Effects of magnesium sulfate on Na ⁺,K ⁺ ‐ATPase and intracranial pressure level after cerebral ischemia

ARTICLE

Auteur(s) : Mehmet Erkan Ustun¹, Hulagu Bariskaner², Alper Yosunkaya³, Mehmet Gurbilek⁴, Necdet Dogan⁵

Laboratory of Pharmacology Department
¹ Association Professor. University of Selcuk, Meram Medicine Faculty, Neurosurgery Department Meram, Konya Turkey;
² Assistant Professor. University of Selcuk, Meram Medicine Faculty, Pharmacology Department Meram, Konya Turkey;
³ Assistant Professor. University of Selcuk, Meram Medicine Faculty, Anaesthesiology Department Meram, Konya, Turkey;
⁴ Professor. University of Selcuk, Meram Medicine Faculty, Biochemistry Department Meram, Konya, Turkey;
⁵ Professor. University of Selcuk, Meram Medicine Faculty, Pharmacology Department Meram, Konya, Turkey

Introduction

Normally, when energy is available, the postsynaptic response is terminated by reuptake of the excitatoring amino acids (EAAs), mainly glutamate and aspartate by extrusion of Ca^2 +  and Na⁺ from the intracellular space, and restoration of K⁺ and Cl^- gradients across the membranes. Na⁺,K⁺-ATPase activity plays an important role in the restoration of Na⁺ and K⁺ gradients across the membranes [1-3]. However, if energy is lacking due to ischemia or trauma, postsynaptic activation will be prolonged and enhanced and intracellular water content, Na⁺ and Ca^2 +  will also increase [4]. Increased Ca^2 +  triggers Ca-dependent lytic enzymes such as xanthine oxidase, phospholipase and ornithine decarboxylase [5, 6]. These enzymes cause elevation of reactive oxygen species (ROS), proteolysis, DNA degeneration, depletion of membrane phospholipids and destruction of membrane integrity. This results in altered permeability and further Ca^2 +  and Na⁺ influx that causes cytotoxic edema and elevation of intracranial pressure (ICP) [7, 8].

Mg^2 +  has opposite actions to Ca^2 +  in maintaining membrane integrity and permeability and also in transfer, storage and utilization of energy [9-11]. Mg^2 +  is also essential for Na⁺,K⁺-ATPase function. So Mg^2 + , by preventing cytotoxic edema, may be benefical in reducing ICP in ischemic or traumatic brain injury. We planned this study to see the effects of Mg^2 +  on Na⁺,K⁺-ATPase activity and ICP in cerebral ischemia.

Animals and methods

Animals were cared for in the Pharmacology Department of the Medical School of Selcuk University in accordance with the National Institutes of Health (NIH). “Guide for the care and use of laboratory animals”. These protocols were reviewed and approved by the Animal Use Committee of Selcuk University.

Thirty New Zealand male 7 day-old rabbits were used in the experiment. All animals were anesthetised with xylazine HCl 15 mg/kg and ketamine 30 mg/kg intramuscularly. Animals were paralyzed with pancuronium bromide 0.2 mg/kg and mechanically ventilated with oxygen and air (Harvard Apparatus South Natick MA). Body temperature was maintained at 37 ± 0.5 °C and values were recorded through a rectal probe (Pt 100, Yellow Springs Instrument). The trachea was then intubated. For recording systemic arterial blood pressure, the left femoral artery was cannulized and for intravenous injections, the right jugular vein was cannulized with polyethylene catheters. A pressure transducer (Grass PT300) was used for blood pressure, arterial blood gas values were evaluated with a GEM^® Premier Plush. A polygraph (Grass 79H) was used.

Anesthetised rabbits were fixated in a supine position and with a cervical incision, bilateral common carotid arteries were exposed. Anesthetised rabbits were then turned to the prone position. The head was elevated slightly and the posterior neck muscles were separated surgically to expose the atlanto-occipital membrane. A 24-gauge, 2-in catheter was directed through the atlanto-occipital membrane and into the cisterna magna. Entrance into the cisterna magna was confirmed by the appearance of cerebrospinal fluid (CSF) in the catheter. Intracranial pressure (ICP) was continuously monitored through this catheter inserted into the cisterna magna by a strain gauge transducer (Transpac IV, Abbott, Ireland). The level of the external auditory meatus was used as the zero reference for measuring CSF pressure throughout the study and preischemic measurements were taken. Then the scalp was shaved and swabbed with polyvinyl iodine. A 3 cm long vertical incision was made and frontoparietal regions were exposed on each side. Craniectomies were performed with a high-speed drill and rongeurs in both parietal regions. The craniectomies were 1 cm in diameter. The dura remained intact to prevent puncture or tearing of the cerebral cortex. The ICP of the rabbits were measured again in order to see the effect of craniectomy on ICP.

