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Increased Na+,K+-ATPase activity in the rat brain after meningitis induction by Streptococcus pneumoniae

Published online by Cambridge University Press:  24 June 2014

Tatiana Barichello ,
Jaqueline S. Generoso ,
Andreza L. Cipriano ,
Renata Casagrande ,
Allan Collodel ,
Geovana D. Savi ,
Emilene B. S. Scherer ,
Janaína Kolling  and
Angela T. S. Wyse
Tatiana Barichello*
Affiliation:
Laboratório de Microbiologia Experimental and Instituto Nacional de Ciência e Tecnologia Translacional em Medicina, Programa de Pós-Graduaçã em Ciências da Saúde, Unidade Acadêmica de Ciências da Saúde, Universidade do Extremo Sul Catarinense, Criciúma, SC, Brazil
Jaqueline S. Generoso
Affiliation:
Laboratório de Microbiologia Experimental and Instituto Nacional de Ciência e Tecnologia Translacional em Medicina, Programa de Pós-Graduaçã em Ciências da Saúde, Unidade Acadêmica de Ciências da Saúde, Universidade do Extremo Sul Catarinense, Criciúma, SC, Brazil
Andreza L. Cipriano
Affiliation:
Laboratório de Microbiologia Experimental and Instituto Nacional de Ciência e Tecnologia Translacional em Medicina, Programa de Pós-Graduaçã em Ciências da Saúde, Unidade Acadêmica de Ciências da Saúde, Universidade do Extremo Sul Catarinense, Criciúma, SC, Brazil
Renata Casagrande
Affiliation:
Laboratório de Microbiologia Experimental and Instituto Nacional de Ciência e Tecnologia Translacional em Medicina, Programa de Pós-Graduaçã em Ciências da Saúde, Unidade Acadêmica de Ciências da Saúde, Universidade do Extremo Sul Catarinense, Criciúma, SC, Brazil
Allan Collodel
Affiliation:
Laboratório de Microbiologia Experimental and Instituto Nacional de Ciência e Tecnologia Translacional em Medicina, Programa de Pós-Graduaçã em Ciências da Saúde, Unidade Acadêmica de Ciências da Saúde, Universidade do Extremo Sul Catarinense, Criciúma, SC, Brazil
Geovana D. Savi
Affiliation:
Laboratório de Microbiologia Experimental and Instituto Nacional de Ciência e Tecnologia Translacional em Medicina, Programa de Pós-Graduaçã em Ciências da Saúde, Unidade Acadêmica de Ciências da Saúde, Universidade do Extremo Sul Catarinense, Criciúma, SC, Brazil
Emilene B. S. Scherer
Affiliation:
Laboratório de Neuroproteção e Doenças Metabólicas, ICBS, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil
Janaína Kolling
Affiliation:
Laboratório de Neuroproteção e Doenças Metabólicas, ICBS, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil
Angela T. S. Wyse
Affiliation:
Laboratório de Neuroproteção e Doenças Metabólicas, ICBS, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil
*
Professor Tatiana Barichello, Laboratório de Microbiologia Experimental, PPGCS, UNASAU, Universidade do Extremo Sul Catarinense, 88806-000 Criciúma, SC, Brazil. Tel: +55 48 3431 2643; Fax: +55 48 3443 4817; E-mail: tba@unesc.net
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Barichello T, Generoso JS, Cipriano AL, Casagrande R, Collodel A, Savi GD, Scherer EBS, Kolling J, Wyse ATS. Increase Na+,K+-ATPase activity in the rat brain after meningitis induction by Streptococcus pneumoniae.

Background: Pneumococcal meningitis is the most severe infection of the central nervous system with a mortality rate up to 20% and an adverse neurological result in up to 50% of survivors. A complicated series of interactions among the host immune response and oxidants seems to be responsible for meningitis associated brain dysfunctions. Na+,K+-ATPase is an essential enzyme responsible for generating and maintaining the membrane potential necessary for neural excitability, however, the Na+,K+-ATPase activity is altered in several illness;

Objective: The aim of this study is to evaluate the Na+,K+-ATPase activity in hippocampus and cortex of the rats submitted to pneumococcal meningitis.

