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Journal of the American College of Nutrition, Vol. 23, No. 5, 541S-544S (2004)
Published by the American College of Nutrition

Magnesium Gluconate Offers No More Protection than Magnesium Sulphate Following Diffuse Traumatic Brain Injury in Rats

Renee J. Turner, BSc, K. W. DaSilva, C. O’Connor, Corinna van den Heuvel, PhD and Robert Vink, PhD

Department of Pathology, University of Adelaide, Adelaide SA, AUSTRALIA

Address reprint requests to: Robert Vink, Ph.D., Department of Pathology, University of Adelaide, Adelaide SA, AUSTRALIA. E-mail: robert.vink{at}adelaide.edu.au


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 REFERENCES
 
Objective: Previous studies have demonstrated that magnesium salts, including the sulphate and chloride forms, are neuroprotective following traumatic brain injury (TBI). Recently, studies in cardiac ischaemia/reperfusion injury have demonstrated that the gluconate salt of magnesium may provide superior protection against oxidative damage and postischaemic dysfunction than MgSO4. We have therefore compared the efficacy of both MgSO4 and magnesium gluconate (MgGl2) on outcome following diffuse TBI in rats.

Methods: Adult male Sprague-Dawley rats were injured using the 2-metre impact acceleration model of diffuse TBI. At 30 min after injury, animals were administered with either 250µmoles/kg i.v. MgSO4, MgGl2, or equal volume saline vehicle. Thereafter, animals were assessed for motor and cognitive outcome using the rotarod and Barnes maze, respectively, or their brains removed at 3 days after TBI and used for histological examination.

Results: Treatment with either magnesium salt significantly improved functional outcome as compared to vehicle treated controls. Similarly, treatment with either magnesium salt attenuated the degree of histological dark cell change at 3 days after TBI relative to the vehicle treated animals. There were no significant differences between the magnesium treated groups.

Conclusions: We conclude that MgSO4 and MgGl2 are equally neuroprotective following TBI. Our results suggest that MgGl2 may only be more effective in conditions that produce ischaemia, where high concentrations of reactive oxygen species are generated.

Key words: magnesium salts, neuroprotection, traumatic brain injury, ischaemia, rat, reactive oxygen species


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 REFERENCES
 
Traumatic brain injury (TBI) is a leading cause of mortality and morbidity in the industrialized world. Survivors are often left with severe and permanent motor and cognitive deficits that adversely affect their ability to return to work and effectively contribute to society. Research has now shown that delayed secondary injury is largely responsible for the development of irreversible injury after TBI [1]. Of the secondary injury factors identified to date, decline in brain intracellular free Mg concentration is recognized as a ubiquitous feature of TBI associated with the formation of neurological deficits [1,2]. Altering posttraumatic Mg homeostasis has subsequently been shown to modify outcome. For example, experimentally induced Mg deficiency has been shown to exacerbate neurologic deficits after TBI while posttraumatic administration of Mg salts results in a significant improvement in neurologic outcome [2].

While the mechanisms by which Mg salts improve outcome are unknown, several have been proposed including attenuation of glutamate excitoxicity, inhibition of Ca channels, improvement in mitochondrial energy metabolism, attenuation of apoptosis, and reduction in blood brain barrier permeability and oedema formation, amongst others [3]. There have also been reports that suggest Mg may reduce oxidative stress after TBI [4,5]. However, the ability of the Mg sulphate (MgSO4) salt to reduce oxidative stress is somewhat limited and recent evidence suggests that the gluconate form of Mg (MgGl2) may be a more effective treatment against free radicals and oxidative stress [6]. Accordingly, the present study has compared the effects of MgGl2 and MgSO4 on short-term histologic and functional outcome following experimental diffuse traumatic brain injury.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 REFERENCES
 
Adult male Sprague-Dawley rats (n = 51; 350–450g) were group housed on a 12 h night-day cycle and provided with a standard diet of rodent pellets and water ad libitum. Animals were injured using the 2 m impact acceleration model of diffuse TBI described in detail elsewhere [7,8]. Briefly, rats were anaesthetised with halothane and body temperature maintained at 37°. A midline incision was performed to expose the skull and a stainless steel disc (10 mm diameter x 3 mm thick) was fixed to the skull centrally between the bregma and lambda sutures using polyacrylamide adhesive. Following surgery, animals were placed in the prone position on a 10 cm foam block and the foam block placed beneath the injury device. Injury was induced by release of a 450 g brass weight from a height of 2 m onto the steel disc. At 30min following trauma, animals were intravenously administered either 250 µmol/kg of magnesium sulphate (n = 21), magnesium gluconate (n = 21) or equal volume of saline vehicle (n = 18). This dose of Mg has been previously shown to be optimal in this model of injury [8].

