Increased calcium influx triggers and accelerates cortical spreading depression in vivo in male adult rats

Neuroscience Letters 558 (2014) 87–90

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Increased calcium influx triggers and accelerates cortical spreading depression in vivo in male adult rats Daniel Torrente a , Rosângela Figueiredo Mendes-da-Silva b , Andréia Albuquerque Cunha Lopes b , Janneth González a , George E. Barreto a , Rubem Carlos Araújo Guedes b,∗ a b

Departamento de Nutrición y Bioquímica, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá, DC, Colombia Departamento de Nutric¸ão, Centro de Ciências da Saúde, Universidade Federal de Pernambuco, Recife, PE, Brazil

h i g h l i g h t s • • • • •

We topically applied ionophore A23187 in rat cortex to increase calcium influx. This ionophore (10–100 ␮M) dose-dependently accelerates spreading depression (CSD). Topical application of a much higher dose of this compound (2 mM) triggers CSD. Increased Ca2+ influx is suggested as a key element in the CSD induction mechanism. Data stimulate and justify further experiments on brain disorders related to CSD.

a r t i c l e

i n f o

Article history: Received 26 August 2013 Received in revised form 26 October 2013 Accepted 6 November 2013 Keywords: Ca2+ influx Ionophore Cortical spreading depression Rat

a b s t r a c t Cortical spreading depression (CSD) is a depolarization wave associated with neurological disorders such as migraine, cerebral ischemia and traumatic brain injury. The mechanism of action of this phenomenon still remains unclear. Although it is suggested that extracellular K+ accumulation contributes to CSD, other ions may play a relevant role in the mechanism of propagation of the wave. In this context, we hypothesize that Ca2+ may play an important function in the wave propagation. Our results demonstrate that enhancing Ca2+ influx into the cells by topical cortical application of the ionophore A23187 (10 ␮M, 50 ␮M and 100 ␮M solutions) increases the velocity of CSD propagation in a dose-dependent manner, and a much higher dose of this compound (2 mM) triggers CSD. In conclusion, increased Ca2+ influx can be a key element in the induction mechanism of the CSD, and should be assessed in further experimental strategies targeting brain disorders related to CSD. © 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Cortical spreading depression (CSD) is a reversible depolarization wave associated with failure of brain ion homeostasis, release of excitatory amino acids, increased energy metabolism and changes in cerebral blood flow that propagates across the brain gray matter at low velocity, usually in the range of 2–5 mm/min [1,2]. In normal brain CSD usually has to be induced by a perturbation of the brain homeostasis such as electrical, mechanical or chemical stimulation [1]. In neurological disorders such as migraine, cerebral ischemia and traumatic brain injury, CSD may play a key role

∗ Corresponding author at: Departamento de Nutric¸ão, Centro de Ciencias da Saúde, Universidade Federal de Pernambuco, Rua Professor Moraes Rego, S/N Cidade Universitária, 50670-901 Recife, PE, Brazil. Tel.: +55 81 21268936; fax: +55 81 21268473. E-mail addresses: [email protected], [email protected] (R.C.A. Guedes). 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.11.004

as a pathophysiological mechanism in brain damage [2]. Clinical relevance of CSD in other neurological disorder and mechanisms of action in the trigger of this phenomenon still remains unclear [3]. Is has been suggested that extracellular K+ accumulation may initiate CSD [3], making topical KCl application a useful model to trigger this phenomenon on in vivo models [4] and is the most reliable method in the context of reproducible results [5,6]. Although a change in K+ homeostasis is important, other ions may be relevantly involved in the mechanism of propagation of the CSD wave. It is proposed that Ca2+ may play an important function in the wave propagation, since reversible changes in Ca2+ homeostasis are also observed in CSD [7]. Moreover, it is thought that release of K+ into the extracellular space might be mediated by Ca2+ influx to the cell [3], though it is unclear whether Ca2+ is critical for initiation or propagation of CSD. Based on these observations, it is possible that Ca2+ may play a role in the pathophysiology of CSD. In this context, a commonly used molecule to increase the intracellular concentration of Ca2+ is

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the ionophore A23187, which allows the influx of divalent ions into the cells, specifically Ca2+ [8,9]. Owing that Ca2+ may be playing a role in CSD physiopathology, in the present study we used this compound to stimulate extracellular Ca2+ influx and determine its effect on CSD propagation.

