Introduction

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Diffuse axonal injury (DAI) occurs in nearly half of all severe cases of clinical traumatic brain injury and has been associated with high mortality and morbidity (Gennarelli, 1994; Marmarou et al., 1994; Povlishock and Pettus, 1996). Indeed, even with the substantial underdiagnosis of clinical DAI (Mittl et al., 1994) more than 85% of the severe cases of traumatic brain injury resulting from motor vehicle accidents have been shown to involve substantial DAI (Adams et al., 1989). Although it was once believed that the axonal injury occurred as a direct result of the tensile forces of the primary initial trauma, it is now recognized as being a delayed process of progressive intra-axonal changes leading to disconnection (Christman et al., 1994; Povlishock et al., 1997). This delayed process is considered part of the secondary injury cascade that has been shown to occur between hours and days after the traumatic event and which is thought to be largely responsible for many of the neurologic deficits that are observed post-traumatically (McIntosh, 1993). Although a diversity of biochemical and physiological factors have been reported to be involved in the secondary injury processes, including changes in ion concentration, excitatory amino acids, opioid peptides, membrane changes, and energy failure (McIntosh, 1993), what they do have in common is that appropriately targeted therapies for these secondary injury factors can attenuate the injury process and significantly improve neurologic outcome (Faden and Salzman, 1992). Some, like magnesium administration, have been shown to affect more than one of the secondary injury processes (Vink et al., 1991).

Previous results have shown that administration of magnesium salts after either focal or diffuse traumatic brain injury improves neurologic outcome, at least when given as a bolus 30 min after the traumatic event (McIntosh et al., 1989; Smith et al., 1993; Heath and Vink, 1998). However, although these earlier doses of magnesium were neuroprotective, they were chosen on the basis of qualitative data in focal models of brain injury using a variety of magnesium salts (Vink et al., 1988). Therefore, it remains unclear as to what magnesium salt, what dosage, and what route of administration is the optimum for use in brain trauma, especially the more diffuse, nonfocal models of injury. The present study, therefore, uses the sensitive Rotarod motor test to compare the neuroprotective efficacy of a range of different doses of magnesium sulfate and magnesium chloride administered either i.v. or i.m. 30 min after severe, diffuse traumatic brain injury. After the characterization of the magnesium salts, optimum dose, and route of administration, animals were then examined by NMR spectroscopy (MRS) using the most successful of the tested therapeutic regimens to examine the effects on metabolic response which previously has been shown to be associated with improved neurologic outcome (Vink et al., 1991; McIntosh, 1993).

	Materials and Methods

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Animal Preparation and Induction of Injury. All experimental protocols were approved by the James Cook University Experimental Ethics Committee according to guidelines established for the use of experimental animals in research as outlined by the Australian National Health and Medical Research Council. Injury was induced using the closed head injury model of DAI as described in detail elsewhere (Marmarou et al., 1994; Heath and Vink, 1995). Briefly, male Sprague-Dawley rats (350-450 g; n = 108) were fed and watered ad libitum before being initially anesthetized with 50 mg/kg i.p. sodium pentobarbital. A 1-cm incision at the base of the tail facilitated insertion of an arterial catheter used to monitor blood pressure and take blood samples and a right femoral venous catheter was inserted for drug administration and continuous maintenance of anesthesia (8 mg/kg/h). Animals were then intubated and ventilated on room air with a Harvard Rodent Ventilator (Harvard Apparatus, South Natick, MA). Body temperature was maintained throughout with a thermostatically controlled heating pad set at 37°C. After exposing the skull by midline incision, a stainless steel disc measuring 10 mm in diameter and 3 mm in depth was cemented centrally along the coronal suture between the lambda and the bregma with a polyacrylamide adhesive. Animals were then secured in the prone position on a 10 cm-deep foam bed and injury was induced by dropping a 450-g brass weight from a distance of 2 m. The stainless steel disc was then detached from the skull immediately after the injury and the animals were weaned off the ventilator and returned to their cages to recover for 1 week of neurological testing and alternate-day MRS monitoring.

Treatment Protocol. The neuroprotective efficacy of i.v. and i.m. magnesium sulfate and magnesium chloride were compared with the Rotarod test of neurologic motor function. Animals were randomly assigned (n = 6/group) to receive either an i.v. or i.m. bolus of MgSO₄ or MgCl₂ in a saline vehicle given at doses ranging between 100 µmol/kg and 1.25 mmol/kg 30 min after induction of severe traumatic brain injury. After establishing the optimum treatment regimen, this therapy was repeated on an additional group of animals in an MRS study to establish the effects on metabolic outcome.

