Concentration of brain free magnesium following severe brain injury correlates with neurologic motor outcome

Recent studies have shown that brain intracellular free magnesium concentration significantly declines following mild to severe, focal and diffuse traumatic brain injury. However, little is known about how this decline or its attenuation by magnesium salts relates to neurologic outcome. This study uses phosphorus magnetic resonance spectroscopy and rotarod tests to characterise the relationship between brain free magnesium concentration and neurologic motor scores following severe, diffuse traumatic brain injury in rats. An intravenous bolus of MgSO(4) or MgCl(2) (100 mumoles/kg) at 30 min following brain injury significantly attenuated the postinjury brain free magnesium decline. This improved magnesium homeostasis was sustained for the entire postinjury monitoring period (1 week). There was an associated significant improvement in neurologic motor function in magnesium treated rats. Moreover, the brain free magnesium concentration over the one week period was linearly correlated with the neurologic motor function (r=0.70; P < 0.001) as assessed on a daily basis. We propose that brain free magnesium concentration may be used as a prognostic indicator of neurologic motor function after traumatic brain injury.     

Efficacy of competitive vs noncompetitive blockade of the NMDA channel following traumatic brain injury

N-methyl-d-aspartate (NMDA) receptor antagonists have been demonstrated widely to be neuroprotective in cerebral ischemia, hypoxia, and traumatic brain injury. However, although noncompetitive NMDA antagonists have typically proven efficacious under all of these conditions, competitive antagonists have not been shown to be beneficial following moderate traumatic brain injury. The present study has used phosphorus magnetic resonance spectroscopy ([³¹P]MRS) to examine the effects of the competitive antagonist cis-4-(phosphonomethyl) piperidine-2-carboxylic acid (CGS-19755) and the noncompetitive antagonist dextromethorphan on biochemical outcome following fluid percussion-induced traumatic brain injury in rats. Five minutes prior to induction of moderate (2.8±0.2 atm) fluid percussion brain injury, animals received either CGS-19755 (10 mg/kg iv), dextromethorphan (10 mg/kg iv), or equal volume saline vehicle. [³¹P]MRS spectra were then acquired for 4 h post-trauma and intracellular pH, free magnesium concentration, cytosolic phosphorylation potential, and oxidative capacity determined. Both CGS-19755-treated animals and saline treated controls demonstrated significant and sustained declines in intracellular free magnesium concentration and bioenergetic status following trauma. In contrast, administration of dextromethorphan significantly attenuated free magnesium decline and improved bioenergetic state during the post-traumatic monitoring period. These results suggest that the neuroprotective actions of NMDA antagonists following traumatic brain injury are associated with attenuation of free magnesium decline and that such actions seem to be preferentially mediated by noncompetitive blockers.     

A preliminary histopathological study of the effect of agmatine on diffuse brain injury in rats

The present study evaluates the effects of agmatine on histopathological damage following traumatic injury using a clinically relevant model of diffuse brain injury. A total of 27 male Sprague-Dawley rats weighing 200-225g were anaesthetised and subjected to head trauma using Marmarou's impact-acceleration model. The rats were then separated into two main groups: one was treated with agmatine and the other with saline for up to 4 days immediately after head trauma. Rats from both groups were killed 1, 3 or 8 days post-injury. The brains were examined histopathologically and scored according to the axonal, neuronal and vascular changes associated with diffuse brain injury. There were no significant differences between the groups at 1day or 3 days after trauma, but evaluation after 8 days revealed a significant improvement in the group treated with agmatine. Our data indicate that agmatine has a beneficial effect in diffuse brain injury and should be trialled for therapeutic use in the management of this condition.     

Traumatic spinal cord injury in rabbits decreases intracellular free magnesium concentration as measured by 31P MRS

The mechanisms by which traumatic injury to the central nervous system cause irreversible tissue damage remain speculative. Recent reports suggest that a decrease in tissue total and free Mg2+ concentration may be an important factor in the development of such injury after experimental brain trauma. Although total Mg changes have been reported following spinal cord trauma, no studies have examined spinal cord-free Mg2+. In the present study, we have used phosphorus magnetic resonance spectroscopy to determine intracellular free Mg2+ concentration and atomic absorption spectrophotometry to measure total tissue Mg concentration in rabbit spinal cord prior to and following impact trauma. We report that intracellular free Mg2+ concentration decreases from a pre-injury value of 0.80 +/- 0.12 mM (mean +/- S.E.M.) to 0.31 +/- 0.05 mM at 2 h post-trauma. Following injury there was an associated decrease in total tissue Mg and K concentration, but no alterations in tissue Na or water content.     