Methods: Animals received 10 µl sterile saline as a placebo or an equivalent volume of Streptococcus pneumoniae to the concentration of 5 × 109cfu/ml and were killed at 24, 48, 72 and 96 h after meningitis induction. The brain structures, hippocampus and cortex, were immediately isolated on dry ice and stored at −80°C to analyse Na+,K+-ATPase activity.

Results: In the hippocampus, we verified the increase of Na+,K+-ATPase activity at 48, 72 and 96 h (p < 0.05) and in the cortex at 24 h (p < 0.05) after pneumococcal meningitis induction.

Conclusion: The Na+,K+-ATPase activity is under the control of a diversity of intracellular messengers that are able to modulate the function of the particular isozymes in a precise way. Furthermore, we verified that pneumococcal meningitis increased the Na+,K+-ATPase activity in hippocampus and cortex; this increase can be correlated with a compensatory mechanism in illness pathophysiology.

Keywords

brainmeningitisNa+K+-ATPaseStreptococcus pneumoniae
Type
Research Article
Information
Acta Neuropsychiatrica , Volume 24 , Issue 5 , October 2012 , pp. 301 - 305
Copyright
Copyright © Cambridge University Press 2011

Significant outcomes

  • The influence of pneumococcal meningitis in Na+,K+-ATPase activity.

  • Increased Na+,K+-ATPase levels may be related to neuronal damage.

  • Our study helps to understand better the pathophysiology of pneumococcal meningitis.

Limitations

  • Animal model.

  • The results in animal models should be interpreted with caution before correlate with the clinic.

  • This study evaluates the animal model of adult rats.

Introduction

Pneumococcal meningitis is characterised by an intense inflammation of the meninges (Reference Grandgirard and Leib1) affecting pia matter, arachnoid and subarachnoid space; this may result in brain damage in cortical and subcortical structures (Reference Meli, Christen, Leib and Täauber2). Patients who died from bacterial meningitis showed apoptosis in hippocampus and cortical necrosis (Reference Nau, Soto and Bruck3). Furthermore, surviving patients may suffer from permanent neurological sequelae such as deafness, blindness, learning impairments, sensorimotor deficits and seizure disorders (Reference Weisfelt, Van De Beek, Spanjaard, Reitsma and De Gans4,Reference Hoogman, Van De Beek, Weisfelt, De Gans and Schmand5). The bacterial growth inside the subarachnoid space initiates a complex immune response (Reference Coimbra, Voisin, De Saizieu, Lindberg, Wittwer, Leppert and Leib6). A complicated series of interactions among the host immune response, cytokines, chemokine (Reference Plata-Salaman7), matrix metalloproteinases, proteolytic enzymes and oxidants seems to be responsible for brain dysfunctions associated with meningitis (Reference Koedel, Scheld and Pfister8). In previous studies, we verified the increased cytokines and chemokine levels (Reference Barichello, Santos and Savi9), increased oxidative damage to proteins and lipid peroxidation in the first 24 h after pneumococcal meningitis induction (Reference Barichello, Savi and Silva10). Moreover the cytokines stimulates reactive oxygen species (ROS) in mitochondria by altering membrane permeability and by inhibiting the electron transport chain, thereby causing mitochondrial damage (Reference Moe, Marin-Garcia, Konig, Goldenthal, Lu and Feng11,Reference Mariappan, Soorappan, Haque, Sriramula and Francis12) and resulting in the inability to generate energy in the form of adenosine triphosphate (ATP) (Reference Stanley and Hoppel13,Reference Lopez-Campistrous, Hao and Xiang14). Na+,K+-ATPase (E.C.3.6.1.37) is an essential enzyme responsible for generating and maintaining the membrane potential necessary for neural excitability (Reference Iannello, Milazzo and Belfiore15), catalysing the activity uptake of K+ and extrusion of Na+ at the expense of hydrolysing ATP generated from cellular glycolysis and oxidative phosphorylation, thus generating steep concentrations gradients for these ions (Reference Mobasheri, Avila and Cózar-Castellano16). In the neurons, where it may use up to 70% of the cell's total energy consumption, the role of the Na+,K+-ATPase is crucial for basis metabolic requirements and for the specialised functions of nerve impulse transmission (Reference Blanco and Mercer17). Earlier studies have shown that Na+,K+-ATPase activity is altered in ischaemia (Reference Wyse, Streck, Worm, Wajner, Ritter and Netto18), neurodegenerative diseases (Reference Hattori, Kitagawa and Higashida19), with chronic administration of ketamine (Reference Luca, Fraga and Ghedim20), increased in animal models using lipopolysaccharide (Reference Dodrill, Beezhold, Meighan, Kashon and Fedan21) and resulted in increased Na+K+ pump activity (Reference Mccarter, Nierman and James22) in sepsis also. Furthermore, Na+,K+-ATPase activity was inhibited by interleukin-1β (IL-1β) in cardiac myocytes (Reference Kreydiyyeh and Al-Sadi23) and reduced cerebral cortical cell membrane Na+,K+-ATPase activity in meningitis by Escherichia coli in the newborn piglet (Reference Park, Chang and Lee24). According to Sellner et al. (Reference Sellner, Täauber and Leib25), a problem factor that contributes to this insufficient therapeutic in the meningitis treatment is our deficient understanding of the pathogenesis and pathophysiology of the bacterial infection in the central nervous system. Thus, the aim of our study was to evaluate the Na+,K+-ATPase activity in hippocampus and cortex of rats submitted to pneumococcal meningitis.

