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Brain creatine kinase activity after meningitis induced by Streptococcus pneumoniae
Brain Research Bulletin 80 (2009) 85–88
Contents lists available at ScienceDirect
Brain Research Bulletin journal homepage: www.elsevier.com/locate/brainresbull
Research report
Brain creatine kinase activity after meningitis induced by Streptococcus pneumoniae Tatiana Barichello a,∗ , Geruza Z. Silva a , Geovana D. Savi a , Joana M. Torquato a , Ana L. Batista a , Giselli Scaini a , Gislaine T. Rezin a , Patricia M. Santos a , Gustavo Feier b , Emilio L. Streck a a Laboratório de Fisiopatologia Experimental, Programa de Pós-Graduac¸ão em Ciências da Saúde, Unidade Acadêmica de Ciências da Saúde, Universidade do Extremo Sul Catarinense, 88806-000 Criciúma, SC, Brazil b Laboratório de Neurociências, Programa de Pós-Graduac¸ão em Ciências da Saúde, Unidade Acadêmica de Ciências da Saúde, Universidade do Extremo Sul Catarinense, 88806-000 Criciúma, SC, Brazil
a r t i c l e
i n f o
Article history: Received 16 April 2009 Accepted 17 April 2009 Available online 3 May 2009 Keywords: Meningitis Streptococcus pneumoniae Central nervous system Creatine kinase
a b s t r a c t Bacterial meningitis due to Streptococcus pneumoniae is associated with a significant mortality rate and persisting neurologic sequelae including sensory-motor deficits, seizures, and impairments of learning and memory. Creatine kinase (CK) is an effective buffering system of cellular ATP levels in high-energy consuming tissues; a decrease in CK activity is associated with a neurodegenerative pathway that results in neuronal loss. Thus, the aim of this study was to evaluate brain CK activity after pneumococcal meningitis. The animals underwent a magna cistern tap receiving either sterile saline as a placebo or an equivalent volume of a S. pneumoniae suspension; they were killed 6, 12, 24 and 48 h after that, the brain was removed and hippocampus, striatum, cerebellum, cerebral cortex and prefrontal cortex were dissected and used for the determination of CK activity. We verified that CK activity was not altered 6 and 12 h after meningitis. Interestingly, 24 h after the induction of the meningitis we observed a decrease in CK activity. Finally, CK activity was not altered 48 h after meningitis. Although it is difficult to extrapolate our findings to the human condition, the inhibition of brain CK activity may be involved in the pathogenesis of pneumococcal meningitis. © 2009 Elsevier Inc. All rights reserved.
1. Introduction The estimated annual incidence of bacterial meningitis is 4–6 per 100,000 adults and Streptococcus pneumoniae and Neisseria meningitidis are the causative bacteria in 80% of cases [14]. S. pneumoniae is a gram-positive bacterium that may be found as a commensally of the human upper respiratory tract [24]. It has been suggested that pneumococcal meningitis is acquired via colonization of the nasopharynx, followed by bacteremia and invasion of the central nervous system [25]. The virulence factor are pneumolysin, peptidoglycan, pneumococcal surface proteins A and C, and pneumococcal surface adhesin A [24,25]. However, depending on host and bacterial factors not fully understood, pneumococci may spread to the middle ear, lung, or bloodstream and cause diseases such as otitis media, pneumonia, meningitis, and sepsis [24]. Pneumococcal meningitis is associated to a significant mortality rate of up to 30% and persisting neurologic sequelae including sensory-motor deficits, seizures, and impairments of learning and
∗ Corresponding author. Tel.: +55 48 3431 2539; fax: +55 48 3431 2644. E-mail address: [email protected] (T. Barichello). 0361-9230/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2009.04.011
memory in up to 50% of the survivors [31]. This host reaction determines the unfavorable outcome of the disease with neuronal injury including cortical necrosis and hippocampal apoptosis. Different animal models have been used to study the pathophysiology of brain damage in bacterial meningitis. In adult rats, meningitis induced by inoculating of the S. pneumoniae into the cisterna magna reproduces, at least in part, human histopathological findings [4,13,17]. Creatine kinase (CK, EC 2.7.3.2) is important for normal energy homeostasis by exerting several integrated functions, such as temporary energy buffering, metabolic capacity, energy transfer and metabolic control. The brain, like other tissues with high and variable rates of ATP metabolism, presents high phosphocreatine concentration and CK activity [5,26,32]. It is also known that a decrease in CK activity is associated with neurodegenerative pathways that result in neuronal death in brain ischemia, neurodegenerative diseases, bipolar disorder, and other pathological states [1,7,9,27]. In this context, several studies reported decreased CK activity in diseases affecting the central nervous system [1,7,9,27]. Therefore, the aim of this study is to evaluate brain CK activity in animals submitted to meningitis by S. pneumoniae inoculation into the cisterna magna.
