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Amiloride Increases Neuronal Damage after Traumatic Brain Injury in Rats

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

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

	ABSTRACT

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Objective: It is well known that traumatic brain injury (TBI) decreases brain free magnesium (Mg) concentration, and that administration of Mg salts after TBI restores concentration of Mg in brain and improves functional outcome. In the presence of hemorrhage, administration of Mg salts exacerbates the injury process and worsens outcome. An alternative to administration of Mg salts may be to prevent cellular loss of Mg with use of amiloride, which inhibits the Na⁺/Mg²⁺ exchange.

Methods: In the present study, male, adult Sprague-Dawley rats were injured using the impact acceleration model of diffuse TBI and administered either 100 mols/kg i.v. amiloride, or an equal volume of 50% DMSO/saline, 30 minutes (min) after injury.

Results: Amiloride did not improve functional outcome (motor or cognitive outcome) after TBI relative to vehicle treated controls. Histologically, treatment with amiloride significantly increased hippocampal caspase-3 expression (apoptosis), axonal swellings in the medulla and the degree of dark cell change (cell stress) in the cortex. Phosphorus NMR demonstrated that amiloride did not increase free Mg concentration after injury.

Conclusions: Thus, amiloride is ineffective in preventing Mg loss after TBI when administered 30 min after trauma. Moreover, by administering amiloride after the TBI-related Mg decline has already been initiated, it may exacerbate injury by, in part by inhibiting Na⁺/Mg²⁺ antiport and preventing entry of Mg back into the cell, and also by inhibiting other Na⁺ linked transporters.

Key words: apoptosis, brain Mg, traumatic brain injury, hemorrhage, Mg treatment, Amiloride, Na⁺/Mg²⁺ antiport

	INTRODUCTION

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Traumatic brain injury (TBI) is the biggest killer of individuals under 45 years of age and often leaves survivors with life-long motor and cognitive deficits that impair their quality of life. Cell damage that occurs following TBI is caused by both primary and secondary injury mechanisms, the latter being made up of delayed biochemical and physiological factors [1]. This delayed injury therefore provides a window of opportunity for therapeutic intervention. A number of studies have now demonstrated that decline in brain free magnesium (Mg²⁺) concentration is a critical secondary injury factor after TBI, and that this decline can be treated with the administration of Mg²⁺ salts with consequent improvement in neuronal cell survival and an associated improvement in functional outcome [2,3]. However, Mg is a vasodilator and its administration in the presence of hemorrhage may increase the hemorrhage and exacerbate the injury. Indeed, in experimental TBI, administration of Mg²⁺ in the presence of ongoing hemorrhage has been shown to be deleterious to outcome [4]. An alternative to Mg²⁺ therapy may be to inhibit neuronal Mg²⁺ loss. A number of mechanisms may account for Mg²⁺ efflux from a cell, with the Na⁺/Mg²⁺ antiporter being the best described to date [5,6]. Therefore, in the present study we have administered the Na⁺/Mg²⁺ transport inhibitor, amiloride, to rats after TBI and assessed the effects of the intervention on functional outcome, brain free Mg²⁺ and neuronal cell death.

	MATERIALS AND METHODS

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Adult male Sprague-Dawley rats (350–450 g) were group housed on a 12 hour (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 anesthetised with sodium pentobarbital (60 mg/kg) or halothane and body temperature maintained at 37°C. 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. Transient apnea was noted in all animals after trauma, and respiration during this time was maintained by mechanical ventilation. At 30 min following trauma, the rats (n = 17/group) were intravenously administered either 100 µmol/kg of amiloride or an equal volume of DMSO/saline vehicle.

Animals (n = 8/group) 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 18 1 mm rods [9]. Animals were placed on the device and the speed of rotation was 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 18 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.

Further subgroups of animals were used for phosphorus (P) magnetic resonance spectroscopy (n = 6/group) or histology and immunohistochemistry (n = 3/group). For P-spectroscopy, animals were placed centrally within a 7.0 telsa magnetic resonance spectrometer and spectra obtained in 30 min blocks for 4 h using a 9 x 5 mm surface coil placed centrally over the exposed skull as previously described in detail elsewhere [4]. Spectra were analysed for intracellular pH and brain free Mg concentration. For histology and immunohistochemistry, animals were killed 24 h post-TBI and their brains perfusion fixed with 4% paraformaldehyde. Brains were then embedded in paraffin, cut into 5 µm coronal sections and used for hematoxylin and eosin (H&E) staining and caspase-3 immunohistochemistry. Semi-quantitative counts for positive neurones in preselected regions of the cortex and hippocampus were obtained using light microscopy.

