pathogenesis of traumatic brain injury. Histol Histopathol 2002;17:1137–52. [2] Meyer KS, Marion DW, Coronel H, Jaffee MS. Combat-related traumatic brain injury and its implications to military healthcare. Psychiatr Clin North Am 2010;33:783–96. [3] Warden D. Military TBI during the Iraq and Afghanistan wars. J Head Trauma Rehabil 2006;21:398–402.
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M. Bralic et al. / Medical Hypotheses 79 (2012) 358–360
[4] Langlois JA, Rutland-Brown W, Wald MM. The epidemiology and impact of traumatic brain injury: a brief overview. J Head Trauma Rehabil 2006;21: 375–8. [5] Centers for Disease Control and Prevention (CDC). Nonfatal traumatic brain injuries from sports and recreation activities – United States, 2001–2005. MMWR Morb Mortal Wkly Rep 2007;56:733–737. [6] Choi SC, Bullock R. Design and statistical issues in multicenter trials of severe head injury. Neurol Res 2001;23:190–2. [7] Goll DE, Thompson VF, Li H, Wei W, Cong J. The calpain system. Physiol Rev 2003;83:731–801. [8] Vosler PS, Brennan CS, Chen J. Calpain-mediated signaling mechanisms in neuronal injury and neurodegeneration. Mol Neurobiol 2008;38:78–100. [9] Liu J, Liu MC, Wang KK. Calpain in the CNS: from synaptic function to neurotoxicity. Sci Signal 2008;1:re1. [10] Marklund N, Hillered L. Animal modelling of traumatic brain injury in preclinical drug development: where do we go from here? Br J Pharmacol 2011;164:1207–29. [11] Carragher NO. Calpain inhibition: a therapeutic strategy targeting multiple disease states. Curr Pharm Des 2006;12:615–38. [12] Kupina NC, Nath R, Bernath EE, et al. The novel calpain inhibitor SJA6017 improves functional outcome after delayed administration in a mouse model of diffuse brain injury. J Neurotrauma 2001;18:1229–40.
[13] Higuchi M, Tomioka M, Takano J, et al. Distinct mechanistic roles of calpain and caspase activation in neurodegeneration as revealed in mice overexpressing their specific inhibitors. J Biol Chem 2005;280:15229–37. [14] Trinchese F, Fa’ M, Liu S, et al. Inhibition of calpains improves memory and synaptic transmission in a mouse model of Alzheimer disease. J Clin Invest 2008;118:2796–807. [15] Anagli J, Han Y, Stewart L, et al. A novel calpastatin-based inhibitor improves postischemic neurological recovery. Biochem Biophys Res Commun 2009;385: 94–9. [16] Colak A, Kaya M, Karaog˘lan A, et al. Calpain inhibitor AK 295 inhibits calpaininduced apoptosis and improves neurologic function after traumatic spinal cord injury in rats. Neurocirugia (Astur) 2009;20:245–54. [17] Frederick JR, Chen Z, Bevers MB, Ingleton LP, Ma M, Neumar RW. Neuroprotection with delayed calpain inhibition after transient forebrain ischemia. Crit Care Med 2008;36(Suppl. 11):S481–5. [18] Kawamura M, Nakajima W, Ishida A, Ohmura A, Miura S, Takada G. Calpain inhibitor MDL 28170 protects hypoxic-ischemic brain injury in neonatal rats by inhibition of both apoptosis and necrosis. Brain Res 2005;1037:59–69. [19] Cox CD, West EJ, Liu MC, Wang KK, Hayes RL, Lyeth BG. Dicyclomine, an M1 muscarinic antagonist, reduces biomarker levels, but not neuronal degeneration, in fluid percussion brain injury. J Neurotrauma 2008;25: 1355–65.