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2. Materials and methods 2.1. Animals Male Wistar rats (300 g of body weight) were obtained from our breeding colony. The animals were housed five to a cage with food and water available ad libitum and were maintained on a normal 12-h light/dark cycle (lights on at 7:00 a.m.). This study was performed in accordance with the Brazilian Society for Neuroscience and Behavior (SBNeC) recommendations for animal care and with the approval of Ethics Committee from Universidade do Extremo Sul Catarinense. 2.2. Animal model of meningitis All surgical procedures and bacteria administrations were performed under anesthesia consisting of an intraperitoneal administration of ketamine (6.6 mg/kg), xylazine (0.3 mg/kg), and acepromazine (0.16 mg/kg) [4,13,14]. S. pneumoniae (ATCC 6303) was cultured overnight in Todd Hewitt broth and grown to logarithmic phase. In the morning of the experiment the bacteria were washed and resuspended in sterile 0.9% NaCl, and grown to logarithmic phase, 5 × 109 CFU/mL [13,17]. Rats underwent a basilar cistern tap with a 23-gauge needle. The position of the needle was verified by the free flow of clear cerebral spinal fluid. Cerebrospinal fluid was withdrawn and the animals received either 10 L of sterile saline as a placebo (sham) or an equivalent volume of the S. pneumoniae. At the time of inoculation, the animals received fluid replacement (10 mL of saline subcutaneously) and were returned to their cages [16]. Following their recovery from anesthesia, the animals were supplied with food and water ad libitum. Meningitis was documented by a quantitative culture of 5 L of CSF obtained by puncture of the cisterna magna immediately before decapitation and cultured quantitatively on sheep blood agar plates to document that they had meningitis. The number of bacteria in the CSF was determined by plating serial dilutions of 10 L of CSF on blood agar plates [4,12]. The animals received antibiotic therapy beginning at 8 h after induction (ceftriaxone, 100 mg/kg twice a day intraperitoneally); and were killed at predetermined time points (i.e. at 6, 12, 24 and 48 h after inoculation) by decapitation [4,12]. The brain was removed and cerebellum, prefrontal cortex, hippocampus, striatum (including nucleus accumbens), and cerebral cortex (the whole cortex except prefrontal cortex) were isolated and stored at 80 ◦ C. 2.3. Tissue and homogenate preparation
Fig. 1. Creatine kinase (CK) activity in cerebellum of rats after the induction of pneumococcal meningitis. Results are expressed as mean ± S.D. (n = 6) (nmol/min × mg protein). For more details, see Section 2. Different from sham group, *p < 0.05 (Student’s t test).
Fig. 2. Creatine kinase (CK) activity in prefrontal cortex of rats after the induction of pneumococcal meningitis. Results are expressed as mean ± S.D. (n = 6) (nmol/min × mg protein). For more details, see Section 2. Different from sham group, *p < 0.05 (Student’s t test).
Hippocampus, striatum, cerebellum, cerebral cortex and prefrontal cortex were homogenized (1:10, w/v) in SETH buffer, pH 7.4 (250 mM sucrose, 2 mM EDTA, 10 mM Trizma base, 50 IU/mL heparin). The homogenates were centrifuged at 800 × g for 10 min and the supernatants kept at −70 ◦ C until used for enzymes activity determination. The maximal period between homogenate preparation and enzyme analysis was always less than 5 days. Protein content was determined by the method described by Lowry et al. [23] using bovine serum albumin as standard. 2.4. Creatine kinase (CK) activity assay Creatine kinase activity was measured in brain homogenates pre-treated with 0.625 mM lauryl maltoside. The reaction mixture consisted of 60 mM Tris–HCl, pH 7.5, containing 7 mM phosphocreatine, 9 mM MgSO4 and approximately 0.4–1.2 g protein in a final volume of 100 L. After 15 min of pre-incubation at 37 ◦ C, the reaction was started by the addition of 0.3 mol of ADP plus 0.08 mol of reduced glutathione. The reaction was stopped after 10 min by the addition of 1 mol of p-hydroxymercuribenzoic acid. The creatine formed was estimated according to the colorimetric method of Hughes [15]. The color was developed by the addition of 100 L 2% -naphtol and 100 L 0.05% diacetyl in a final volume of 1 mL and read spectrophotometrically after 20 min at 540 nm. Results were expressed as units/min × mg protein.