Neurological Assessment. Animals were blindly assessed for neurologic motor outcome with the Rotarod test as previously described in detail elsewhere (Hamm et al., 1994). This test has been found to be the most sensitive for detection of acute motor deficits after impact acceleration-induced traumatic brain injury (Heath and Vink, 1995). Briefly, Rotarod scores were based on performance on a Rotarod device (James Cook University, Townsville, QLD), which consisted of a motorized rotating assembly of 18 rods (1 mm in diameter) upon which the animals were placed. To walk as the rods rotated beneath them, the animals were required to grip a rod, thus introducing a grip test component to the assessment. Rotational speed of the device was increased from 0 to 30 rpm in intervals of 3 rpm for 10-s periods. The duration in seconds and rpm score was recorded at the point at which the animal either completed the 2-min task, fell from the rods, or gripped the rods and spun for two consecutive revolutions rather than actively walking on the rungs. Animals were pretrained on the device over 1 week (twice a day) before induction of injury and the mean value in seconds over this period was used as a preinjury baseline.

Phosphorus MRS. Having established the optimum magnesium salt, dose, and route of administration, this therapy was repeated in a group of animals in an MRS study to establish effects on metabolic outcome. Before injury, continuously for 8 h after injury, then subsequently at 2, 4, and 6 days after injury, animals from the optimum treatment group and a saline control group (n = 6/group) were placed in a specially designed Plexiglas holder and phosphorus MRS spectra were obtained with a 7.0 tesla magnet interfaced with a Varian spectrometer console as previously described in detail elsewhere (Heath and Vink, 1996; Vink et al., 1996). Briefly, a 5-mm × 9-mm surface coil was placed centrally over the skull and skin and temporal muscle were retracted well clear of the coil to ensure that there was no contribution from these tissues to the brain spectrum. After adjusting the B1 field to less than 0.25 ppm at the half-height of the proton water signal, phosphorus spectra were obtained with a 90° pulse width set at a 2-mm cortical depth in 30-min blocks at a spectral width of 4000 Hz containing 2048 data points. Previous studies have shown that the sensitive volume of this type of coil extends to approximately a one-diameter depth (5 mm) directly under the coil (Vink et al., 1987). The accumulated free induction decays were analyzed after convolution difference (20/500 Hz exponential filter), with the peaks being integrated after fitting with the spectrometer computer software and peak frequencies determined with the computer peak picking program.

Data Analysis. Intracellular pH was determined from the chemical shift of the inorganic phosphate peak relative to phosphocreatine (PCr) with the equation

(1)

Similarly, free magnesium concentration was determined from the chemical shift difference between the  and  peaks of ATP (Gupta et al., 1978) with the equation

(2)

where _- is the chemical shift difference between the  and  peaks of ATP. The K_d for MgATP was initially assumed to be 50 µM at pH 7.2 and 0.15 M ionic strength, and was corrected for pH by multiplying by a correction factor calculated as (Bock et al., 1987)

(3)

Cytosolic phosphorylation potential (PP) was determined according to the equation

(4)

where  represents all of the ionic forms of the free species. The concentration of ADP was calculated from the creatine kinase equilibrium equation after correcting the equilibrium constant for pH and free magnesium concentration as previously described in detail (Lawson and Veech, 1979; Vink et al., 1994). Concentrations of the other metabolites were determined from the integrated peak areas of the respective MRS peaks, assuming that preinjury, the normal values for PCr and ATP were 4.72 and 2.59 µmol/g, respectively, and that the total creatine pool remained constant at 10.83 µmol/g (Veech et al., 1979; Siesjo, 1981). Brain water content was assumed to be 80%, with the intracellular compartment accounting for 78% of the total water (Siesjo, 1981).