Contents lists available at SciVerse ScienceDirect
Medical Hypotheses journal homepage: www.elsevier.com/locate/mehy
Introduction of calpain inhibitors in traumatic brain injury: A novel approach? Marina Bralic a,⇑, Valter Stemberga b, Sanja Stifter c a
Department of Neurology, Clinical Hospital Centre Rijeka, Cambierieva 17 Rijeka, Croatia Department of Forensic Medicine and Criminalistics, School of Medicine, University of Rijeka, Croatia c Department of Pathology, School of Medicine, University of Rijeka, Croatia b
a r t i c l e
i n f o
Article history: Received 19 March 2012 Accepted 23 May 2012
a b s t r a c t Traumatic brain injury (TBI) is a major cause of death and disability throughout the world. In recent years, researchers focused on the pathological significance of calcium accumulation in the brain after TBI. Neuronal calcium homeostasis disturbances may result in the activation of calpain a ubiquitous calcium-sensitive protease. The calpain family has a well-established causal role in neuronal cell death following acute brain injury: their activation has been observed to progressively increase after either contusive or diffuse brain trauma in animals, suggesting calpain to be a mediator of early neuronal damage. We hypothesize that pretreatment with the calpain inhibitors in population at objective risk (military soldiers’ pre combat) in appropriate dose would open therapeutic time window expected to prevent and reduce extensive brain damage by providing optimal TBI neuroprotection. Additional therapeutic strategy for TBI, based on calpain modulating actions such as pretreatment with calpain inhibitors has been proposed. Since calpain overexpression has been well established in acute neuronal injury and further subsequent neurodegeneration, from a clinical viewpoint, we speculate that if this hypothesis proves correct pretreatment inhibitors introduction may become a therapeutic option for different brain pathologies to be approached and treated with. Ó 2012 Elsevier Ltd. All rights reserved.
Introduction Traumatic brain injury (TBI) is a major direct and indirect cause of death and disability throughout the world. It is a serious neuronal disorder commonly caused by car accidents, sports related events or violence [1]. Solely in the United States, approximately 52,000 people die and 80,000 people suffer permanent disability as the consequence of TBI. TBI accounts for 25% of all combat casualties as the leading cause of death (approaching 50% incidence) among wounded soldiers [2,3]. TBI represents a major public health problem both in military service and civil life. Preventive measures are highly recommended as stated by some authors in attempt to reduce the risk and number of TBI cases. The primary injury to the brain initiates a secondary injury process that spreads through multiple molecular mechanisms in the pathogenesis of TBI. TBI is also the leading cause of death and disability in young population less than 24 years old [4]. The Centers for Disease Control and Prevention (CDC) estimate that at least 5.3 million Americans currently have a long-term
⇑ Corresponding author. Tel.: +385 51 658 311; fax: +385 51 334 606. E-mail address: [email protected] (M. Bralic). 0306-9877/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mehy.2012.05.034
requirement for assistance with daily living activities as a result of TBI [5]. The events after TBI are generalized into four categories and we are focusing on primary injury that disrupts brain tissues while all discussed throughout the paper potentially impacts the secondary injury and neurodegeneration following to some extent TBI [1]. Despite its prevalence and severity effective treatment has not been developed yet for this form of injury. Many investigators have pointed out that the absence of reliable brain injury biochemical markers could have contributed to these failures [6]. There are currently no approved drug treatments for acute TBI injury although a large number of clinical trials are investigating potential therapies. In recent years, main stream research has focused on the pathological significance of calcium accumulation in the brain after TBI. Disturbances in neuronal calcium homeostasis may result in the activation of calpain a ubiquitous calcium-sensitive protease [7]. Calpain family is at the crossroad between physiology and pathology: they are essential for normal physiologic neuronal function and their transient activation triggers numerous cell signaling and remodeling events in brain parenchyma [8]. However, alterations in calcium homeostasis lead to persistent, pathologic activation of calpain in a number of neurodegenerative diseases. It has been confirmed that sustained calpain activation
M. Bralic et al. / Medical Hypotheses 79 (2012) 358–360
produced by trauma is associated with neuron death and axonal degeneration in multiple models of TBI. Further, the calpain family has a well-established causal role in neuronal cell death following acute brain injury: their activation has been observed within minutes to hours after either contusive or diffuse brain trauma in animals, suggesting calpain as an early mediator of neuronal damage [9,10]. Hypothesis We propose additional therapeutic strategy for TBI, based on calpain modulating actions such as pretreatment with calpain inhibitors. Our hypothesize is that the pretreatment with calpain inhibitors in population at risk (combat military) in appropriate dose and therapeutic time window (pre combat) may be expected to prevent extensive brain damage and provide optimal neuroprotection of TBI when it is expected to occur significantly. Discussion Current state of art recognizes the complexity and significant contribution of calpain induced molecular pathways in the settings of brain injury, though