Fig. 3. Creatine kinase (CK) activity in hippocampus of rats after the induction of pneumococcal meningitis. Results are expressed as mean ± S.D. (n = 6) (nmol/min × mg protein). For more details, see Section 2. Different from sham group, *p < 0.05 (Student’s t test).
2.5. Statistical analysis Data were analyzed by Student’s t test and are expressed as mean ± standard deviation. All analyses were performed using the Statistical Package for the Social Science (SPSS) software version 16.0.
3. Results In the present work, we evaluated CK activity in hippocampus, striatum, cerebellum, cerebral cortex and prefrontal cortex of rats submitted to meningitis by S. pneumoniae inoculation into the cisterna magna. Our results demonstrated that CK activity was not altered 6 and 12 h after meningitis. Interestingly, 24 h after the induction of the meningitis we observed a decrease in CK activity in cerebellum, prefrontal cortex, hippocampus, striatum and cerebral cortex (Figs. 1–5, respectively). Finally, CK activity was not altered 48 h after meningitis.
Fig. 4. Creatine kinase (CK) activity in striatum of rats after the induction of pneumococcal meningitis. Results are expressed as mean ± S.D. (n = 6) (nmol/min × mg protein). For more details, see Section 2. Different from sham group, *p < 0.05 (Student’s t test).
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cognitive impairment reported in patients affected by meningitis [31]. Although it is difficult to extrapolate our findings to the human condition, the inhibition of brain CK activity may be involved in the pathogenesis of pneumococcal meningitis. Conflict of interest The authors declare that they have not had any financial, personal or other relationships that have influenced the work. Acknowledgements Fig. 5. Creatine kinase (CK) activity in cerebral cortex of rats after the induction of pneumococcal meningitis. Results are expressed as mean ± S.D. (n = 6) (nmol/min × mg protein). For more details, see Section 2. Different from sham group, *p < 0.05 (Student’s t test).
4. Discussion The creatine/phosphocreatine/CK system is important for normal energy homeostasis by exerting several integrated functions, such as temporary energy buffering, metabolic capacity, energy transfer and metabolic control [1,5,26,32]. The brain of adult rats, like other tissues with high and variable rates of ATP metabolism, presents high phosphocreatine concentration and CK activity. It is well described that inhibition of CK activity has been implicated in the pathogenesis of a number of diseases, especially in the brain [7,9,27]. In present work we reported the effects of pneumococcal meningitis on brain CK activity. We showed that CK was not affected 6 and 12 h after meningitis, but was inhibited at 24 h; 48 h after meningitis the enzyme returned to normal activity. Several works suggest that neuronal injury in bacterial meningitis is a consequence of the direct toxicity of bacterial components and inflammatory and oxidative mechanisms [2,6,10,19,20,21]. However, few studies report brain energy metabolism parameters in meningitis. Ghielmetti et al. [11] showed that cortical ATP, ADP and total adenine nucleotides were decreased by approximately 25% in infant rats infected with S. pneumoniae. The CK molecule presents many cysteine residues in its structure [1,5,26,32]. Sulfhydryl groups of the enzyme can be a target for oxidation by nitric oxide and other free radicals leading to CK activity inhibition [33]. In this context, we also speculate that oxidative stress may be involved in the mechanism of CK activity inhibition. We demonstrated the inhibition of CK activity 24 h after the induction of meningitis in all brain areas evaluated in the present work. We have also previously observed that an increase in the oxidation of sulfhydryl groups and formation of carbonyl groups occurs in the brain of rats after meningitis [3]. For this reason we hypothesize that free radicals formed up to 24 h may be related to the inhibition of CK activity. The activity of CK returned to normal levels 48 h after meningitis; we hypothesize that the cells recovered antioxidant defenses, new CK molecules were synthesized, and/or the enzyme recovered from the insult. Another study showed that the apoptosis in the dentate gyrus of infant mouse infected with S. pneumoniae showed a peak 30 h after infection and was followed by a decrease in apoptosis, returning to normal levels in 40 h [31]. These data are in accordance to our results, where a decrease in the activity of the enzyme occurred 24 h after the induction of meningitis, close to the maximum peak of apoptosis, and was reestablished at 48 h. It has also been demonstrated that the creatine/phosphocreatine/CK circuit is involved in processes that involve habituation, spatial learning and seizure susceptibility [18,28,29]. In the present work we showed that CK was inhibited after meningitis in areas that are crucial for cognitive processes [8,30,22]. So, we also speculate that diminished CK may be involved in the
