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Neuroprotective properties of l -carnosine in the brain slices exposed to autoblood in the hemorrhagic stroke model in vitro
Regulatory Peptides 167 (2011) 65–69
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Regulatory Peptides j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / r e g p e p
Neuroprotective properties of L-carnosine in the brain slices exposed to autoblood in the hemorrhagic stroke model in vitro A.Kh. Khama-Murad, A.A. Mokrushin, L.I. Pavlinova ⁎ Pavlov Institute Physiology, Russian Academy Sciences, Saint-Petersburg, Russia
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Article history: Received 16 April 2009 Received in revised form 1 December 2009 Accepted 25 November 2010 Available online 9 December 2010 Keywords: Surviving brain slices Edema Focal potentials L-carnosine
a b s t r a c t Neuroprotective properties of L-carnosine have been studied in our in vitro model on olfactory cortex slices of hypertensive rats under a long autoblood (blood clot) influence. Application of L-carnosine (5 mg/ml) on slices before autoblood influence leads to restoration of the activity of glutamatergic and GABA-ergic receptors inhibited in the presence of autoblood and interferes with swelling of slices. L-Carnosine protects a bioelectric activity of nervous cells in case of long influence of autoblood and also renders an anti edema effect. This model of hemorrhagic stroke may provide a perspective for investigating the mechanisms of neuroprotection. Published by Elsevier B.V.
1. Introduction Intracerebral hemorrhage as a result of bleeding leads to neuronal injury through primary factors, including oxidative stress and excitotoxicity [1,2], as well as secondary factors that include edema formation or swelling of a nervous tissue due to increase in the water content [3–5]. Intracerebral hemorrhage has no effective treatment. The neuronal cells of the central nervous system are susceptible to various forms of stroke including hemorrhage. Pharmaceutical pretreatment of stroke may prevent or reduce cellular injury and facilitate subsequent brain recovery. Carnosine (beta-alaninyl-Lhistidine), a specific constituent of excitable tissues of vertebrates and an endogenously synthesized dipeptide, acts as a free radical scavenger [6,7]. Besides it protects nervous cells as was shown in various in vitro models [8–10]. Carnosine exhibits a significant antioxidant protecting effect in case of the brain damaged by ischemic injury and hypobaric hypoxia [11–17]. It has gained great interest because of its potential use as a neuroprotective compound in clinical practice. The elaboration of neuroprotective drugs against insult requires specially developed pharmacological model in vitro. Brain slices are widely used to study ischemia [18,19]. Earlier we proposed a new in vitro model of hemorrhagic stroke which enables to reveal an excitotoxic effect of autoblood and blood clot on bioelectrical activity of neurons mediated by NMDA, AMPA and GABAergic receptors in the surviving olfactory cortex slices [20,21]. Protective effects of carnosine
⁎ Corresponding author. Tel.: +7 812 6842039. E-mail address: [email protected] (L.I. Pavlinova). 0167-0115/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.regpep.2010.11.007
on electrogenesis and edema formation in the injured brain tissue were investigated in this study. 2. Materials and methods 2.1. Tissue preparation and hemorrhagic stroke modeling All animals used in this study were treated with observance of recommendations on ethics of work with the animals offered European Communities Council Direction (86/609 EEC). Olfactory cortex slices were prepared from brain of hypertensive SHR rat males (approximately 200 g body weight) taking into consideration that high blood pressure is known to represent a rather reliable prognostic factor of outcome of intracerebral hemorrhage [22]. After decapitation, slices about 400 μm in thickness prepared within 1 min were placed into artificial cerebrospinal fluid (ACSF), consisted of (in mM): NaCl— 124; KCl—5; CaCl2—2.6; KH2PO4—1.24; MgSO4—1.2; NaHCO3—3; glucose—10; Tris–HCl—23; equilibrated with O2, with osmolarity of 295–305 mOsm (OMT-5-01, “Burevestnic”, Russia) for preincubation during 30 min. The concentrations of Ca2+ and Mg2+ were optimized for maximal synaptic activity in olfactory cortex. The temperature was 37 °C, pH 7.2–7.3. After incubation in ACSF at the interval (30, 60, 120, and 360 min) the control slices were placed in perfusion chamber to record the electrophysiological parameters (amplitudes of AP LOT, AMPA and NMDA EPSP, IPSPslow) and were taken for water content determination. To simulate hemorrhagic injury other group of slices was placed in autoblood (3 ml) for electrophysiological parameters and water content study at above indicated terms. The term 360 min was used as maximal, because this interval is corresponded to “time
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window” of the reversibility of the damaged nervous cells when electrogenesis in olfactory slices could be restored [20]. For determination of the L-carnosine protective properties and for comparison its effects with such ones of pharmaceutical preparation the slices were pretreated with L-carnosine (5 mg/ml of the incubation medium) or vitamins C (1.2 mg/ml) and D (0.4 mg/ml), or gliatilin (0.02 μl/ml) during 30 min and then placed in autoblood. Orthodromic stimulation of the lateral olfactory tract (LOT) was conducted using rectangular pulses with a duration of 0.05–0.1 ms and an intensity of 1–3 V with aid of the electrical stimulator (ESU-1, Russia) through platinum bipolar electrodes. Frequency of electric LOT stimulation was 0.003 Hz. Focal potentials (FPs) from slices were recorded using glass microelectrodes filled