Materials and methods

Infecting organism

Streptococcus pneumoniae (serotype 3) was cultured overnight in 10 ml of Todd Hewitt Broth, diluted in fresh medium and grown to logarithmic phase. The culture was centrifuged for 10 min at (5000 ×g) and re-suspended in sterile saline to the concentration of the 5 × 109 cfu/ml. The accuracy of the inoculum size was confirmed by quantitative cultures (Reference Grandgirard, Schürch, Cottagnoud and Leib26,Reference Barichello, Pereira and Savi27). The bacterial concentration of 1 × 103 can be identified in cerebral spinal fluid (CSF) by Gram stain (Reference La Scolea and Dryja28).

Animal model of meningitis

Male Wistar rats (250–300 g body weight) from our breeding colony were used for the experiments. All the procedures were approved by the Animal Care and UNESC's Experimentation Committee, Brazil, and followed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) revised in 1996. All the surgical procedures and bacterial inoculations 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) (Reference Barichello, Santos and Savi9,Reference Barichello, Savi and Silva10,Reference Grandgirard, Steiner, Täauber and Leib29). Rats underwent a cisterna magna tap with a 23-gauge needle. The animals received either 10 µl of sterile saline as a placebo (n = 7) or an equivalent volume of S. pneumoniae suspension in the meningitis group (n = 37). At the inoculation time, animals received fluid replacement (2 ml of saline subcutaneously) and were subsequently returned to their cages. Following their recovery from anesthesia, animals were supplied with food and water ad libitum. Meningitis was documented by a quantitative culture of 5 µl of CSF obtained by puncture of the cistern magna (Reference Barichello, Santos and Savi9). Animals were decapitated at 24, 48, 72 and 96 h after meningitis induction. The brain structures, hippocampus and cortex, were immediately isolated on dry ice and stored at −80°C to analyse of Na+,K+-ATPase activity.