All data were analyzed using analysis of variance and Student Newman Keuls post-hoc tests. Significance level was taken as p < 0.05.

	RESULTS

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Following injury, all animals demonstrated a significant decline in rotarod performance from pre-injury scores (Fig. 1). Consistent with previous results, animals displayed improved performance over time with repeated exposure to the task [8]. No significant differences in mean rotarod scores were observed between the amiloride and vehicle-treated groups at any time over the 7-day assessment period. Similar results were observed in the Barnes maze cognitive tests where all animals demonstrated profound cognitive deficits on day 1 post-TBI, and a gradual improvement in function with repeated exposure to the task (data not shown). Again, no differences were observed between the treatment groups.

View larger version (22K): [in this window] [in a new window]   Fig. 1. Rotarod performance of rats prior to and after traumatic brain injury.

View larger version (115K): [in this window] [in a new window]   Fig. 2. Dark cell change in H&E stained sections of hippocampus and cortex in (A) sham animals; (B) vehicle treated animals; and (C) amiloride treated animals.

	DISCUSSION

 
The current experiment has demonstrated that amiloride did not improve functional outcome when administered 30 min after induction of traumatic brain injury in rats, and exacerbated neuronal injury as determined by H&E staining and caspase-3 immunoreactivity. Amiloride also did not increase post-traumatic concentration of brain free Mg. While confirming that a sustained decline in brain Mg is associated with functional deficits after brain trauma, amiloride’s inability of to restore Mg concentration was unexpected, given that the compound is a blocker of Na/Mg²⁺ antiport [5,6].

The interpretation of DCC remains controversial [7]. However, if DCC represents damaged and stressed neurons, then the lack of improvement in motor outcome may be explained by an adverse effect of amiloride on neurones. The concept that amiloride may have an adverse affect on neurones was supported by the increase in caspase-3 immunoreactivity observed in the present studies. Similarly, amiloride therapy at least doubled the number of axonal swellings after TBI, indicating that disruption of axons at the level of the medulla may also have prevented an improvement in neurological outcome in these animals.

Our findings contradict reports that amiloride may be neuroprotective [12]. In these in vitro studies, amiloride was shown to protect primary cerebellar granule cells from glutamate-induced delayed neuronal death, presumably by inhibiting glutamate n-methyl-D-aspartate receptor activity. However, the protection was highly dependent upon the time-point at which the inhibitor was added to the culture medium. When added concurrently with glutamate, amiloride was highly protective. In contrast, when added after glutamate exposure, the neuroprotective effect was markedly reduced. This suggests that there may be a strict time dependence associated with the protective properties of amiloride. When applied to our own in vivo results, it suggests that amiloride was administered too late after TBI to be effective. The fact that brain intracellular free Mg concentration is already low at 30 minutes after injury, and that amiloride failed to improve this low Mg concentration, supports the concept that amiloride was administered too late to block Mg loss through the Na⁺/Mg²⁺ exchanger.

Why post-traumatic amiloride administration exacerbated neuronal cell death after TBI is unclear, although there are several possibilities. Amiloride is a non-specific Na⁺ transport inhibitor and inhibition of other Na⁺ transporters may have proven detrimental following injury. Block of the Na⁺/H⁺ exchange may have prevented H⁺ efflux from cells, thereby decreasing intracellular pH. This is unlikely since we did not observe acidosis in the magnetic resonance studies. Alternatively, block of the Na⁺/Ca²⁺ exchanger may exacerbate injury by preventing Ca²⁺ efflux from cells, thus markedly increasing intracellular Ca²⁺ concentration and exacerbating Ca-induced injury. Finally, inhibition of the Na⁺/Mg²⁺ exchanger after Mg has already left the cells may prevent later Mg re-entry, thus preventing Mg from participating in intracellular neuronal repair.

	CONCLUSIONS

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Post-traumatic amiloride administration did not improve neurological outcome following TBI in rats. Moreover, it increased neuronal injury as indicated by the increased dark cell change, axonal swellings and caspase-3 immunoreactivity in both the hippocampus and cortex. We conclude that administration of amiloride at 30 mins after trauma is too late to prevent Mg²⁺ efflux from cells and may in fact exacerbate injury through prevention of Mg²⁺ influx at later time points. Further studies investigating the administration of amiloride prior to injury may provide information as to whether Mg²⁺ efflux through the Na/Mg antiport contributes to the Mg²⁺ decline after TBI.

	ACKNOWLEDGMENTS

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We thank Robert Beveridge for his technical assistance with the magnetic resonance experiments.

Received August 5, 2004.

	REFERENCES

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