This work was supported by grants from Conselho Nacional de Pesquisa e Desenvolvimento (CNPq) and Universidade do Extremo Sul Catarinense (UNESC). References [1] R.H. Andres, A.D. Ducray, U. Schlattner, T. Wallimann, H.R. Widmer, Functions and effects of creatine in the central nervous system, Brain Res. Bull. 76 (2008) 329–343. [2] A. Aycicek, A. Iscan, O. Erel, M. Akcali, S. Selek, Total antioxidant/oxidant status in meningism and meningitis, Pediatr. Neurol. 35 (2006) 382–386. [3] T. Barichello, F.C. Petronilho, G.Z. Silva, B. Souza, G.D. Savi, G. Feier, J. Quevedo, F. Dal-Pizzol, Oxidative damage in the rat hippocampus and cortex after meningitis induced by Streptococcus pneumoniae, BMC Proc. 2 (2008) 6. [4] C.L. Bellac, R.S. Coimbra, F. Simon, H. Imboden, S.L. Leib, Gene and protein expression of galectin-3 and galectin-9 in experimental pneumococcal meningitis, Neurobiol. Dis. 28 (2007) 175–183. [5] S.P. Bessman, C.L. Carpenter, The creatine–creatine phosphate energy shuttle, Annu. Rev. Biochem. 54 (1985) 831–865. [6] S. Christen, M. Schaper, J. Lykkesfeldt, C. Siegenthaler, Y.D. Bifrare, S. Banic, S.L. Leib, M.G. Täuber, Oxidative stress in brain during experimental bacterial meningitis: differential effects of alpha-phenyl-tert-butyl nitrone and N-acetylcysteine treatment, Free Radical Biol. Med. 31 (2001) 754–762. [7] C.M. Comim, G.T. Rezin, G. Scaini, P.B. Di-Pietro, M.R. Cardoso, F.C. Petronilho, C. Ritter, E.L. Streck, J. Quevedo, F. Dal-Pizzol, Mitochondrial respiratory chain and creatine kinase activities in rat brain after sepsis induced by cecal ligation and perforation, Mitochondrion 8 (2008) 313–318. [8] M. D’Esposito, From cognitive to neural models of working memory, Philos. Trans. R. Soc. Lond. B: Biol. Sci. 362 (2007) 761–772. [9] P.B. Di-Pietro, M.L. Dias, G. Scaini, M. Burigo, L. Constantino, R.A. Machado, F. Dal-Pizzol, E.L. Streck, Inhibition of brain creatine kinase activity after renal ischemia is attenuated by N-acetylcysteine and deferoxamine administration, Neurosci. Lett. 434 (2008) 139–143. [10] J. Gerber, M. Lotz, S. Ebert, S. Kiel, G. Huether, U. Kuhnt, R. Nau, Melatonin is neuroprotective in experimental Streptococcus pneumoniae meningitis, J. Infect. Dis. 191 (2005) 783–790. [11] M. Ghielmetti, H. Ren, S.L. Leib, M.G. Täuber, S. Christen, Impaired cortical energy metabolism but not major antioxidant defenses in experimental bacterial meningitis, Brain Res. 976 (2003) 139–148. [12] D. Grandgirard, C. Schürch, P. Cottagnoud, S.L. Leib, Prevention of brain injury by the non-bacteriolytic antibiotic daptomycin in experimental pneumococcal meningitis, Antimicrob. Agents Chemother. 51 (2007) 2173–2178. [13] D. Grandgirard, O. Steiner, M.G. Täuber, S.L. Leib, An infant mouse model of brain damage in pneumococcal meningitis, Acta Neuropathol. 114 (2007) 609–617. [14] M. Hoogman, D. Van de Beek, M. Weisfelt, J. Gans, B. Schmand, Cognitive outcome in adults after bacterial meningitis, J. Neurol. Neurosurg. Psychiatry 78 (2007) 1092–1096. [15] B.P. Hughes, A method for estimation of serum creatine kinase and its use in comparing creatine kinase and aldolase activity in normal and pathologic sera, Clin. Chim. Acta 7 (1962) 597–604. [16] J.E. Irazuzta, R.K. Pretzlaff, B. Zingarelli, V. Xue, F. Zemlan, Modulation of nuclear factor-kB activation and decreased markers of neurological injury associated with hypothermic therapy in experimental bacterial meningitis, Crit. Care Med. 30 (2002) 2553–2559. [17] J.E. Irazuzta, R.K. Pretzlaff, B. Zingarelli, Caspases inhibition decreases neurological sequelae in meningitis, Crit. Care Med. 5 (2008) 1603–1606. [18] C.R. Jost, C.E. Van der Zee, H.J. Zandt, F. Oerlemans, M. Verheij, F. Streijger, J. Fransen, A. Heerschap, A.R. Cools, B. Wieringa, Creatine kinase B-driven energy transfer in the brain is important for habituation and spatial learning behaviour, mossy fibre field size and determination of seizure susceptibility, Eur. J. Neurosci. 15 (2002) 1692–1706. [19] S. Kastenbauer, U. Koedel, B.F. Becker, H.W. Pfister, Oxidative stress in bacterial meningitis in humans, Neurology 58 (2002) 186–191. [20] M. Klein, U. Koedel, H.W. Pfister, Oxidative stress in pneumococcal meningitis: a future target for adjunctive therapy? Prog. Neurobiol. 80 (2006) 269–280. [21] U. Koedel, H.W. Pfister, Oxidative stress in bacterial meningitis, Brain Pathol. 9 (1999) 57–67.