clinical appreciation and therapeutic impact is still limited. Calpain inhibition has emerged as an attractive prophylactic therapeutic approach since the correlation between prolonged calpain activation and trauma-induced neurodegeneration is well established and documented [11]. Additionally, observed relatively low basal levels of calpain activity in unaffected brain are suggestive that short-term inhibition of calpain secretion when there is an objectively higher risk for brain injury to be expected in near future may yield with fewer collateral since their effects antagonize with other common targets such as glutamate receptors. So far, the majority of studies have been conducted on animal models, aimed to reveal the role of calpain in the treatment of TBI. They confirmed that inhibition of calpain after trauma may attenuate cell death and improve functional outcome [12]. But short pretraumatic calpain inhibition as we suggest might be justify in cases of expected trauma to proceed. The cell death mainly induced by intrinsic apoptotic pathways has been recognized as dominant. This is the major cause of prolonged and by nature chronic, sub chronic as well as sub lethal neuronal damage leading to the progressive neurodegeneration [1]. Consequently, it has been shown that calpain inhibition is associated with functional decrease and behavioral deficits of manifested axonal pathology due to cell death in animal models of TBI. Post-traumatically delivered calpain inhibition reduces degradation of multiple neuronal or non neuronal substrate proteins such as cytoskeletal proteins and signaling molecules, reducing neurodegeneration after brain injury but the extent of neuronal deficit reduction significantly correlates with real time period of calpain inhibitor introduction [11]. This is actually well functional in animal models but our opinion is that human population in significant risk of brain injury should be premedicated as prophylaxis directly using specific calpain inhibitors or indirectly via other targets that modulate intracellular free calcium levels. Short term effects and fast half life degradation point of calpain inhibitors predispose them to be optimal precondition in population where we expect brain injury to occur. The effectiveness of pretreatment therapy has not been explored yet, may be due to assumption that the physiological roles
359
of calpain might be in jeopardy. But current vivid development in this field gives us impression that the time to come forward and step out with novel approaches should not be dismissed. We are aware that use of drugs calpain inhibitors is a source of concern because inhibition might affect its physiological function, causing the adverse side effects. According to studies conducted on genetically modified mice overexpressing the endogenous inhibitor calpastatin this seems unlikely to happen. Namely, the mice showed normal cytoskeletal components and synaptic markers as well as undisturbed development, fertility, morphology, motility, and life span when in normal conditions [13]. Thus, it is likely that calpain has a regulatory or signaling function in cells rather than a digestive function such as the lysosomal proteases, and therefore, undesired side effects by calpain inhibitors might be of limited potential. Furthermore, the calpain inhibitors E64 and BDA-410 showed no effects on synaptic function, cognitive behavior, and locomotion in mice used in model experiments of Alzheimer’s disease. In fact, calpain is likely to cleave polypeptides at a limited number of sites, leaving large, often catalytically active fragments [14]. Previous studies have demonstrated neuroprotective effects of postischemic calpain inhibitor therapy in animal models [15]. These points out cumulative effect of apoptotic cell death in progressive neurodegenerative milieu. Calpain inhibitor AK 295, tested in a murine model has confirmed efficacy on spinal cord structure, neurological function, and apoptosis after spinal cord injury [16]. Frederick and colleagues have demonstrated the effectiveness of delayed calpain inhibition after transient forebrain ischemia on rat model [17]. A study conducted by Kawamura et al. showed that calpain inhibitor MDL 28170 protects hypoxic-ischemic brain injury in neonatal rats by inhibition of both apoptosis and necrosis [18]. A recent study has demonstrated that pretreatment with the M1-specific muscarinic cholinergic receptor antagonist dicyclomine attenuated 145 kDa spectrin BDP levels in the CSF at 24 h after moderate lateral fluid percussion in rats [19]. Clinical implications and future directions We suggest novel potential use of calpain inhibitors, but the mechanism of brain protection remains to be elucidated, and several key questions are to be addressed such as whether a calpain inhibitor therapy results in sustained neuroprotection and its optimal duration, time point of application or solely dosage determination necessary for perquisite result. Since calpain overexpression has been well established in acute neuronal injury and further subsequent neurodegeneration, from a clinical viewpoint, we speculate that if this hypothesis proves to be correct pretreatment therapy may become a therapeutic option for different brain pathologies in acute and chronic progressive stage to be treated with. Conflicts of interest statement None declared. References [1] Ray SK, Dixon CE, Banik NL. Molecular mechanisms in the pathogenesis of traumatic brain injury. Histol Histopathol 2002;17:1137–52. [2] Meyer KS, Marion DW, Coronel H, Jaffee MS. Combat-related traumatic brain injury and its implications to military healthcare. Psychiatr Clin North Am 2010;33:783–96. [3] Warden D. Military TBI during the Iraq and Afghanistan wars. J Head Trauma Rehabil 2006;21:398–402.