	Results

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Dose Response. The Rotarod test was used to determine the effects of the magnesium salts on post-traumatic functional outcome. Similar to previous studies, all animals demonstrated a decline in Rotarod scores after induction of severe traumatic brain injury (Heath and Vink, 1995; 1996). However, as early as 24 h after injury, all treatment groups receiving an i.m. or i.v. bolus of magnesium recorded improved Rotarod scores compared with controls. In i.m. MgSO₄-treated animals (Fig. 1), a dose of 750 µmol/kg resulted in the greatest improvement in Rotarod score as compared with nontreated control animals. This was particularly apparent between days 4 and 7 after injury. By determining the area under the curve between days 1 and 7 for each dose, dose-response effects of the drug could be summarized as a neuroscore relative to uninjured animals (Fig. 2). The 750 µmole/kg i.m. MgSO₄ dose improved Rotarod performance to 91.9 ± 4.5% of normal (uninjured) function compared with untreated animals that only had 56.5 ± 4.1% of normal motor function. After i.v. administration of MgSO₄, the optimum dose was 250 µmol/kg (Fig. 2). Similar results were obtained with the MgCl₂ salt, with optimum i.m. and i.v. doses of 750 µmol/kg and 250 µmol/kg, respectively. These optima were the same as the optimum dosages for the MgSO₄ salt. Although both the MgSO₄ and MgCl₂ salts resulted in similar neurologic outcomes by day 7 after trauma, there was a tendency for the MgSO₄-treated animals to perform better at earlier time points than the MgCl₂-treated animals. This effect could not be attributed to added protection afforded by the sulfate anion because equimolar administration of Na₂SO₄ had no beneficial effects on outcome (Fig. 3). MgSO₄ was therefore chosen for further characterization with phosphorus MRS to determine effects on biochemical outcome.

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Rotarod performance of animals over 7 days following induction of severe traumatic brain injury. Groups (n = 6) were administered different i.m. dosages (250 µmol/kg, 500 µmol/kg, 750 µmol/kg, 1 mmol/kg, and 1.25 mmol/kg) of magnesium sulfate 30 min after injury.

View larger version (61K): [in this window] [in a new window]   Fig. 2.   Percentage of neuroscore relative to uninjured animals obtained in animals following induction of severe traumatic brain injury. Groups (n = 6) were administered an i.m. (250-1250 µmol/kg) or i.v. (100-500 µmol/kg) dose of either MgSO4 or MgCl2 at 30 min after injury. Control animals received no treatment. *p < .05 versus nontreated controls.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Rotarod performance of animals (n = 6/group) over 7 days following induction of severe traumatic brain injury. Both no treatment () and sodium sulfate-treated () animals demonstrate significantly lower Rotarod scores compared with animals receiving the optimal dosage i.m. MgSO4 750 µmol/kg (). All points in the magnesium sulfate treatment group are significantly different from sulfate and control groups (p < .05).

Phosphorus MRS. The optimum i.m. dose of MgSO₄ was administered as a bolus 30 min after injury. Brain intracellular pH, as determined from the relative chemical shift of P_i to PCr, did not change significantly from preinjury levels (7.10 ± 0.01) over the 6-day post-traumatic MRS monitoring period (7.12 ± 0.02). These values were similar to those obtained in control (untreated) animals (7.10 ± 0.02). Similarly, ATP concentration, as determined from the intensity of the ATP peaks, did not change significantly at any time in either treated or control animals (results not shown). This is consistent with previous studies demonstrating that ATP concentration does not change significantly after severe brain injury (Heath and Vink, 1995; 1996). The lack of any significant ATP change after trauma enabled the calculation of brain intracellular free magnesium from the chemical shifts of the ATP resonances. Before injury, brain free magnesium concentration in the control and MgSO₄ treatment groups was 0.47 ± 0.02 mM and 0.50 ± 0.03 mM, respectively (Fig. 4). These values are similar to recent reports of free magnesium concentration in the brain in a number of species (Altura and Gupta, 1992; Kauppinen et al., 1992; Helpern et al., 1993; Headrick et al., 1994). By 30 min after injury, there was a highly significant (p < .001) decline in free magnesium to less than 60% of preinjury levels in both groups of animals. This significant decline persisted for the entire 6-day monitoring period in untreated animals. In contrast, intracellular free magnesium in magnesium-treated animals increased from a 30 min minimum of 0.33 ± 0.02 mM to a mean post-treatment value of 0.46 ± 0.03 mM. This value was not significantly different from preinjury values and was sustained for the remainder of the observation period (Fig. 4).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Brain intracellular free magnesium concentration over 6 days following impact acceleration-induced traumatic brain injury in rats. Groups (n = 6/group) were administered either i.m. MgSO4 750 µmol/kg () or no treatment (). *p < .05 versus no treatment.

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View larger version (25K): [in this window] [in a new window]   Fig. 5.   Relationship between brain-free magnesium concentration (), brain PP (), and Rotarod scores following severe traumatic axonal brain injury. Points represent mean values and scores obtained in the same animals on the same days (0, 1, 2, 4, and 6) in both the magnesium-treated and control groups.