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[22] H. Lou, Etiology and pathogenesis of attention-deficit hyperactivity disorder (ADHD); significance of prematurity and perinatal hypoxic-haemodynamic encephalopathy, Acta Paediatr. 85 (1996) 1266–1271. [23] O.H. Lowry, N.G. Rosebough, A.L. Farr, R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193 (1951) 265–275. [24] A. Martner, C. Dahlgren, J.C. Paton, A.E. Wold, Pneumolysin released during Streptococcus pneumoniae autolysis is a potent activator of intracellular oxygen radical production in neutrophils, Infect. Immun. 76 (2008) 4079–4087. [25] D.N. Meli, S. Christen, S.L. Leib, M.G. Täuber, Current concepts in the pathogenesis of meningitis caused by Streptococcus pneumonia, Curr. Opin. Infect. Dis. 15 (2002) 253–257. [26] T. Schnyder, H. Gross, H. Winkler, H.M. Eppenberger, T. Wallimann, Crystallization of mitochondrial creatine kinase. Growing of large protein crystals and electron microscopic investigation of microcrystals consisting of octamers, J. Biol. Chem. 266 (1991) 5318–5322. [27] E.L. Streck, G. Amboni, G. Scaini, P.B. Di-Pietro, G.T. Rezin, S.S. Valvassori, G. Luz, F. Kapczinski, J. Quevedo, Brain creatine kinase activity in an animal model of mania, Prog. Neuropsychopharmacol. Biol. Psychiatry 31 (2007) 887–891.
[28] F. Streijger, C.R. Jost, F. Oerlemans, B.A. Ellenbroek, A.R. Cools, B. Wieringa, C.E. Van der Zee, Mice lacking the UbCKmit isoform of creatine kinase reveal slower spatial learning acquisition, diminished exploration and habituation, and reduced acoustic startle reflex responses, Mol. Cell. Biochem. 256 (257) (2004) 305–318. [29] F. Streijger, F. Oerlemans, B.A. Ellenbroek, C.R. Jost, B. Wieringa, C.E. Van der Zee, Structural and behavioural consequences of double deficiency for creatine kinases BCK and UbCKmit, Behav. Brain Res. 157 (2005) 219–234. [30] W.A. Suzuki, Making new memories: the role of the hippocampus in new associative learning, Ann. N. Y. Acad. Sci. 1097 (2007) 1–11. [31] D. van de Beek, B. Schman, J. de Gans, M. Weisfelt, H. Vaessen, J. Dankert, M. Vermeulen, Cognitive impairment in adults with good recovery after bacterial meningitis, J. Infect. Dis. 186 (2002) 1047–1052. [32] T. Wallimann, M. Wyss, D. Brdiczka, K. Nicolay, H.M. Eppenberger, Intracellular compartmentation, structure and function of creatine kinase isoenzymes in tissues with high and fluctuating energy demands: the ‘phosphocreatine circuit’ for cellular energy homeostasis, Biochem. J. 281 (1992) 21–40. [33] H. Wolosker, R. Panizzutti, S. Engelender, Inhibition of creatine kinase by Snitrosoglutathione, FEBS Lett. 392 (1996) 274–276.