2. Materials and methods 2.1. Animals Adult male Wistar rats, weighing 280–350 g were used in this study. The animals were housed in a room with controlled temperature (23 ± 2 ◦ C) and maintained on a 12-h light/dark cycle (lights on at 7:00 a.m.). The animals were handled in accordance with the standards of the Ethics Committee for Animal Research, of the Universidade Federal de Pernambuco, Brazil, which comply with the “Principles of Laboratory Animal Care” (NIH; Bethesda, USA).

2.2. Cortical topical ionophore A23187 treatment Ionophore A23187 (Sigma, St. Louis, USA) was diluted in DMSO and solutions at (␮M) 10, 50 and 100 were topically applied in three groups of male adult rats (n = 5, n = 6 and n = 5, respectively). This topical application was performed over the intact dura-mater, on two circular portions (3–4 mm diameter) of the cortical surface, where the recording electrodes were placed. After 1–2 h of baseline CSD recording, ionophore was applied during the last 15 min of the 30 min interval between two consecutive CSD-eliciting KClstimulations. At the end of the 15 min topical application, the treated region was dried out with a piece of cotton immediately before the next CSD episode was elicited. CSD propagation velocities recorded after topical application of ionophore were compared to mean values obtained in the same animals during the baseline period. As a rule, topical ionophore application was repeated three times (see Fig. 1A). After the third ionophore application, the treated cortical region was abundantly washed out with Ringer solution and the CSD recording continued during 1–2 h (recovery period). To determine the effect of ionophore at higher concentrations, 5 animals that recovered from 10 and 50 ␮M ionophore were topically treated with a 2 mM solution.

2.3. CSD recording For the CSD recording, the rats were anesthetized i.p. with a mixture of 1000 mg/kg urethane plus 40 mg/kg chloralose, and three trephine holes were drilled on the right side of the skull. These holes were aligned in the frontal-occipital direction and were parallel to the midline. CSD was elicited at 30 min intervals by 1-min topical application of 2% KCl solution (approximately 270 mM) to the anterior hole (2 mm in diameter) drilled at the frontal region. The two other holes (3–4 mm in diameter) on the parieto-occipital region served as recording places. The direct-current (DC) slow potential change accompanying CSD were continuously recorded for about 7.5 h, using two Ag–AgCl agar-Ringer electrodes (one in each hole) against a common reference electrode of the same type placed on the nasal bones (see diagram in the lower part of Fig. 1B). The amplitude and duration of the negative DC potential change typical of CSD, as well as its propagation velocity, were calculated. The CSD velocity of propagation was calculated from the time required for a CSD wave to pass the distance between the two cortical recording points. During the recording period, rectal temperature was maintained at 37 ± 1 ◦ C by means of a heating blanket. At the end of the session, the animal was killed with an overdose of anesthetic.

Fig. 1. (A) diagram of the protocol for ionophore topical cortical application during the CSD recording session. After a control (baseline) recording period, ionophore was applied three times (rectangle), and this was followed by a recovery period. The equidistant vertical dark lines indicate KCl stimulation at 30-min intervals, necessary to elicit CSD. (B) Slow potential change (P) recordings in adult rats (80–120 days old), showing the effect of topical applications of ionophore (10, 50 and 100 ␮M) on the latency for a CSD to cross the distance between the recording points 1 and 2 shown in the skull diagram. This diagram also shows the common reference electrode (R) on the nasal bones, and the stimulation point where KCl was applied for 1 min (horizontal black bars at the beginning of P1 traces). The vertical bars correspond to 10 mV (negative upward). The recovery from the topical molecule effect was reached 30–60 min after ionophore removal. Note the amplitude increase, in the 100 ␮M ionophore application (right column), on the recording point 2 (where ionophore was applied), but not on the point 1 (used as control).