The pathobiology of moderate diffuse traumatic brain injury as identified using a new experimental model of injury in rats

Experimental models of traumatic brain injury have been developed to replicate selected aspects of human head injury, such as contusion, concussion, and/or diffuse axonal injury. Although diffuse axonal injury is a major feature of clinical head injury, relatively few experimental models of diffuse traumatic brain injury (TBI) have been developed, particularly in smaller animals such as rodents. Here, we describe the pathophysiological consequences of moderate diffuse TBI in rats generated by a newly developed, highly controlled, and reproducible model. This model of TBI caused brain edema beginning 20 min after injury and peaking at 24 h post-trauma, as shown by wet weight/dry weight ratios and diffusion-weighted magnetic resonance imaging. Increased permeability of the blood-brain barrier was present up to 4 h post-injury as evaluated using Evans blue dye. Phosphorus magnetic resonance spectroscopy showed significant declines in brain-free magnesium concentration and reduced cytosolic phosphorylation potential at 4 h post-injury. Diffuse axonal damage was demonstrated using manganese-enhanced magnetic resonance imaging, and intracerebral injection of a fluorescent vital dye (Fluoro-Ruby) at 24-h and 7-day post-injury. Morphological evidence of apoptosis and caspase-3 activation were also found in the cerebral hemisphere and brainstem at 24 h after trauma. These results show that this model is capable of reproducing major biochemical and neurological changes of diffuse clinical TBI.     

Temporal characterisation of pro- and anti-apoptotic mechanisms following diffuse traumatic brain injury in rats.

Few studies have characterised apoptosis in a brain injury model that causes a significant degree of diffuse axonal injury. Such characterisation is essential from a clinical viewpoint since diffuse axonal injury is a major component of human head injury. The present study therefore, examines the expression of active and proactive caspase-3, and the bax, bcl-2 and bcl-x members of the bcl-2 family, to characterise the temporal profile of apoptosis in a model of traumatic brain injury in rats that produces significant diffuse axonal injury. Pentobarbital anaesthetised male Sprague-Dawley rats were injured using the 2m impact-acceleration model of diffuse traumatic brain injury. After injury, diffuse trauma resulted in an increased bax expression followed by induction of caspase-3. The increase in caspase-3 was simultaneous with an increase in anti-apoptotic bcl-2 expression. Bcl-x levels were increased after induction of caspase-3 and the increased levels of bcl-x were sustained to the end of the 5-day observation period. Increased active caspase-3 expression was associated with the appearance of TUNEL positive cells. These cells were detected in different brain regions at different times, with some regions showing no apoptotic cells until 3 days after injury. No TUNEL positive cells were detected at 7 and 14 days after injury. DNA electrophoresis confirmed that DNA fragmentation was maximal at 3 days after injury. Increased active caspase-3 levels were also significantly correlated with increased bcl-2 levels (r=0.80; P<0.001) suggesting that the apoptotic cascade after diffuse traumatic brain injury is a carefully controlled cellular homeostatic response. Pharmacological manipulation of this balance may offer a therapeutic approach for preventing cell death and improving outcome after diffuse traumatic brain injury.     

Accumulation of amyloid beta and tau and the formation of neurofilament inclusions following diffuse brain injury in the pig.

Brain trauma in humans increases the risk for developing Alzheimer disease (AD) and may induce the acute formation of AD-like plaques containing amyloid beta (A beta). To further explore the potential link between brain trauma and neurodegeneration, we conducted neuropathological studies using a pig model of diffuse brain injury. Brain injury was induced in anesthetized animals via nonimpact head rotational acceleration of 110 degrees over 20 ms in the coronal plane (n = 15 injured, n = 3 noninjured). At 1, 3, 7, and 10 days post-trauma, control and injured animals were euthanized and immunohistochemical analysis was performed on brain sections using antibodies specific for A beta, beta-amyloid precursor protein (betaPP), tau, and neurofilament (NF) proteins. In addition to diffuse axonal pathology, we detected accumulation of A beta and tau that colocalized with immunoreactive betaPP and NF in damaged axons throughout the white matter in all injured animals at 3-10 days post-trauma. In a subset of brain injured animals, diffuse A beta-containing plaque-like profiles were found in both the gray and white matter, and accumulations of tau and NF rich inclusions were observed in neuronal perikarya. These results show that this pig model of diffuse brain injury is characterized by accumulations of proteins that also form pathological aggregates in AD and related neurodegenerative diseases.     

