Available online http://ccforum.com/content/9/1/R18 ResearchFebruary 2005 Vol 9 No 1 Open Access Effect of magnesium sulfate administration on blood–brain barrier in a rat model of intraperitoneal sepsis: a randomized controlled experimental study Figen Esen1, Tulin Erdem2, Damla Aktan2, Mukadder Orhan3, Mehmet Kaya4, Haluk Eraksoy5, Nahit Cakar1 and Lutfi Telci1 1Professor, University of Istanbul, Istanbul Faculty of Medicine, Department of Anesthesiology and Intensive Care, Istanbul, Turkey 2Staff Anesthesiologist, University of Istanbul, Istanbul Faculty of Medicine Department of Anesthesiology and Intensive Care, Istanbul, Turkey 3MD, University of Istanbul, Istanbul Faculty of Medicine Department of Anesthesiology and Intensive Care, Istanbul, Turkey 4Professor, University of Istanbul, Istanbul Faculty of Medicine Department of Physiology, Istanbul, Turkey 5Professor, University of Istanbul, Istanbul Faculty of Medicine, Department of Infectious Disease and Clinical Microbiology, Istanbul, Turkey Corresponding author: Figen Esen, [email protected] Received: 1 September 2004 Critical Care 2005, 9:R18-R23 (DOI 10.1186/cc3004) Revisions requested: 23 September 2004 This article is online at: http://ccforum.com/content/9/1/R18 Revisions received: 14 October 2004 © 2004 Esen et al.; licensee BioMed Central Ltd. Accepted: 25 October 2004 This is an Open Access article distributed under the terms of the Published: 23 November 2004 Creative Commons Attribution License (http://creativecommons.org/ licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Introduction Permeability changes in the blood–brain barrier (BBB) and their possible contribution to brain edema formation have a crucial role in the pathophysiology of septic encephalopathy. Magnesium sulfate has been shown to have a protective effect on BBB integrity in multiple experimental models. In this study we determine whether magnesium sulfate administration could have any protective effects on BBB derangement in a rat model of sepsis. Methods This randomized controlled experimental study was performed on adult male Sprague– Dawley rats. Intraperitoneal sepsis was induced by using the infected fibrin–thrombin clot model. To examine the effect of magnesium in septic and sham-operated rats, a dose of 750 µmol/kg magnesium sulfate was given intramuscularly immediately after surgery. Control groups for both infected and sham- operated rats were injected with equal volume of saline. Those rats surviving for 24 hours were anesthetized and decapitated for the investigation of brain tissue specific gravity and BBB integrity by the spectrophotometric assay of Evans blue dye extravasations. Another set of experiments was performed for hemodynamic measurements and plasma magnesium level analysis. Rats were allocated into four parallel groups undergoing identical procedures. Results Sepsis significantly increased BBB permeability to Evans blue. The dye content of each hemisphere was significantly lower in the magnesium-treated septic rats (left hemisphere, 0.00218 ± 0.0005; right hemisphere, 0.00199 ± 0.0007 [all results are means ± standard deviation]) than in control septic animals (left hemisphere, 0.00466 ± 0.0002; right hemisphere, 0.00641 ± 0.0003). In septic animals treated with magnesium sulfate, specific gravity was higher (left hemisphere, 1.0438 ± 0.0007; right hemisphere, 1.0439 ± 0.0004) than in the untreated septic animals (left hemisphere, 1.0429 ± 0.0009; right hemisphere, 1.0424 ± 0.0012), indicating less edema formation with the administration of magnesium. A significant decrease in plasma magnesium levels was observed 24 hours after the induction of sepsis. The dose of magnesium that we used maintained the baseline plasma magnesium levels in magnesium-treated septic rats. Conclusions Magnesium administration attenuated the increased BBB permeability defect and caused a reduction in brain edema formation in our rat model of intraperitoneal sepsis. Keywords: blood–brain barrier, brain edema, magnesium, sepsis, septic encephalopathy BBB = blood–brain barrier; EB = Evans blue; SG = specific gravity. R18 Critical Care February 2005 Vol 9 No 1 Esen et al. Introduction NaCl]) and 160 µl (100 units/ml) of sterile human thrombin to Patients with severe sepsis often manifest symptoms of a 5 ml syringe. The resulting clot was then incubated at room encephalopathy. Acute alterations in mental status, which temperature for 30 min before implantation into the abdominal occur fairly frequently in septic patients, have been shown to cavity. The Escherichia coli strain was isolated from an intra- be associated with poor prognosis [1]. However, not much is abdominal collection from a patient with secondary peritonitis. known