2.4. Statistics CSD amplitudes and durations before and after topical application of the ionophore were compared with the paired t-test. CSD velocities were compared using ANOVA followed by post hoc (Tukey) test. Differences were considered significant when p < 0.05 3. Results 3.1. Ionophore decreases latency and increases CSD velocity in male rats To assess the effect of the Ca+2 influx on the CSD parameters, we applied the ionophore topically at different concentrations (10, 50 and 100 ␮M) during 15 min over the intact dura-mater at one of the trephine holes used for the electrophysiological recordings. Our results showed that 10 ␮M and 50 ␮M ionophore did not affect the amplitude and duration of the CSD, whereas 100 ␮M ionophore

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Table 1 Amplitudes (in mV) and durations (in s) of the negative DC slow potential shift of spreading depression in the rat cortex, elicited before, during and after (recovery period) topical cortical application of 10 ␮M, 50 ␮M and 100 ␮M solutions of ionophore A23187. Each rat was tested with a single ionophore concentration. Data are expressed as mean ± standard deviation. Before ionophore

During

After ionophore

CSD amplitude (mV) 12.06 ± 5.15 10 ␮M 10.32 ± 8.31 50 ␮M 11.32 ± 6.52 100 ␮M

11.85 ± 1.75 11.32 ± 7.26 30.85 ± 4.90*

11.33 ± 3.57 10.85 ± 9.64 30.67 ± 7.83*

CSD duration (s) 10 ␮M 62.56 ± 14.98 50 ␮M 60.87 ± 4.09 100 ␮M 65.94 ± 14.13

64.79 ± 10.30 56.22 ± 7.74 64.52 ± 14.15

62.76 ± 6.02 57.19 ± 8.92 65.14 ± 16.35

* Indicate values significantly higher than the corresponding ‘before’ value (p < 0.05; paired t-test).

increased the wave amplitude, which did not return to the basal values (Table 1). CSD wave latency and velocity were unaffected by 10 ␮M ionophore (Fig. 1B, left column). On the other hand, at higher concentration (50 ␮M), it decreased the latency for KCl to elicit a CSD wave and increased the CSD propagation velocity (17.7%) crossing the distance between the two recording points; after ionophore removal, the CSD latency and propagation velocity returned to the pretreatment levels (Fig. 1B, middle column). Although 100 ␮M ionophore decreased wave latency and increased velocity by 20.7%, CSD propagating wave did not return to control levels after ionophore removal (Fig. 1B, right column). The quantitative data of the CSD velocities are illustrated in Fig. 2, panel A. 3.2. Ionophore A23187 induces CSD in male rats In order to analyze the effect of high intracellular Ca2+ concentration, and observe whether this concentration in the cell could play an important role in CSD, we used topical application of 2 mM ionophore in 5 animals that recovered from 10 and 50 ␮M ionophore (see Section 2). Our result showed that at this higher concentration, the ionophore immediately induces CSD in all rats tested. The application of ionophore on the recording points P1 and P2 induced the CSD wave first in electrode 1 and 2, respectively (Fig. 2B). This finding demonstrated that the use of this ionophore mimics the effect of the 2% KCl, used to elicit the CSD. Since different concentrations of ionophore (10, 50, 100 ␮M and 2 mM) were dissolved in 1%, 2.5%, 5% and 10% DMSO, we further determined the effect of this solvent in CSD wave propagation; our results showed that topical application of DMSO at various concentration did not induce any significant change in the wave (data not shown).

Fig. 2. (A) Enhancements of CSD-velocity in adult rats, after topical cortical application of Ionophore A23187 solution during the 15 min immediately preceding CSD elicitation with KCl. A23187 was topically applied to a circular area (3–4 mm diameter) of the parietal cortical surface (recording place) on the intact dura-mater. Ionophore solutions were used with the following concentrations (␮M) 10, 50 and 100; Control: baseline CSD velocities, before topical treatment. Ionophore: maximal increase of CSD velocities obtained after topical application. Recovery: CSD velocities after cortical removal of the ionophore. Data are expressed as mean ± SEM; * indicates significant difference compared to control condition. (B) Induction of CSD by 2 mM topical ionophore application indicated by the horizontal bars; Ionophore P1: represents the application of the drug in the electrode position 1 (see diagram in Fig. 1B). Ionophore P2: represents the application of the drug in the electrode position 2.