Then rabbits were randomly divided into three groups. In group 1 (n = 10) (control group) only craniectomy was performed for determining baseline levels and ischemia was not applied. In groups 2 (untreated group) (n = 10) and 3 (MgSO₄- treated group) (n = 10) bilateral common carotid arteries were clamped for 60 min to produce transient cerebral ischemia [12, 13]. Before the clamps were opened ICP was recorded. Group 2 received saline while in group 3, within 5 min after the clamps were opened, magnesium sulfate was administered 100 mg/kg via the jugular vein as a bolus injection. At the end of 60 min reperfusion, ICP was recorded again and approximately 0.25 g of brain samples were resected from both parietal regions (a total of 0.5 g) in all groups. Rabbits were sacrificed after the resections. The samples were stored below – 70 C until the homogenization procedure.

Biochemical studies

Brain cell membrane preparation

Brain cell membranes were prepared according to the method described by Harik et al. [14]. Frozen cerebral cortices were homogenized in 10 mM Tris-HCl buffer containing 0.32 M sucrose and 0.5 mM EDTA (pH 7.40). The homogenate was centrifuged at 1 000 g for 10 min. The supernatant was centrifuged again at 4 000 × g for 60 min. The final pellet was resuspended in the same Tris/EDTA buffer and used as the membrane fraction.

Determination of Na⁺,K⁺-ATPase activity

The rate of ATP hydrolysis was determined using a reaction mixture containing 50-100 µg of the membrane protein with and without ouabain [15]. The samples were incubated for 5 min at 37 ^oC prior to starting the reaction. The reaction was initiated by the addition of an ATP solution (adjusted to pH 7.4) and stopped after 5 min by the addition of 0.5 mL of 12.5% trichloroacetic acid. Samples then were centrifuged at 2 000 × g for 10 min. Aliquots of the supernatant were taken for an analysis of the inorganic phosphate using the method of Fiske and Subbarow [16]. Controls for the zero time period were prepared identically, expect that the reaction was stopped immediately after the addition of ATP to determine the concentration of Pi present in the assay salt solutions. The activity of Na⁺,K⁺-ATPase was determined by subtracting the enzyme activity in the presence of ouabain from the total activity in the absence of ouabain. Enzyme activity was expressed as µmol. Pi mg-l protein h-l.

Statistical analysis

One-way analysis of variance and Tuckey-HSD tests were used for the evaluation of the Na⁺,K⁺-ATPase results. The differences in ICP values between the three groups were analyzed with Kruskal-Wallis ANOVA and pre and postischemic recordings with the Bonferroni adjusted Mann-Whitney U test. Two-way ANOVA for repeated measures and the unpaired t test were used for evaluating arterial blood gas and hemodynamic results between the groups. The correlation between postischemic or postcraniectomy (120^th min) ICP values and Na⁺,K⁺-ATP ase was evaluated with the Pearson correlation test. P < 0.05 was considered significant.

Results

The Mean Arterial Pressure, pH, blood gases, body temperature and heart rate were similar in all groups before ischemia. There were significant differences in MAP, pH, blood gases and heart rate values between Group 1 and the other groups after 60 min ischemia (P < 0.05). Significant increases in PaCO₂ values and heart rate were found in Groups 2 and 3 in comparison with Group 1 at 120 min after ischemia (P < 0.05) (table I).

Table I. The mean arterial pressure (MAP), pH values, arterial oxygen (pO₂), carbon dioxide pressures (pCO₂), body temperature (^oC) and Heart rate (HR) of all groups (mean ± SD)

The tissue Na⁺,K⁺-ATPase (µmol.Pi.mg-l.protein.h-1) mean ± SD levels of each group are as follows: group 1- Na⁺,K⁺-ATPase; 6.42 ± 1.68, group 2- Na⁺,K⁺-ATPase; 2.58 ± 0.96, group 3 Na⁺,K⁺-ATPase; 5.16 ± 1.42. There were significant differences (P < 0.001) between Na⁺,K⁺-ATPase levels of group 1 and 2. The levels of group 1 were considered as the baseline levels for Na⁺,K⁺-ATPase. The Na⁺,K⁺-ATPase levels of group 2 were significantly different from group 3 (P < 0.05).

ICP (cmH₂O) levels before and after craniectomy of all groups were similar. Preischemic and postischemic ICP levels (60^th min) of groups 2 and 3 were significantly different (P < 0.001). Postischemic ICP levels at the 60^th min and 120^th min were significantly different only in group 3 (P < 0.05) (figure 1). The 120^th postischemic min ICP level of group 3 was similar to the preischemic level. The correlation (r) and P values were – 0.407 and 0.05 between Na⁺,K⁺-ATPase and ICP values (figure 2).

Discussion

The results of the present study demonstrate that postischemic treatment with Mg^2 +  after experimental cerebral ischemia (Cl) significantly attenuated the increase of ICP and significantly improved Na⁺,K⁺-ATPase activity. Na + -K + -ATPase activity has been shown to decline in cerebral ischemia and also in spinal cord injury model [17, 18]. Mg^2 +  is essential for Na + -K + -ATPase function, it effects metabolism, particularly phosphorylation reactions which generate ATP [19]. Mg^2 +  markedly inhibits high-energy phosphate breakdown during anoxia [18, 20]. It also activates phosphatase which hydrolyses and transfers organic phosphate groups and reactions which involve ATP [21]. Mg^2 +  activates the synthesis of membrane phospholipids and maintains the membrane integrity [11, 22]. Therefore, it can prevent Ca^2 +  influx due to the depletion of membrane phospholipids.