Tissue preparation

For preparation of synaptic plasma membrane and determination of Na+,K+-ATPase activity, hippocampus was homogenised in 10 volumes of 0.32 mM sucrose solution containing 5.0 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and 1.0 mM ethylenediaminetetraacetic acid, pH 7.4. Synaptic plasma membranes were prepared according to the method of Jones and Matus (Reference Jones and Matus30), with some modifications (Reference Wyse, Wajner, Brusque and Wannmacher31). The homogenate was centrifuged at 1000 ×g for 20 min; the supernatant was removed and centrifuged at 12.000 ×g for 20 min. The pellet was then suspended in hypotonic buffer (5.0 mM Tris–HCl buffer, pH 8.1), incubated at 0°C for 30 min, and applied on a discontinuous sucrose density gradient consisting of successive layers of 0.3, 0.8 and 1.0 M. After centrifugation at 69.000 ×g for 2 h, the fraction at the 0.8–1.0 M sucrose interface was taken as the membrane enzyme preparation.

Na+,K+-ATPase activity assay

The reaction mixture for Na+,K+-ATPase activity assay contained 5.0 mM MgCl2, 80.0 mM NaCl, 20.0 mM KCl and 40.0 mM Tris–HCl, pH 7.4, in final volume of 200 ml. The reaction was initiated by addition of ATP to a final concentration of 3.0 mM. Controls were carried out under the same conditions with the addition of 1.0 mM ouabain. Na+,K+-ATPase activity was calculated by the difference between the two assays, as described by Wyse et al. (Reference Wyse, Streck, Worm, Wajner, Ritter and Netto18). Released inorganic phosphate (Pi) was measured by the method of Chan et al. (Reference Chan, Delfer and Junger32). Specific activity of the enzyme was expressed as nmol Pi released per min per mg of protein. All samples were run in duplicates.

Protein measurement

Protein was measured by the Bradford method (Reference Bradford33) with bovine serum albumin used as standard.

Statistics

The variables were showed by mean ± SEM of five to six animals in each group. Differences among groups were evaluated using the variance analysis followed by Student–Newman–Keuls post hoc test. p-Values <0.05 was considered statistically significant.

Results

Na+,K+-ATPase activity was evaluated in hippocampus and cortex from adult Wistar rats submitted by pneumococcal meningitis. Our results showed that meningitis increased in Na+,K+-ATPase activity at 48, 72 and 96 h (p < 0.05; F = 14.992) in the hippocampus after meningitis induction (Fig. 1); furthermore, in the cortex, there were an increase in Na+,K+-ATPase activity only at 24 h (p < 0.05; F = 2.662) after meningitis induction when compared with the control group (Fig. 1).

Fig. 1. Na+,K+-ATPase activity in hippocampus (a) and cortex (b) after Streptococcus pneumoniae meningitis induction. Na+,K+-ATPase activity was calculated by released Pi and the specific activity of the enzyme was expressed as nmol Pi released per min per mg of protein. Results show the mean ± SEM of five to six animals in each group. Symbols indicate statistically significant when compared with sham group *p < 0.05.