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M. Bralic et al. / Medical Hypotheses 79 (2012) 358–360
[4] Langlois JA, Rutland-Brown W, Wald MM. The epidemiology and impact of traumatic brain injury: a brief overview. J Head Trauma Rehabil 2006;21: 375–8. [5] Centers for Disease Control and Prevention (CDC). Nonfatal traumatic brain injuries from sports and recreation activities – United States, 2001–2005. MMWR Morb Mortal Wkly Rep 2007;56:733–737. [6] Choi SC, Bullock R. Design and statistical issues in multicenter trials of severe head injury. Neurol Res 2001;23:190–2. [7] Goll DE, Thompson VF, Li H, Wei W, Cong J. The calpain system. Physiol Rev 2003;83:731–801. [8] Vosler PS, Brennan CS, Chen J. Calpain-mediated signaling mechanisms in neuronal injury and neurodegeneration. Mol Neurobiol 2008;38:78–100. [9] Liu J, Liu MC, Wang KK. Calpain in the CNS: from synaptic function to neurotoxicity. Sci Signal 2008;1:re1. [10] Marklund N, Hillered L. Animal modelling of traumatic brain injury in preclinical drug development: where do we go from here? Br J Pharmacol 2011;164:1207–29. [11] Carragher NO. Calpain inhibition: a therapeutic strategy targeting multiple disease states. Curr Pharm Des 2006;12:615–38. [12] Kupina NC, Nath R, Bernath EE, et al. The novel calpain inhibitor SJA6017 improves functional outcome after delayed administration in a mouse model of diffuse brain injury. J Neurotrauma 2001;18:1229–40.
[13] Higuchi M, Tomioka M, Takano J, et al. Distinct mechanistic roles of calpain and caspase activation in neurodegeneration as revealed in mice overexpressing their specific inhibitors. J Biol Chem 2005;280:15229–37. [14] Trinchese F, Fa’ M, Liu S, et al. Inhibition of calpains improves memory and synaptic transmission in a mouse model of Alzheimer disease. J Clin Invest 2008;118:2796–807. [15] Anagli J, Han Y, Stewart L, et al. A novel calpastatin-based inhibitor improves postischemic neurological recovery. Biochem Biophys Res Commun 2009;385: 94–9. [16] Colak A, Kaya M, Karaog˘lan A, et al. Calpain inhibitor AK 295 inhibits calpaininduced apoptosis and improves neurologic function after traumatic spinal cord injury in rats. Neurocirugia (Astur) 2009;20:245–54. [17] Frederick JR, Chen Z, Bevers MB, Ingleton LP, Ma M, Neumar RW. Neuroprotection with delayed calpain inhibition after transient forebrain ischemia. Crit Care Med 2008;36(Suppl. 11):S481–5. [18] Kawamura M, Nakajima W, Ishida A, Ohmura A, Miura S, Takada G. Calpain inhibitor MDL 28170 protects hypoxic-ischemic brain injury in neonatal rats by inhibition of both apoptosis and necrosis. Brain Res 2005;1037:59–69. [19] Cox CD, West EJ, Liu MC, Wang KK, Hayes RL, Lyeth BG. Dicyclomine, an M1 muscarinic antagonist, reduces biomarker levels, but not neuronal degeneration, in fluid percussion brain injury. J Neurotrauma 2008;25: 1355–65.