Contents lists available at ScienceDirect
Brain Research Bulletin journal homepage: www.elsevier.com/locate/brainresbull
Research report
Brain creatine kinase activity after meningitis induced by Streptococcus pneumoniae Tatiana Barichello a,∗ , Geruza Z. Silva a , Geovana D. Savi a , Joana M. Torquato a , Ana L. Batista a , Giselli Scaini a , Gislaine T. Rezin a , Patricia M. Santos a , Gustavo Feier b , Emilio L. Streck a a Laboratório de Fisiopatologia Experimental, Programa de Pós-Graduac¸ão em Ciências da Saúde, Unidade Acadêmica de Ciências da Saúde, Universidade do Extremo Sul Catarinense, 88806-000 Criciúma, SC, Brazil b Laboratório de Neurociências, Programa de Pós-Graduac¸ão em Ciências da Saúde, Unidade Acadêmica de Ciências da Saúde, Universidade do Extremo Sul Catarinense, 88806-000 Criciúma, SC, Brazil
a r t i c l e
i n f o
Article history: Received 16 April 2009 Accepted 17 April 2009 Available online 3 May 2009 Keywords: Meningitis Streptococcus pneumoniae Central nervous system Creatine kinase
a b s t r a c t Bacterial meningitis due to Streptococcus pneumoniae is associated with a significant mortality rate and persisting neurologic sequelae including sensory-motor deficits, seizures, and impairments of learning and memory. Creatine kinase (CK) is an effective buffering system of cellular ATP levels in high-energy consuming tissues; a decrease in CK activity is associated with a neurodegenerative pathway that results in neuronal loss. Thus, the aim of this study was to evaluate brain CK activity after pneumococcal meningitis. The animals underwent a magna cistern tap receiving either sterile saline as a placebo or an equivalent volume of a S. pneumoniae suspension; they were killed 6, 12, 24 and 48 h after that, the brain was removed and hippocampus, striatum, cerebellum, cerebral cortex and prefrontal cortex were dissected and used for the determination of CK activity. We verified that CK activity was not altered 6 and 12 h after meningitis. Interestingly, 24 h after the induction of the meningitis we observed a decrease in CK activity. Finally, CK activity was not altered 48 h after meningitis. Although it is difficult to extrapolate our findings to the human condition, the inhibition of brain CK activity may be involved in the pathogenesis of pneumococcal meningitis. © 2009 Elsevier Inc. All rights reserved.
1. Introduction The estimated annual incidence of bacterial meningitis is 4–6 per 100,000 adults and Streptococcus pneumoniae and Neisseria meningitidis are the causative bacteria in 80% of cases [14]. S. pneumoniae is a gram-positive bacterium that may be found as a commensally of the human upper respiratory tract [24]. It has been suggested that pneumococcal meningitis is acquired via colonization of the nasopharynx, followed by bacteremia and invasion of the central nervous system [25]. The virulence factor are pneumolysin, peptidoglycan, pneumococcal surface proteins A and C, and pneumococcal surface adhesin A [24,25]. However, depending on host and bacterial factors not fully understood, pneumococci may spread to the middle ear, lung, or bloodstream and cause diseases such as otitis media, pneumonia, meningitis, and sepsis [24]. Pneumococcal meningitis is associated to a significant mortality rate of up to 30% and persisting neurologic sequelae including sensory-motor deficits, seizures, and impairments of learning and
∗ Corresponding author. Tel.: +55 48 3431 2539; fax: +55 48 3431 2644. E-mail address: [email protected] (T. Barichello). 0361-9230/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2009.04.011
memory in up to 50% of the survivors [31]. This host reaction determines the unfavorable outcome of the disease with neuronal injury including cortical necrosis and hippocampal apoptosis. Different animal models have been used to study the pathophysiology of brain damage in bacterial meningitis. In adult rats, meningitis induced by inoculating of the S. pneumoniae into the cisterna magna reproduces, at least in part, human histopathological findings [4,13,17]. Creatine kinase (CK, EC 2.7.3.2) is important for normal energy homeostasis by exerting several integrated functions, such as temporary energy buffering, metabolic capacity, energy transfer and metabolic control. The brain, like other tissues with high and variable rates of ATP metabolism, presents high phosphocreatine concentration and CK activity [5,26,32]. It is also known that a decrease in CK activity is associated with neurodegenerative pathways that result in neuronal death in brain ischemia, neurodegenerative diseases, bipolar disorder, and other pathological states [1,7,9,27]. In this context, several studies reported decreased CK activity in diseases affecting the central nervous system [1,7,9,27]. Therefore, the aim of this study is to evaluate brain CK activity in animals submitted to meningitis by S. pneumoniae inoculation into the cisterna magna.
