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Journal of the American College of Nutrition, Vol. 23, No. 5, 538S-540S (2004)
Published by the American College of Nutrition

A Substance P Antagonist Increases Brain Intracellular Free Magnesium Concentration after Diffuse Traumatic Brain Injury in Rats

Robert Vink, PhD, J. J. Donkin, M. I. Cruz, Alan J Nimmo, PhD and Ibolja Cernak

Department of Pathology, University of Adelaide, Adelaide SA (R.V., J.J.D.), AUSTRALIA
School of Pharmacy and Molecular Sciences, James Cook University, Townsville QLD (A.J.N., I.C.), AUSTRALIA
Department of Neuroscience, Georgetown University, Washington DC (M.I.C.)

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
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 REFERENCES
 
Objective: Magnesium (Mg) deficiency has been shown to increase substance P release and induce a pro-inflammatory response that can be attenuated with the administration of a substance P-antagonist. Neurogenic inflammation has also been implicated in traumatic brain injury (TBI), a condition where brain intracellular free magnesium (Mgf) decline is known to occur and has been correlated with functional outcome. We therefore examined whether a substance P antagonist restores brain intracellular free magnesium concentration following TBI.

Methods: Male, adult Sprague-Dawley rats were injured using the Cernak impact acceleration model of diffuse TBI. At 30 min after injury, animals were administered either 0.25 mg/kg i.v. n-acetyl tryptophan or equal volume saline. Prior to and 4 h after induction of injury, phosphorus magnetic resonance spectra were acquired using a 7-tesla magnet interfaced with a Bruker console. Mgf was calculated from the chemical shift of the beta ATP. Before injury, Mgf was 0.51 ± 0.05 mM (SEM).

Results: By 4 hr after injury, Mgf had significantly declined to 0.27 ± 0.02 mM in saline treated rats. In contrast, rats treated with n-acetyl tryptophan had a Mgf of 0.47 ± 0.06 mM at 4 h after injury, which was not significantly different from preinjury values. There were no significant differences in pH between the treatment groups.

Conclusion: It seems that any beneficial effect of a substance P antagonist on functional outcome following TBI may be related to improvement in brain Mg homeostasis induced by the compound.

Key words: brain trauma, brain Mg, inflammation, functional outcome, rats, substance P-antagonist


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 REFERENCES
 
Two mechanisms are associated with the development of neuronal cell death and neurological deficits after traumatic brain injury (TBI) [1]. The first is known as primary injury and encompasses all the mechanical events that occur at the time of the trauma, including tissue tearing, laceration, axon stretching and shearing, amongst others. Other than preventative measures, little can be done for primary injury. On the other hand, the second mechanism is made up of biochemical and physiological factors that form an injury cascade which is amenable to pharmacological intervention because it manifests over hours to days after the primary event. Several factors have been identified as playing a role in this secondary injury cascade [2], none the least being the magnesium ion (Mg2+).

The brain intracellular free Mg concentration has been shown to decline by between 40 and 60% in a number of TBI models, and is now considered a ubiquitous feature of traumatic brain injury [3]. Consistent with this, post-injury treatment with Mg salts has been shown to restore brain free Mg concentration [4], reduce neuronal cell damage [5,6], and improve functional outcome following TBI [3], including both motor and cognitive components. In the same way, seemingly unrelated treatments that have improved functional outcome after TBI all share the ability to restore brain intracellular free Mg concentration [7], suggesting that a compound’s ability to restore brain free Mg concentration may be critical to its success as a neuroprotective agent.

Recent studies have identified that neurogenic inflammation plays a role in edema formation and development of functional deficits following TBI [8]. Whether this neurogenic inflammation is related to the Mg decline after TBI is unknown, although studies in heart have shown that Mg deficiency results in a substance P-mediated neurogenic inflammation that can be inhibited by a substance P antagonist [9]. Subsequent studies in TBI have since demonstrated that administration of a substance P antagonist improves both motor and cognitive outcome after TBI [10]. Since drugs that improve outcome after TBI have been shown to also improve brain free Mg concentration [7], we sought to determine whether the substance P antagonist used in our previous studies may restore brain free Mg concentration after TBI as one of its possible mechanism of action.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 REFERENCES
 