Nitric oxide levels in rat cortex,hippocampus, cerebellum,and brainstem after impact acceleration head injury

Nitric oxide (NO) is a potential mediator of secondary brain injury in the settings of cerebral ischemia and inflammation. Traumatic brain injury (TBI) alters the levels of stable end products of NO metabolism. We investigated these changes and attempted to identify brain regions that were unique with regard to NO production in the period immediately after TBI. The experiment involved assaying nitrite-nitrate concentrations in the rat cortex, cerebellum, hippocampus, and brainstem after impact-acceleration head injury. Five rats comprised the sham-operated (control) group, five sustained mild head injury (MHI), and five sustained severe head injury (SHI). There was a uniform decline in the tissue concentrations of NO metabolites in all four brain regions in both injured groups. There were no significant differences in the concentrations of NO metabolites among the various sites tested in the MHI group; however, there appeared to be a relationship between degree of decline in NO levels and amount of trauma sustained by a given region in the SHI group. In these rats, NO dropped to the lowest levels in the brain region where the direct trauma was most severe. The results suggest that nitrite-nitrate levels in these four brain regions fall below normal in the first 5 min after impact trauma. This decrease may, in part, be related to reduced activity of all nitric oxide synthase isoforms, which would cause a drop in the levels of NO metabolites. We believe that this decline may be linked to, and may even cause, the global decrease in cerebral blood flow that occurs in the initial stages of TBI.     

Traumatic spinal cord injury in rabbits decreases intracellular free magnesium concentration as measured by P MRS

The mechanisms by which traumatic injury to the central nervous system cause irreversible tissue damage remain speculative. Recent reports suggest that a decrease in tissue total and free Mg²⁺ concentration may be an important factor in the development of such injury after experimental brain trauma. Although total Mg changes have been reproted following spinal cord trauma, no studies have examined spinal cord-free Mg²⁺. In the present study, we have used phosphorus magnetic resonance spectroscopy to determine intracellular free Mg²⁺ concentration and atomic absorption spectrophotometry to measure total tissue Mg concentration in rabbit spinal cord prior to and following impact trauma. We report that intracellular free Mg²⁺ concentration decreases from a pre-injury value of 0.80 ± 0.12mM (mean ±S.E.M.) to 0.31 ± 0.05mM at 2 h post-trauma. Following injury there was a associated decrease in total tissue Mg and K concentration, but no alterations in tissue Na or water content.     

Neurogenic inflammation is associated with development of edema and functional deficits following traumatic brain injury in rats

The present study has used capsaicin-induced neuropeptide depletion to examine the role of neurogenic inflammation in the development of edema and functional deficits following traumatic brain injury (TBI). Adult, male rats were treated with capsaicin (neuropeptide-depleted) or equal volume vehicle (controls) 14 days prior to induction of moderate/severe diffuse TBI. Injury in vehicle treated control animals resulted in acute (4-5 h) edema formation, which was confirmed as being vasogenic in origin by diffusion weighted magnetic resonance imaging and the presence of increased permeability of the blood-brain barrier (BBB) to Evans blue dye. There was also a significant decline in brain magnesium concentration, as assessed by phosphorus magnetic resonance spectroscopy, and the development of profound motor and cognitive deficits. In contrast, capsaicin pre-treatment resulted in a significant reduction in post-traumatic edema formation (p < 0.001), BBB permeability (p < 0.001), free magnesium decline (p < 0.01) and both motor and cognitive deficits (p < 0.001). We conclude that neurogenic inflammation may play an integral role in the development of edema and functional deficits following TBI, and that neuropeptides may be a novel target for development of interventional pharmacological strategies.     

Increased mortality in female rats after brain trauma is associated with lower free Mg2+.