4. Discussion CSD represents a pathophysiological phenomenon that could increase brain damage in migraine, cerebral ischemia and TBI [2] and clarifying the mechanisms of action of this phenomenon may help to design new approaches for the treatment of these disorders. In the present study we found that topical cortical ionophore application increased the velocity of CSD propagation and a higher dose of this compound triggered CSD, suggesting a role for Ca2+ influx into the cells in CSD. Calcium ionophore A23187 had been previously used to investigate the regulatory activities, and metabolism of Ca2+ in biological systems [10,11]. Effects of this compound are attributed to the electrically neutral exchange of Ca2+ for 2H. Although this compound can transport other divalent ions, such as Mg+ , it has a higher selectivity for calcium [8,9]. In the present work, we hypothesized

that the relationship between intracellular and extracellular calcium concentration was playing a role in propagating CSD in brains stimulated with ionophore. For example, calcium entry activates Ca2+ -dependent K+ channels, releasing K+ into the extracellular space [3], thus contributing to a faster accumulation of extracellular K+ . Moreover, this could be accompanied by membrane depolarization due to Ca2+ influx and K+ outflux, and consequently activation of voltage dependent Ca2+ channel leading to an increase in Ca2+ influx that activates proteins to release excitatory amino acids (Glutamate) [12]. In this context, this may explain a possible alternative pathological event associated to CSD, along with accumulation of extracellular K+ [3,13]. Interestingly, 10 ␮M ionophore did not exert effect on CSD parameters (amplitude, duration and propagation velocity). It might be explained owing to the exchange of Ca2+ into the membrane in that may be slower and could not trigger the

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mechanisms above mentioned. Furthermore, cells present mechanisms for decreasing intracellular Ca2+ levels and might buffer the amount of Ca2+ that crossed the membrane [14,15]. The CSD triggering effect of 2 mM ionophore may be similar to that suggested above; however, the difference relies on that the administration of higher ionophore concentrations (2 mM) might induce cycles of repetitive and frequent depolarizations and repolarizations in cell membrane, thus inducing CSD [3]. This hypothesis works under the premise that Ca2+ influx and K+ outflow release excitatory amino acids into the extracellular space and excite others CNS cells, thus propagating the CSD wave. Although the most important mechanism triggering CSD are related to K+ and glutamate [16,17], other studies proposed that Ca2+ might play an important role in this phenomena [18,19]. Also, it is assumed that different ion concentration could massively depolarize cells that in turn will release glutamate, exciting and propagating this stimulus into the neighboring cells [20]; altogether, these observations suggest that Ca2+ influx to the cell might explain this successive depolarization process. In conclusion, influx of extracellular Ca2+ facilitated CSD velocity, and higher influx of calcium triggers the CSD. These results suggest that Ca2+ has a key role in the induction mechanism of the CSD, and should be considered in future experimental strategies targeting brain disorders related to CSD. Acknowledgments This work was supported in part by PUJ grants IDs 5024 and 4367, and PROLAB IBRO/LARC CNPq – Convenio Brazil/Colombia to GEB, and by CNPq (INCT de Neurociência Translacional; No. 573604/2008-8); Edital MCT/CNPq 14/2010 (No. 477456/2010-3) and IBN-Net/Finep (No. 4191) to RCAG. References [1] A.A.P. Leão, Spreading depression of activity in the cerebral cortex, J. Neurophysiol. 7 (1944) 359–390. [2] M. Lauritzen, J.P. Dreier, M. Fabricius, J.A. Hartings, R. Graf, A.J. Strong, Clinical relevance of cortical spreading depression in neurological disorders: migraine, malignant stroke, subarachnoid and intracranial hemorrhage, and traumatic brain injury, J. Cereb. Blood Flow Metab. 31 (2011) 17–35.

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