Adult male Sprague-Dawley rats (n = 12; 400–420 g) were injured using the Cernak impact acceleration model of diffuse TBI as described in detail elsewhere [11]. Briefly, rats were anesthetised with isoflurane and body temperature maintained at 37°C. A midline incision was performed to expose the skull and a circular 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. Injury was induced using a hydraulically controlled, high-velocity impactor that is targeted to contact the steel disc cemented onto the rodent skull. The targeting is facilitated using a laser guide that ensures that the 10 mm impactor and the steel disc are parallel. The distance the impactor travels after contacting the steel disc is under user control thus varying the injury severity. In the current experiments, we used a distance of 18 mm, which results in moderate to severe injury. The head is decelerated after impact using a molded, gel-filled base upon which the animals head is supported during injury. At 30 min following trauma, animals were intravenously administered either 0.25 mg/kg of the substance P antagonist, n-acetyl tryptophan, (n = 6) or an equal volume of saline vehicle (n = 6).

Prior to and following injury, animals were placed centrally within a 7.0 telsa magnetic resonance spectrometer interfaced with a Bruker console and phosphorus spectra obtained in 30 min blocks for 4 h using a 9 x 5 mm surface coil placed centrally over the exposed skull. Acquired spectra were analysed for intracellular pH and brain free Mg concentration as previously described in detail elsewhere [4]. All data were analyzed using analysis of variance and Student Newman Keuls post-hoc tests. Significance level was taken as p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
REFERENCES
 
Prior to injury, brain pH in all animals was 7.13 ± 0.04 (mean ± SEM) while brain intracellular free Mg was 0.51 ± 0.05 mM. After injury, brain ATP concentration and pH did not change significantly in either treatment group. In contrast, there was a significant decline (p < 0.05) in brain intracellular free Mg concentration to 0.27 ± 0.02 in vehicle treated control animals by 4 hrs post-trauma (Fig. 1). These values are consistent with previous reports in rodent TBI [3,4,11]. Animals administered the substance P antagonist, n-acetyl tryptophan, at 30 min after injury demonstrated a brain intracellular free Mg concentration of 0.47 ± 0.06 at 4 hours after injury, which was significantly greater (p < 0.05) than that recorded in saline treated control animals, but not significantly different from the pre-injury brain free Mg concentration.



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  		Fig. 1. Brain intracellular free Mg concentration prior to and 4 hrs following severe diffuse traumatic brain injury in rats. Animals were administered n-acetyl tryptophan (NAT, substance P antagonist) or equal volume saline at 30 minutes after induction of injury. * p < 0.05.

 

   DISCUSSION
 
Previous studies of post-ischemic myocardial injury [9] have shown that Mg deficiency induces neurogenic inflammation that is mediated, in large part, by substance P. Moreover, administration of a substance P antagonist reduced the myocardial injury and improved outcome following ischemia [9]. Substance P is a neuropeptide localized to sensory nerve fibres whose release causes increased vascular permeability and vasogenic edema formation [12]. TBI also causes increased vascular permeability and vasogenic edema formation, and recent studies have identified that this event is associated with neuropeptide release [8], and in particular, substance P [13]. Substance P has also been implicated in depression and emesis [14], memory and learning [15], excitoxicity [16] and induction of immediate early genes [17]. Accordingly, there was the possibility that inhibition of posttraumatic substance P activity with a substance P antagonist may improve functional outcome. This hypothesis was subsequently confirmed in preliminary studies [10].

We have previously demonstrated that pharmacological interventions that improve functional outcome after TBI generally all improve brain free Mg concentration [7]. While the improvement in brain free Mg concentration may simply be a reflection of reduced neuronal injury, the fact that administration of Mg alone also improves functional outcome [3] suggests that restored brain Mg homeostasis may be a mechanism of action by which neuroprotective effects are mediated. In the present study, we have shown that administration of a substance P antagonist also improves brain free Mg concentration after TBI, raising the possibility that some of its neuroprotective actions may also be mediated by the Mg2+. Indeed, Mg has been shown to have effects on blood brain barrier permeability and vasogenic edema formation [18], learning and memory [19], affective disorders [20], and excitoxicty [21]. Nonetheless, substance P has a number of additional central nervous system effects that are unrelated to any known action of Mg [22,23]. Accordingly, substance P antagonists may be a novel multifactorial drug intervention for TBI that incorporates Mg-dependent and Mg-independent neuroprotective actions.

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

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This Article
Right arrow Abstract Freely available
Right arrow Figures Only
Right arrow Full Text (PDF)
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Vink, R.
Right arrow Articles by Cernak, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Vink, R.
Right arrow Articles by Cernak, I.

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