Male and female Sprague-Dawley rat siblings (200-350 g) were monitored by phosphorus magnetic resonance spectroscopy for 4 h after moderate (2.8 atmospheres) fluid-percussion-induced traumatic brain injury. Following injury, two of nine male animals died whereas 100% of all female rats (n = 16) died (p < 0.01). Prior to injury, brain free magnesium concentration in males was 0.58 +/- 0.05 mM and in females 0.41 +/- 0.09. After injury, mean brain free magnesium concentration in males declined to 0.32 +/- 0.06 whereas mean brain free magnesium concentration in ventilated females (n = 6) after injury declined to 0.17 +/- 0.03 (p < 0.05). There were no significant differences between groups with respect to any other measured variables. We conclude that female rats are more susceptible to irreversible injury after brain trauma, and that this increased susceptibility to injury may be related to brain free magnesium levels.     

The morphological and neurochemical effects of diffuse brain injury on rat central noradrenergic system

The central noradrenergic system is widely distributed throughout the brain and is closely related to spontaneous motility and level of consciousness. The study presented here evaluated the morphological as well as neurochemical effects of diffuse brain injury on the central noradrenergic system in rat. Adult male Sprague-Dawley rats were subjected to impact-acceleration brain injury produced with a weight-drop device. Morphological changes in locus coeruleus (LC) neurons were examined by using immunohistochemistry for dopamine-beta-hydroxylase, and norepinephrine (NE) turnover in the cerebral cortex was measured by high performance liquid chromatography with electrochemical detection. The size of LC neurons increased by 11% 24 h after injury but had decreased by 27% seven days after injury. Axons of noradrenergic neurons were swollen 24 h and 48 h after injury but the swelling had dwindled in seven days. NE turnover was significantly reduced seven days after injury and remained at a low level until eight weeks after injury. These results suggest that focal impairment of axonal transport due to diffuse brain injury causes cellular changes in LC and that the neurochemical effect of injury on the central noradrenargic system lasts over an extended period of time. Chronic suppression of NE turnover may explain the sustained behavioral and psychological abnormalities observed in a clinical situation.     

Upregulation of ICAM-1 and MCP-1 but not of MIP-2 and sensorimotor deficit in response to traumatic axonal injury in rats

The pathophysiology of traumatic axonal injury (TAI) is only partially understood. In this study, we investigated the inflammatory response as well as the extent of neurological deficit in a rat model of traumatic brain injury (TBI). Forty-two adult rats were subjected to moderate impact-acceleration brain injury and their brains were analyzed immunohistochemically for ICAM-1 expression and neutrophil infiltration from 1 hr up to 14 days after trauma. In addition, the chemotactic factors MIP-2 and MCP-1 were measured in brain homogenates by ELISA. For evaluating the neurological deficit, three sensorimotor tests were applied for the first time in this model. In the first 24 hr after trauma, the number of ICAM-1 positive vessels increased up to 4-fold in cortical and subcortical regions compared with sham operated controls (P < 0.05). Maximal ICAM-1 expression (up to 8-fold increase) was detected after 4 days (P < 0.001 vs. 24 hr), returning to control levels in all brain regions by 7 days after trauma. MCP-1 was elevated between 4 hr and 16 hr post-injury as compared with controls. In contrast, neither neutrophil infiltration nor elevation of MIP-2, both events relevant in focal brain injury, could be detected. In all neurological tests, a significant deficit was observed in traumatized rats as compared with sham operated animals from Day 1 post-injury (grasping reflex of the hindpaws: P < 0.001, vibrissae-evoked forelimb placing: P = 0.002, lateral stepping: P = 0.037). In conclusion, after moderate impact acceleration brain injury ICAM-1 upregulation has been demonstrated in the absence of neutrophil infiltration and is paralleled by a selective induction of chemokines, pointing out that individual and distinct inflammatory events occur after diffuse vs. focal TBI.     