	Discussion

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The present study has demonstrated that magnesium salts administered after severe traumatic brain injury result in a significant improvement in neurologic outcome as determined by the Rotarod test. Complementary MRS studies demonstrated that this motor improvement was associated with an increased brain intracellular free magnesium concentration and bioenergetic state, both of which were linearly correlated with Rotarod scores. These findings are consistent with a number of previous studies which have shown that different magnesium salts are cerebroprotective after focal traumatic brain injury (McIntosh et al., 1989; Smith et al., 1993; Heath and Vink, 1998). We have extended these earlier findings to establish that both magnesium chloride and magnesium sulfate given at the same dose are equally protective in a severe DAI model of brain trauma and have characterized the optimum dose for bolus administration at 30 min after injury.

Experimental studies of moderate focal brain injury have shown that low-concentration bolus doses of i.v. MgSO₄ or MgCl₂ before and up to 1 h after brain injury restore intracellular free magnesium concentration and improve post-traumatic neurologic deficits (Vink et al., 1991). In the present study, we have used a model of severe, diffuse traumatic axonal injury to demonstrate that such treatment with magnesium salts is also effective in significantly improving motor function and bioenergetic state in this clinically relevant form of diffuse brain injury. Equimolar doses of both i.m. and i.v. magnesium sulfate or magnesium chloride were effective in attenuating post-traumatic motor deficits, suggesting that it is the magnesium cation that is responsible for the neurologic improvement as opposed to the anion. That magnesium does indeed enter the brain intracellular space after brain injury was demonstrated in the associated magnetic resonance spectroscopy study. Magnesium sulfate administration rapidly increased the concentration of the free ion and resulted in a significant improvement in the bioenergetic state as reflected in the PP. Moreover, this improvement was sustained for the remainder of the 1-week observation period despite magnesium being administered as a single bolus at 30 min after trauma. This suggests that the events which lead to the decline in brain intracellular free magnesium concentration occur within the first few hours after trauma. Administration of magnesium at this time will restore the magnesium homeostasis disrupted by the traumatic event and have a lasting effect on both the cell bioenergetic state and motor outcome. Indeed, the linear correlation between brain-free magnesium and motor outcome suggests that the sooner restoration of magnesium homeostasis can take place, the higher the likelihood of an improvement in post-traumatic motor performance.

Although the mechanisms by which magnesium is protective are unknown, several possibilities have been variously proposed. These include effects on metabolism, particularly phosphorylation reactions which generate ATP (Vink et al., 1994) membrane integrity and permeability (Gunther et al., 1994), the functioning of the Na⁺/K⁺ ATPase with subsequent effects on edema (McIntosh et al., 1990), and effects on neurotransmitter release (Rothman, 1983). Our present results certainly support the idea that effects on energy metabolism can have marked effects on the outcome after neurotrauma. Indeed, we demonstrate that PP is highly correlated with neurologic motor outcome measured in the same animals on the same day. This suggests that the recovery mechanisms after trauma may be energy-dependent and that an improvement in brain energy status with magnesium salts may facilitate these processes. These processes would conceivably include membrane synthesis, protein synthesis, DNA synthesis, Na⁺/K⁺ ATPase function, and stabilization of ion gradients. All of these processes have been reported to be adversely affected by traumatic brain injury (McIntosh, 1993).

In addition to effects on energy metabolism, the effects of the magnesium ion on calcium ion flux has also received considerable attention in recent years. The magnesium ion is known be an antagonist of calcium channels in general (Iseri and French, 1984) and in particular, to act as a voltage-dependent blocker of the N-methyl-D-aspartate (NMDA) channel (Mayer et al., 1984; Nowak et al., 1984). It is the specific effects of the ion on the activity of the NMDA channel that has been recently reported to be critical to the outcome after central nervous system trauma (Zhang et al., 1996). These authors demonstrate in an in vitro study that the magnesium block of the NMDA channel is reduced after neural injury, and that this reduction may be linked to either a decline in magnesium levels or a change in the structure of the NMDA channel. Indeed, increasing the magnesium level in culture was neuroprotective.

Although a number of mechanistic possibilities exist, the present in vivo findings support that a neuroprotective role for magnesium exists after severe diffuse traumatic brain injury. Moreover, this is the first study to use Rotarod motor tests to establish dose-response curves for effective magnesium based pharmacotherapies after traumatic brain injury.