Animals were allowed to recover and were subsequently assessed for motor and cognitive outcome over the next 7 days using the rotarod and Barne’s Maze tasks, respectively. The rotarod consists of a rotating assembly of eighteen 1mm rods [9]. Animals were placed on the device and required the speed of rotation increased from 0 rpm to a maximum of 30 rpm, with each speed maintained for 10 s. The time at which the animals completed the 2 min task, fell off completely or gripped the rungs for 2 consecutive turns without walking was recorded. Cognitive outcome was assessed using the Barne’s Maze which consists of a 1.22 m diameter raised circular platform with eighteen holes, 9.5 cm in diameter, evenly spaced around the circumference [10,11]. Animals were required to locate the escape tunnel beneath one of the holes in response to aversive sound and light stimuli. The latency of each animal to locate and enter the escape tunnel was recorded. Animals were trained in both tasks for a 5-day period pre-injury.

A subgroup of animals (n = 3/group) was perfusion fixed using 4% paraformaldehyde at 3 days post-injury. The fixed intact brain was then removed and stored in 4% paraformaldehyde for 3 days and then transferred into 10% formaldehyde until required for histological analysis. Brains were embedded in paraffin and cut into 5 µm coronal sections using a Vibratome. Sections were subsequently stained with haematoxlin and eosin (H&E) and examined for dark cell change (cell stress) by light microscopy.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
REFERENCES
 
Prior to injury, mean rotarod score in all animals was 117 ± 3 sec (mean ± SEM). After injury, there was a significant (p < 0.001; ANOVA) decline in rotarod scores in all treatment groups, reaching a minimum at 1 day after trauma (Fig. 1). In vehicle treated animals, there was no significant improvement in rotarod score over the remainder of the 7-day assessment period. In contrast, both MgGl2 and MgSO4 treated animals demonstrated a significant improvement in motor performance over time such that their rotarod scores on day 7 of the assessment task were no longer significantly different from pre-injury scores. This is similar to that previously described for Mg salts after TBI [8]. The rotarod performance of the Mg treated animals was significantly greater (p < 0.01) than that of the vehicle treated controls, but no difference was apparent between the two Mg salts. Similarly, Mg treatment improved rate of cognitive recovery in the cognitive task, but no significant difference was noted between the two Mg salts (results not shown).



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  		Fig. 1. Rotarod (motor) score after severe traumatic brain injury in rats. Treatment with either magnesium sulphate or magnesium gluconate resulted in a significant improvement in motor outcome relative to vehicle treated animals (p < 0.01).

 
In cortical sections obtained on day 3 after TBI (Fig. 2), vehicle treated animals demonstrated a high degree of neuronal dark cell change indicative of cell stress. With administration of either MgGl2 or MgSO4, there was a profound decrease in the degree of dark cell change noted in both the cortex and the hippocampus of injured animals. There were no significant differences between the Mg salts with respect to the degree of protection against dark cell change after TBI.



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  		Fig. 2. H&E stained sections of the cortex after traumatic brain injury in rats. (A) Vehicle treated animal showing marked dark cell change and misshapen neurones; (B) magnesium gluconate treated animals; (C) magnesium sulphate treated animals.

 

   DISCUSSION
 
The present study has demonstrated that both MgGl2 and MgSO4 significantly attenuate acute functional deficits following severe, diffuse traumatic brain injury in rats, consistent with the ability of both salts to attenuate cortical and hippocampal dark cell change (cell stress). While Mg salts have been widely shown to reduce neuronal cell death and improve acute functional deficits after TBI [3,12–14], this is the first study to examine the effects of MgGl2 in TBI and compare it with the more widely used MgSO4. Our previous studies have compared MgSO4 with MgCl2 and found no significant difference between these two salts [8].