Magnesium protects against neurological deficit after brain injury

The biochemical factors that mediate secondary or delayed damage to the central nervous system (CNS) remain speculative. We have recently demonstrated that brain injury in rats causes a rapid decline in brain intracellular free magnesium (Mg2+) and total magnesium concentrations that is significantly correlated with the severity of injury. In order to further investigate the relationship between Mg2+ and brain injury, we examined the effect of Mg2+ treatment on posttraumatic neurological outcome following fluid-percussion brain injury (2.0 atm) in rats. Since administration of ATP-MgCl2 has been shown to be beneficial in a variety of models of organ ischemia, we also examined the efficacy of ATP-MgCl2 or ATP alone in the treatment of experimental brain injury. Animals treated with low (12.5 mumol) or high (125 mumol) dose MgCl2 at 30 min postinjury showed a significant dose-dependent improvement in neurological function when compared to saline-treated controls. Treatment with ATP-MgCl2 (12.5 mumol) or ATP alone (12.5 mumol) caused no significant improvement in chronic neurological outcome. MgCl2-treated animals showed no change in postinjury mean arterial blood pressure (MAP), whereas animals treated with either ATP-MgCl2 or ATP alone showed a transient but significant fall in MAP (P less than 0.01) during the drug-infusion period. Our results suggest that postinjury treatment with MgCl2 is effective in limiting the extent of neurological dysfunction following experimental traumatic brain injury in the rat.     

Quantitative evaluation of microscopic injury with diffusion tensor imaging in a rat model of diffuse axonal injury

Diffuse axonal injury (DAI) is the predominant effect of severe traumatic brain injury and contributes significantly to neurological deficits. However, it is difficult to diagnose or characterize non-invasively with conventional imaging. Our study provides significant validation of a visual and statistical diffusion tensor imaging (DTI) technique as compared with pathological and electron microscopic study in a rat DAI model at multiple predilection sites and time points following trauma. Two DTI parameters, fractional anisotropy (FA) and axial diffusivity (AD), were significantly reduced from 12 h to 5 days post-trauma, corresponding to pathological axonal injury. At 7 days post-trauma, FA remained decreased, whereas AD pseudo-normalized and radial diffusivity increased. The temporal alterations in DTI parameters were observed in multiple predilection sites, and the extent of the changes in these parameters correlated significantly with the severity of histologically visualized axonal injury, as assessed by integrated optical density of immunochemically stained injured axons with quantitative stereology. Although anatomical T2-weighted magnetic resonance images showed no abnormal signals in microscopic lesions, we detected and characterized axonal injury directly by DTI at each time point. These results demonstrate that DTI has significant potential as a non-invasive tool with which to quantitatively diagnose and evaluate microstructural injury in the experimental and clinical assessment of DAI. This method can assist in accurate evaluation of the extent of axonal injury, detection of severe predilection foci, determination of approximate time of injury, and monitoring of the pathogenic condition at the early post-injury stage.     

Soluble amyloid precursor protein alpha reduces neuronal injury and improves functional outcome following diffuse traumatic brain injury in rats

Amyloid precursor protein (APP) has previously been shown to increase following traumatic brain injury (TBI). Whereas a number of investigators assume that increased APP may lead to the production of neurotoxic Abeta and be deleterious to outcome, the soluble alpha form of APP (sAPPalpha) is a product of the non-amyloidogenic cleavage of amyloid precursor protein that has previously been shown in vitro to have many neuroprotective and neurotrophic functions. However, no study to date has addressed whether sAPPalpha may be neuroprotective in vivo. The present study examined the effects of in vivo, posttraumatic sAPPalpha administration on functional motor outcome, cellular apoptosis, and axonal injury following severe impact-acceleration TBI in rats. Intracerebroventricular administration of sAPPalpha at 30 min posttrauma significantly improved motor outcome compared to vehicle-treated controls as assessed using the rotarod task. Immunohistochemical analysis using antibodies directed toward caspase-3 showed that posttraumatic treatment with sAPPalpha significantly reduced the number of apoptotic neuronal perikarya within the hippocampal CA3 region and within the cortex 3 days after injury compared to vehicle-treated animals. Similarly, sAPPalpha-treated animals demonstrated a reduction in axonal injury within the corpus callosum at all time points, with the reduction being significant at both 3 and 7 days postinjury. Our results demonstrate that in vivo administration of sAPPalpha improves functional outcome and reduces neuronal cell loss and axonal injury following severe diffuse TBI in rats. Promotion of APP processing toward sAPPalpha may thus be a novel therapeutic strategy in the treatment of TBI.     