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2. Materials and methods 2.1. Animals Male Wistar rats (300 g of body weight) were obtained from our breeding colony. The animals were housed five to a cage with food and water available ad libitum and were maintained on a normal 12-h light/dark cycle (lights on at 7:00 a.m.). This study was performed in accordance with the Brazilian Society for Neuroscience and Behavior (SBNeC) recommendations for animal care and with the approval of Ethics Committee from Universidade do Extremo Sul Catarinense. 2.2. Animal model of meningitis All surgical procedures and bacteria administrations were performed under anesthesia consisting of an intraperitoneal administration of ketamine (6.6 mg/kg), xylazine (0.3 mg/kg), and acepromazine (0.16 mg/kg) [4,13,14]. S. pneumoniae (ATCC 6303) was cultured overnight in Todd Hewitt broth and grown to logarithmic phase. In the morning of the experiment the bacteria were washed and resuspended in sterile 0.9% NaCl, and grown to logarithmic phase, 5 × 109 CFU/mL [13,17]. Rats underwent a basilar cistern tap with a 23-gauge needle. The position of the needle was verified by the free flow of clear cerebral spinal fluid. Cerebrospinal fluid was withdrawn and the animals received either 10 L of sterile saline as a placebo (sham) or an equivalent volume of the S. pneumoniae. At the time of inoculation, the animals received fluid replacement (10 mL of saline subcutaneously) and were returned to their cages [16]. Following their recovery from anesthesia, the animals were supplied with food and water ad libitum. Meningitis was documented by a quantitative culture of 5 L of CSF obtained by puncture of the cisterna magna immediately before decapitation and cultured quantitatively on sheep blood agar plates to document that they had meningitis. The number of bacteria in the CSF was determined by plating serial dilutions of 10 L of CSF on blood agar plates [4,12]. The animals received antibiotic therapy beginning at 8 h after induction (ceftriaxone, 100 mg/kg twice a day intraperitoneally); and were killed at predetermined time points (i.e. at 6, 12, 24 and 48 h after inoculation) by decapitation [4,12]. The brain was removed and cerebellum, prefrontal cortex, hippocampus, striatum (including nucleus accumbens), and cerebral cortex (the whole cortex except prefrontal cortex) were isolated and stored at 80 ◦ C. 2.3. Tissue and homogenate preparation
Fig. 1. Creatine kinase (CK) activity in cerebellum of rats after the induction of pneumococcal meningitis. Results are expressed as mean ± S.D. (n = 6) (nmol/min × mg protein). For more details, see Section 2. Different from sham group, *p < 0.05 (Student’s t test).
Fig. 2. Creatine kinase (CK) activity in prefrontal cortex of rats after the induction of pneumococcal meningitis. Results are expressed as mean ± S.D. (n = 6) (nmol/min × mg protein). For more details, see Section 2. Different from sham group, *p < 0.05 (Student’s t test).
Hippocampus, striatum, cerebellum, cerebral cortex and prefrontal cortex were homogenized (1:10, w/v) in SETH buffer, pH 7.4 (250 mM sucrose, 2 mM EDTA, 10 mM Trizma base, 50 IU/mL heparin). The homogenates were centrifuged at 800 × g for 10 min and the supernatants kept at −70 ◦ C until used for enzymes activity determination. The maximal period between homogenate preparation and enzyme analysis was always less than 5 days. Protein content was determined by the method described by Lowry et al. [23] using bovine serum albumin as standard. 2.4. Creatine kinase (CK) activity assay Creatine kinase activity was measured in brain homogenates pre-treated with 0.625 mM lauryl maltoside. The reaction mixture consisted of 60 mM Tris–HCl, pH 7.5, containing 7 mM phosphocreatine, 9 mM MgSO4 and approximately 0.4–1.2 g protein in a final volume of 100 L. After 15 min of pre-incubation at 37 ◦ C, the reaction was started by the addition of 0.3 mol of ADP plus 0.08 mol of reduced glutathione. The reaction was stopped after 10 min by the addition of 1 mol of p-hydroxymercuribenzoic acid. The creatine formed was estimated according to the colorimetric method of Hughes [15]. The color was developed by the addition of 100 L 2% -naphtol and 100 L 0.05% diacetyl in a final volume of 1 mL and read spectrophotometrically after 20 min at 540 nm. Results were expressed as units/min × mg protein.