We have examined the effects of MgGl2 on outcome following TBI since it has been shown to be more protective in cardiac studies than other Mg salts [6,15]. Although the precise mechanisms are unknown, Mak et al. [15] demonstrated that MgGl2 inhibited lipid peroxidation in a concentration dependent manner. The effects of MgSO4 and MgCl2 on lipid peroxidation were ≤ 20% effective compared to MgGl2. MgCl2 also significantly inhibited the formation of hydroxyl radicals in the Fenton-reaction system and blocked ‘Fe-catalysed’ deoxyribose degradation, with the anion influencing the anti-oxidant effect [15]. Specifically, the Gl2– in MgGl2 reduced oxidative injury in isolated membrane preparations exposed to exogenous free radicals. An increase in the survival and proliferation of cultured endothelial cells, in addition to a decrease in glutathione loss, during exogenous free radical stress has also been observed [6]. There has been no published data demonstrating the role of Mg2+ salts on the inhibition of ROS in TBI. Considering the potentially crucial role of free radical generation in neuronal cell death following TBI, MgGl2 may be superior to MgSO4 and MgCl2.

However, our present results demonstrate that there was no significant difference on outcome after TBI irrespective of whether the form of Mg administered was the sulphate or gluconate salt. On the one hand, this would confirm that it is the Mg cation that is critical for neuroprotective function in TBI, and that this effect is independent of the salt used. However, the Gl– has far greater free radical scavenging ability than either the sulphate or chloride forms [6]. It may therefore be concluded that ROS production in TBI may not be as crucial to the development of motor deficits as current dogma suggests. This is consistent with the observation that antioxidants and free radical scavengers have not been successful in clinical trials of TBI [16]. Alternatively, it may be that the level of injury used in the current study was insufficient to cause profound oxidative stress. Certainly, there is no ischaemia or haemorrhage present in this model when used at the severity adopted in this study. However, haemorrhage and ischaemia are common in more severe forms of trauma, particularly those that result in focal injuries [17]. Accordingly, MgGl2 may only be more effective than the other Mg salts in severities of TBI that produce ischaemia, where high concentrations of reactive oxygen species are generated.

Received August 5, 2004.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 REFERENCES
 

  1. McIntosh TK: Neurochemical sequelae of traumatic brain injury.Cereb Brain Metab Rev6 :109 –162,1994 .[Medline]
  2. Vink R, Cernak I: Regulation of brain intracellular free magnesium following traumatic injury to the central nervous system.Front Biosci5 :656 –665,2000 .
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  4. Ustun ME, Duman A, Ogun CO, Vatansev H, Ak A: Effects of nimodipine and magnesium sulfate on endogenous antioxidant levels in brain tissue after experimental head trauma.J Neurosurg Anesthesiol13 :227 –232,2001 .[Medline]
  5. Cernak I, Savic VJ, Kotur J, Prokic V, Veljovic M, Grbovic D: Characterization of plasma magnesium concentration and oxidative stress following graded traumatic brain injury in humans.J Neurotrauma17 :53 –68,2000 .[Medline]
  6. Murthi SB, Wise RM, Weglicki WB, Komarov AM, Kramer JH: Mg-gluconate provides superior protection against postischemic dysfunction and oxidative injury compared to Mg-sulfate.Mol Cell Biochem245 :141 –148,2003 .[Medline]
  7. Foda MAA, Marmarou A: A new model of diffuse brain injury in rats; Part II: morphological characterization.J Neurosurg80 :301 –313,1994 .[Medline]
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  13. Bareyre FM, Saatman KE, Raghupathi R, McIntosh TK: Postinjury treatment with magnesium chloride attenuates cortical damage after traumatic brain injury in rats.J Neurotrauma17 :1029 –1039,2000 .[Medline]
  14. Saatman KE, Bareyre FM, Grady MS, McIntosh TK: Acute cytoskeletal alterations and cell death induced by experimental brain injury are attenuated by magnesium treatment and exacerbated by magnesium deficiency.J Neuropathol Exp Neurol60 :183 –194,2001 .[Medline]
  15. Mak IT, Komarov AM, Kramer JH, Weglicki WB: Protective mechanisms of Mg-gluconate against oxidative endothelial cytotoxicity.Cell Mol Biol (Noisy-le-grand)46 :1337 –1344,2000 .
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This Article
Right arrow Abstract Freely available
Right arrow Figures Only
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Services
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Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
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Right arrow Citing Articles via Google Scholar
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Right arrow Articles by Turner, R. J.
Right arrow Articles by Vink, R.
Right arrow Search for Related Content
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Right arrow PubMed Citation
Right arrow Articles by Turner, R. J.
Right arrow Articles by Vink, R.

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