Discussion

Bacterial cell wall components into the CSF are well known as inflammatory host response inducers (Reference Koedel, Scheld and Pfister8); so, the neuronal injury is mediated by the release of ROS, proteases, cytokines and excitatory amino acids, and is executed by the activation of transcription factors, caspases and others proteases (Reference Nau and Bruck34). In previous studies, we verified that the tumour necrosis factor-alpha levels increased in the first hours and then there was also an increase of the IL-1β, IL-6 and cytokine-induced neutrophil chemoattractant-1 levels (Reference Barichello, Pereira and Savi27), lipid peroxidation, protein carbonylation and decreased the protein integrity in hippocampus and cortex after pneumococcal meningitis induction (Reference Barichello, Savi and Silva10). However, in the mitochondrial respiratory chain these levels showed increases in the activities in hippocampus and cortex (Reference Barichello, Savi and Simões35). Na+,K+-ATPase is a key regulator of cellular ion homeostasis (Reference Mobasheri, Avila and Cózar-Castellano16). Our study showed that pneumococcal meningitis increased the Na+,K+-ATPase activity in two brain regions, in the hippocampus and cortex of adult rats in different times. In addition, the injury in bacterial meningitis is different in these brain areas and has been characterised by tissue necrosis in the cortical hemispheres and by apoptotic cell death in the hippocampal dentate gyrus (Reference Nau, Soto and Bruck3). In other severe neurological disorders, such as autism, the Na+,K+-ATPase activity also increased in the frontal cortex and cerebellum (Reference Ji, Chauhan, Brown and Chauhan36). Furthermore, recent studies have reported an association of cytokines with autism (Reference Molloy, Morrow and Meinzen-Derr37,Reference Ashwood and Wakefield38,Reference Croonenberghs, Bosmans, Deboutte, Kenis and Maes39) and increased the Na+,K+-ATPase activity in response to increased intracellular calcium (Reference Ji, Chauhan, Brown and Chauhan36). Lipopolysaccharide also increased Na+,K+-ATPase expression in airway epithelium (Reference Dodrill, Beezhold, Meighan, Kashon and Fedan21). Nevertheless, it is well known that lipopolysaccharide also increases the production of cytokines, chemokines, reactive oxygen and nitrogen species (Reference Basu and Fenton40). Patients in shock or sepsis have a high production rate of lactic acid that has traditionally been attributed to poor tissue perfusion and hypoxia (Reference Hotchkiss and Karl41). The increased glycolysis in these situations has been associated to increased activity of membrane ion pumps such as the Na+,K+-ATPase (Reference Mccarter, Nierman and James22). McCarter et al. (Reference Mccarter, Nierman and James22) showed that increased muscle lactate during sepsis correlates with evidence of elevated muscle Na+,K+-ATPase, but not with the evidence of impaired oxidative metabolism. Furthermore, plasma concentration of the lactate and epinephrine, a known stimulator of Na+,K+ pump, were increased in septic rats by E. coli(Reference Jones, Westfall and Sayeed42). Lactic acid is recognised as an important virulence factor for multiple streptococcal species (Reference Kling, Cavicchio, Sollinger, Madoff, Schnitzer and Kinane43) and can be associated with the increase of Na+,K+-ATPase activity. Their activity is under the control of a diversity of intracellular messengers that are able to modulate the function of the particular isozymes in a precise way (Reference Blanco and Mercer17). The sodium pump is not simply an ion transporter because the Na+,K+-ATPase isozymes have kinetic properties that are single; isozyme-specific regulation may be important in adapting Na+ pump function to the necessities of each cell (Reference Blanco and Mercer17,Reference Scheiner-Bobis44), and it is responsible for generating and maintaining membrane potential and thus disturbances in its activity could have grave consequences for neuronal functioning (Reference Mobasheri, Avila and Cózar-Castellano16). So, the present findings showed that pneumococcal meningitis increased the Na+,K+-ATPase activity; thus, toxic substances and proteases produced by bacteria can cause damage to neurons directly. For example, hemolysin and pneumolysin released by the bacteria are capable of causing damage to neurons, probably by promoting the influx of extracellular Ca+2(Reference Nau and Bruck34). Na+,K+-ATPase activity also increased in response to increased intracellular calcium (Reference Ji, Chauhan, Brown and Chauhan36). Our results should be interpreted with caution before correlation with the clinic; however, the rat model allows for a refined assessment of clinical and neurological symptoms (Reference Brandt45).

Acknowledgements

This research was supported by grants from CNPq, FAPESC, UNESC, Instituto Nacional de Ciência e Tecnologia Translacional em Medicina (INCT-TM) and L'Oréal-UNESCO Brazil Fellowship for Women in Science 2011.

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Figure 0

Fig. 1. Na+,K+-ATPase activity in hippocampus (a) and cortex (b) after Streptococcus pneumoniae meningitis induction. Na+,K+-ATPase activity was calculated by released Pi and the specific activity of the enzyme was expressed as nmol Pi released per min per mg of protein. Results show the mean ± SEM of five to six animals in each group. Symbols indicate statistically significant when compared with sham group *p < 0.05.