about the exact mechanism of brain injury in sepsis. The bacteria were inoculated into a brain heart infusion broth Studies have suggested that septic encephalopathy might (DIFCO Laboratories, Detroit, MI, USA) and incubated over- involve a disturbance of plasma and brain neutral amino acid night at 35°C. The count of E. coli was adjusted to 1.8 × 109 transport across the blood–brain barrier (BBB), similar to colony-forming units/ml with McFarland standard 6. After mak- those seen in porto-systemic encephalopathy. This process ing a 0.5 cm midline abdominal incision, the peritoneum was has been related to the breakdown of the BBB because opened and the prepared clot was injected into the peritoneal patients with septic encephalopathy have high protein levels in cavity directly from the syringe. Sham-operated rats had a ster- the cerebrospinal fluid [2]. Recently, derangements in the ile clot injected into their peritoneal cavity. To examine the BBB causing perivascular edema have been demonstrated in effect of magnesium in septic and sham-operated rats, a dose sepsis-induced pigs [3]. of 750 µmol/kg MgSO4 was given intramuscularly immediately after surgery. Control groups for both infected and sham-oper- Protective effects of magnesium sulfate (MgSO4) against ated rats were injected with an equal volume of saline. BBB breakdown after severe insulin-induced hypoglycemia have been reported in animals [4]. Similar effects of magne- After surgery, the animals were given 50 µl/g per hour of saline sium on BBB were also evident in a diffuse traumatic brain subcutaneously and were allowed to wake up while breathing injury model in rats [5-7]. spontaneously. They were returned to their cages and were allowed free access to water. Those rats surviving for 24 hours In summary, MgSO4 was shown to have a protective effect on after the surgery were anesthetized and decapitated for the BBB integrity in multiple experimental models. We hypothe- investigation of brain tissue specific gravity (SG) and BBB sized that MgSO4 will also protect against BBB derangements integrity. observed in sepsis and tested the hypothesis in a rat model of sepsis induced by an intraperitoneally inserted infected fibrin– We used the method described by Mikawa and colleagues [9] thrombin clot. to determine BBB integrity by Evans blue (EB) dye. EB dye (4 ml/kg, 2%) was administered intravenously and allowed to cir- Methods culate for 60 min. The animals were then perfused with saline One hundred and twenty-six male Sprague–Dawley rats through the left ventricle at a pressure of 110 mmHg until weighing 320–440 g were used in this study. Rats were pur- colorless fluid was obtained from the right atrium. Afterwards, chased from the Institute for Experimental Research and Appli- the brains were removed and dissected. Each hemisphere cation (Istanbul Medical Faculty), and were cared for before was weighed and the samples were then homogenized in 3.5 and during all stages of the experimental protocol in compli- ml phosphate-buffered saline and vortex-mixed for 2 min after ance with the applicable institutional guidelines and regula- the addition of 2.5 ml of 60% trichloroacetic acid to precipitate tions of the Institute for Experimental Medicine Research and protein. The samples were then cooled for 30 min and centri- Application. fuged for 30 min at 1000 r.p.m. The absorbance of the super- natants for EB dye was measured at 610 nm with a Rats were prepared for surgery under anesthesia with intra- spectrophotometer. EB dye content is expressed as µg/mg of muscular 100 µg/g ketamine (Parke-Davis, Morris Plains, NJ, brain tissue against a standard curve. USA) and 20 µg/g xylazine hydrochloride Rompun 2% (Bayer, Munich, Germany) and allowed to breathe spontaneously. The The method defined by Marmarou and colleagues was used loss of corneal reflex and no movement in response to a painful for the determination of SG [10]. We obtained 1 mm3 samples stimulus confirmed maintenance of adequate anesthesia for taken from the right and left hemispheres of each animal. Sam- the experimental procedure. The rats were subsequently rand- ples were placed into linear density gradient columns of kero- omized into one of four groups: sham control (C), sham control sene and bromobenzene. A calibration curve was determined MgSO4-treated (C-Mg), septic (S) and septic with MgSO4 (S- for each column by using anhydrous K2SO4 solutions of Mg). known SG (1.045, 1.040, 1.035 and 1.025). Brain tissue SG values were subsequently determined with this calibration Intraperitoneal sepsis was induced with the infected fibrin– curve. thrombin clot model described by Mathiak and colleagues [8]. Fibrin–thrombin clots were formed by adding