Fig. 3. Creatine kinase (CK) activity in hippocampus of rats after the induction of pneumococcal meningitis. Results are expressed as mean ± S.D. (n = 6) (nmol/min × mg protein). For more details, see Section 2. Different from sham group, *p < 0.05 (Student’s t test).
2.5. Statistical analysis Data were analyzed by Student’s t test and are expressed as mean ± standard deviation. All analyses were performed using the Statistical Package for the Social Science (SPSS) software version 16.0.
3. Results In the present work, we evaluated CK activity in hippocampus, striatum, cerebellum, cerebral cortex and prefrontal cortex of rats submitted to meningitis by S. pneumoniae inoculation into the cisterna magna. Our results demonstrated that CK activity was not altered 6 and 12 h after meningitis. Interestingly, 24 h after the induction of the meningitis we observed a decrease in CK activity in cerebellum, prefrontal cortex, hippocampus, striatum and cerebral cortex (Figs. 1–5, respectively). Finally, CK activity was not altered 48 h after meningitis.
Fig. 4. Creatine kinase (CK) activity in striatum of rats after the induction of pneumococcal meningitis. Results are expressed as mean ± S.D. (n = 6) (nmol/min × mg protein). For more details, see Section 2. Different from sham group, *p < 0.05 (Student’s t test).
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cognitive impairment reported in patients affected by meningitis [31]. Although it is difficult to extrapolate our findings to the human condition, the inhibition of brain CK activity may be involved in the pathogenesis of pneumococcal meningitis. Conflict of interest The authors declare that they have not had any financial, personal or other relationships that have influenced the work. Acknowledgements Fig. 5. Creatine kinase (CK) activity in cerebral cortex of rats after the induction of pneumococcal meningitis. Results are expressed as mean ± S.D. (n = 6) (nmol/min × mg protein). For more details, see Section 2. Different from sham group, *p < 0.05 (Student’s t test).
4. Discussion The creatine/phosphocreatine/CK system is important for normal energy homeostasis by exerting several integrated functions, such as temporary energy buffering, metabolic capacity, energy transfer and metabolic control [1,5,26,32]. The brain of adult rats, like other tissues with high and variable rates of ATP metabolism, presents high phosphocreatine concentration and CK activity. It is well described that inhibition of CK activity has been implicated in the pathogenesis of a number of diseases, especially in the brain [7,9,27]. In present work we reported the effects of pneumococcal meningitis on brain CK activity. We showed that CK was not affected 6 and 12 h after meningitis, but was inhibited at 24 h; 48 h after meningitis the enzyme returned to normal activity. Several works suggest that neuronal injury in bacterial meningitis is a consequence of the direct toxicity of bacterial components and inflammatory and oxidative mechanisms [2,6,10,19,20,21]. However, few studies report brain energy metabolism parameters in meningitis. Ghielmetti et al. [11] showed that cortical ATP, ADP and total adenine nucleotides were decreased by approximately 25% in infant rats infected with S. pneumoniae. The CK molecule presents many cysteine residues in its structure [1,5,26,32]. Sulfhydryl groups of the enzyme can be a target for oxidation by nitric oxide and other free radicals leading to CK activity inhibition [33]. In this context, we also speculate that oxidative stress may be involved in the mechanism of CK activity inhibition. We demonstrated the inhibition of CK activity 24 h after the induction of meningitis in all brain areas evaluated in the present work. We have also previously observed that an increase in the oxidation of sulfhydryl groups and formation of carbonyl groups occurs in the brain of rats after meningitis [3]. For this reason we hypothesize that free radicals formed up to 24 h may be related to the inhibition of CK activity. The activity of CK returned to normal levels 48 h after meningitis; we hypothesize that the cells recovered antioxidant defenses, new CK molecules were synthesized, and/or the enzyme recovered from the insult. Another study showed that the apoptosis in the dentate gyrus of infant mouse infected with S. pneumoniae showed a peak 30 h after infection and was followed by a decrease in apoptosis, returning to normal levels in 40 h [31]. These data are in accordance to our results, where a decrease in the activity of the enzyme occurred 24 h after the induction of meningitis, close to the maximum peak of apoptosis, and was reestablished at 48 h. It has also been demonstrated that the creatine/phosphocreatine/CK circuit is involved in processes that involve habituation, spatial learning and seizure susceptibility [18,28,29]. In the present work we showed that CK was inhibited after meningitis in areas that are crucial for cognitive processes [8,30,22]. So, we also speculate that diminished CK may be involved in the
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