Changes in free fatty acids, phospholipids, and cholesterol following impact injury to the rat spinal cord

Free fatty acids (FFA), phospholipid, and cholesterol levels were measured in spinal cord samples from rats subjected to low (25 g-cm), moderate (50 g-cm), or severe (100 g-cm) impact trauma to the T10 spinal segment. All degrees of injury caused early (15 min) declines in total phospholipids after trauma; phospholipid levels remained significantly below controls in rats subjected to moderate and severe injuries for up to 3 days, whereas phospholipids had returned to baseline values by 4 hr in the low injury group. Rapid and persistent decreases in cholesterol levels were observed for all injury groups. Severe trauma was associated with biphasic increases in FFA levels: levels were elevated at 5 and 15 min post-trauma and had declined by 30 min; a second elevation was observed at 1 hr, progressively increasing to reach a maximum at 24 hr, before declining over the next 6 days. Low and moderate injuries caused similar early total FFA increases; later increases were significantly smaller than in the severely injured group. Among the free fatty acids, significant increases were observed in palmitate, stearate, oleate, linoleate, linolenate, arachidonate, and docosahexaenoate. These findings indicate that traumatic spinal cord injury results in early, transient, postinjury membrane phospholipid hydrolysis, the magnitude of which is relatively independent of the severity of injury. More delayed and sustained lipid hydrolysis also occurs after trauma, the magnitude of which is related to the severity of injury.     

Neurogenesis and glial proliferation are stimulated following diffuse traumatic brain injury in adult rats

Although increased neurogenesis has been described in rodent models of focal traumatic brain injury (TBI), the neurogenic response occurring after diffuse TBI uncomplicated by focal injury has not been examined to date, despite the pervasiveness of this distinct type of brain injury in the TBI patient population. Here we characterize multiple stages of neurogenesis following a traumatic axonal injury (TAI) model of diffuse TBI as well as the proliferative response of glial cells. TAI was induced in adult rats using an impact-acceleration model, and 5-bromo-2'-deoxyuridine (BrdU) was administered on days 1-4 posttrauma or sham operation to label mitotic cells. Using immunohistochemistry for BrdU combined with phenotype-specific markers, we found that proliferation was increased following TAI in the subventricular zone of the lateral ventricles and in the hippocampal subgranular zone, although the ultimate production of new dentate granule neurons at 8 weeks was not significantly enhanced. Also, abundant proliferating and reactive astrocytes, microglia, and polydendrocytes were detected throughout the brain following TAI, indicating that a robust glial response occurs in this model, although very few new cells in the nonneurogenic brain regions became mature neurons. We conclude that diffuse brain injury stimulates early stages of a neurogenic response similar to that described for models of focal TBI.     

Degenerative changes in traumatic brain injury: post-injury magnetic resonance identified ventricular expansion compared to pre-injury levels

Magnetic resonance (MR) scans obtained 42 days and 10 months post-injury were compared to scans obtained in similar planes three months prior to injury. In comparison to pre-injury scans, post-injury MR scan analysis demonstrated significant ventricular volume increase which is considered a measure of the degree of diffuse axonal injury. Most important, the trauma induced degenerative effects appeared to be quite complete by 42 days post-injury as there was little further degeneration that occurred between the 6 week and 10 month post-injury scans. This study demonstrates that in humans the majority of gross trauma-induced degenerative changes are complete by 6 weeks post-trauma.     

弥漫性轴索损伤后海马生长抑素样神经元的变化

目的 :弥漫性轴索损伤 (DAI)能导致伤后认知障碍。本实验通过建立实验性 DAI动物模型 ,以了解在 DAI伤后与认知功能关系密切海马生长抑素 (Ss)样神经元的变化。方法 :采用 Marmarou打击装置建立 DAI动物模型 ,免疫组织化学染色以显示海马 Ss样神经元。结果 :1海马 Ss样神经元在重伤组、轻伤组、对照组有显著差异 (P<0 .0 1)。 2损伤后二周组神经元减少与一周组比较有显著差异 (P<0 .0 1或 P<0 .0 5 )。结论 :1DAI后海马 Ss样神经元的减少可能是伤后认知障碍 ,甚至是 DAI后植物生存的主要病理改变之一。 2伤后的迟发性细胞死亡在该种神经元的减少中起着重要作用。