Depletion of Mg^2 +  exposes the neurons to the toxic effect of the EAAs and impaired Na⁺, K⁺ and Ca^2 +  gradients promote further damage to the injured brain [9]. In addition, intracellular Mg^2 +  has also been shown to have a voltage-gating role in NMDA receptors and to regulate the release of EAAs [23]. Since excessive activation of EAA receptors has been implicated in the onset of neuronal damage associated with cerebral ischemia, treatment with MgSO₄ may function by limiting excitotoxin-induced secondary neuronal damage [24, 25]. A variety of competitive and noncompetitive antagonists block the NMDA-gated channel. Several of these NMDA receptor antagonists have been investigated for their potential as treatments to decrease the effect of EAAs and reduce toxicity after brain injury [26, 28]. Although some noncompetitive NMDA receptor antagonists (MK-801, ketamine and phencylidine) have been shown to have a more potent protective effect after brain trauma, their severe side effects limit their usefulness in humans [26]. By comparison, as an NMDA receptor antagonist, Mg^2 +  is appealing as a safe therapeutic agent for the injured brain.

We used 7 day old rabbits because magnesium sulfate passes the immature blood-brain barrier better [29, 31]. Magnesium sulfate given prior to birth to pre-eclamptic mothers and mothers in preterm labour has, in retrospect, been found to be associated with a decreased incidence of both intraventricular haemorrhage and cerebral palsy, this may be due to the immature BBB in babies [32]. We used a dose of 100 mg/kg magnesium sulfate [33-35]. In our study, increases in PaCO₂ levels 60 min after ischemia in the untreated (Group II) and treated (Group III) groups were similar. This may indicate that cerebral ischemia induces a transient disturbance of pulmonary gas exchange. As described previously, the decrease in elevated ICP after ischemia with MgSO₄ treatment is due to some extent to the attenuated brain edema formation. There is no literature about the normal range of ICP in the rabbit. But the ICP levels of the control group and preischemic levels of groups 2 and 3 give us an idea about the normal levels and can be used as a guide because the increase after ischemia both in groups 2 and 3 is over two folds. Also, in group 3 the significant decrease in ICP 120 min after ischemia is important. The Mg^2 +  presumably decreased edema formation by a direct effect on regulation of normal intracellular Na⁺ and K⁺ gradients by improving the Na⁺-K⁺-ATPase activity, the impairment of which might enhance posttraumatic edema formation [9], secondly by an NMDA receptor antagonist effect which inhibits Ca^2 +  influx and protects neurons from the deletorius effect of EAAs and thus reduces cytotoxic brain edema [36, 37].

Intracellular Mg^2 +  levels have been shown to decrease rapidly after central nervous system injuries. This decrease in Mg^2 +  may adversely affect the various cellular processes that depend on Mg^2 + , thus leading to further cell injury and death [11, 27]. In vivo pretreatment with Mg^2 +  attenuated its decline in the brain tissue, significantly improved neurological outcome [38], and protected against irreversible damage after spinal ischemia [39], whereas preinjury dietary depletion of Mg^2 +  resulted in lower Mg^2 +  concentrations in the brain and worsened the neurological outcome [38]. Postinjury and postischemic intravenous Mg^2 +  treatment improved histological changes and neurological outcome in several studies [40]. Mg^2 +  treatment attenuated the increase of lactate and MDA in other studies [41, 42] and also attenuated [19, 36, 43-51] the decrease of endogen antioxidant activity in brain tissue after traumatic head injury in rabbits [52]. In contrast, in some studies, it has been postulated that Mg^2 +  treatment was not markedly neuroprotective after ischemia [13, 53] and it may be due to insufficient crossing of MgSO₄ through the blood-brain barrier [54]. Brewer et al. showed that i.v. MgSO₄ infusion, although significantly increasing plasma ionized Mg^2 +  concentration, does not increase ventricular CSF ionized Mg^2 +  concentration [54]. Esen et al. [37] postulated that magnesium seems to attenuate the BBB permeability defect. In another study, it has been demonstrated that in a subaracnoid hemorrhage model, i.v. administration of MgSO₄ dilated the spactic artery to 75% of the baseline level but topical administration dilated it to 150% [55]. So although i.v. administration of MgSO₄ has been reported to be beneficial, intrathecal administration will result in a high concentration in CSF which may be much more beneficial.

Conclusion

MgSO₄ is effective in suppressing the decrease in Na⁺,K⁺-ATPase levels in brain tissue and attenuating the increased ICP after cerebral ischemia. Recently a clinical study [56] showed some beneficial effects of magnesium but further clinical studies are warranted.

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