with 1 M NaCl (resistance 1–5 MΩ) conducted with amplifier (Russia). The recording point was located in the focus of maximal activity at a depth of 270–300 μm. The indifferent silver electrode was located in the chamber. FPs were amplified and then computer-processed using special home-made software. The rate of perfusion of ACSF in perfusion chamber was 2 ml/min. The following individual components of FPs were analyzed: the compound action potential of the lateral olfactory tract (AP LOT, presynaptic component FP), and the non-N-methyl-D-aspartate (nNMDA) and N-methyl-D-aspartate (NMDA) receptor components of the excitatory postsynaptic potentials (EPSPs); the first of these is activated by [alpha]-amino-3-hydroxy-5-methylisoxalol-4-propionic acid (AMPA), the second with NMDA [23–26] referred to as AMPA and NMDA EPSPs; as well as slow (GABABergic) inhibitory postsynaptic potential (IPSPs) which is generated at GABAB receptors activation (Fig. 1). Amplitudes of these components were measured from the isoline to the peak level [23]. The amplitudes of AMPA EPSP were measured over a 1.5–2 ms window centered at the peak of the response. Peak NMDA EPSP was measured as the average potential observed in a 7–8 ms window. Pharmacological evidence of AMPA and NMDA components of focal potential were performed by adding 25 μM CNQX and/or 50 μM APV respectively to the ACSF as previously described [23]. The technique of controlled experiment is used to standardize conditions of individual experiments. To test own neurotrophic influence of L-carnosine on parameters of glutamatergic and GABAergic synaptic cell activity in olfactory cortex slices we tested the following L-carnosine concentrations: 50 μM, 250 μM, 500 μM, 2 mM, 10 mM and 20 mM. To test protective properties of L-carnosine we determined the level of the cell injury and the efficacy of an L-carnosine protection in the neuronal activity recovery by comparing the FP amplitudes in slices pretreated with L-carnosine (5 mg/ml or 20 mM) after 360 min
Fig. 1. Representative response of bioelectrical activity recorded in olfactory cortex slice (FP). AP LOT, action potential of LOT fibers; AMPA EPSP and NMDA EPSP, components of excitatory postsynaptic potential; IPSPs, slow (GABABergic) inhibitory postsynaptic potential; dotted line represents isoline; arrows show the measuring modes of amplitudes of individual FP components.
of autoblood (blood clot) action with the control values obtained on separate group of slices. 2.2. Determination of total tissue water Percent water and swelling were determined in olfactory cortex slices by wet and dry weight measurements before and after autoblood exposure as described [18,19]. Slices after preincubation in the control medium or after pretreatment with L-carnosine (5 mg/ml of ACSF); vitamin C (1.2 mg/ml); vitamin D (0.4 mg/ml) or gliatilin (0.02 μl/ml) were placed on a filter paper and the excess fluid quickly removed, without extracting water from the tissue. Then slices were weighed on a small piece of preweighed aluminum foil (wet weight, g). The slices were weighed again after incubation with autoblood or in ACSF for 360 min (wet weight, g). After that slices were dried in an oven at 85 °C overnight and re-weighed (dry weight, g). The water content (W) of the slice was calculated as W (wet weight − dry weight) (dry weight−1) and given as g H2O (g dw− 1). The changes in W quantified the swelling of the slice tissue and expressed in Fig. 5 as percentage to control level. All chemical components were from Sigma except gliatilin and vitamins C and D. Gliatilin (choline acetate) commonly used in clinic for treatment of the ischemic and hemorrhagic strokes was from Italpharmeco C. A., Italy; Vitamins C and D were pharmaceutics. The data were processed statistically using nonparametric Wilcoxon– Mann–Whitney U test. The differences were significant at p ≤0.05. 3. Results 3.1. Neurotrophic effects of L-carnosine Carnosine is a dipeptide which is highly concentrated in mammalian olfactory sensory neurons. The biological function of carnosine in the nervous system remains unclear. Besides prevalent evidence concerning carnosine's protective role, it has been suggested that it as co-transmitter or modulator could be implicated in some forms of functional plasticity [27]. Early it was shown that L-carnosine (μM concentrations) evokes inward currents itself in olfactory bulb neurons in cultured slices [28]. At the same time in analogous neuronal culture L-carnosine did not evoke a membrane current or affect currents evoked by glutamate, GABA or glycine even though applied in mM concentrations [29]. In the present study we previously investigated the modifications of the evoked cell activity in control slices in the presence of different L-carnosine concentrations (Fig. 2). L-Carnosine showed dual effect on synaptic activity in neurons of olfactory cortex. In response to incubation with L-carnosine (50 μM) the amplitudes of AP LOT (18%, U = 10, n = 12, p ≤ 0.05); AMPA EPSP (35%, U = 12, n = 12, p ≤ 0.05); IPSPs (34%, U = 9, n = 12, p ≤ 0.05) were increased in control tissue. The change of the NMDA EPSP amplitude was unreliable. The raising of the L-carnosine concentrations (250 μM, 500 μM, 2 mM, 10 mM, and 20 mM) decreased the amplitudes of all FPs components to control level. Under the same conditions L-carnosine induced the modification of synaptic plasticity and facilitated long-term posttetanic potentiation. Electrical tetanization of LOT was accompanied with LTP induction generally prevailed over LTD [30]. Since higher L-carnosine concentrations locate in linear section of dose–response curve and these concentrations correspond to the intrinsic level in structures of the mammalian olfactory sensory analyzer, we used 20 mM of L-carnosine (Fig. 2) in subsequent study. 3.2. Amplitudes of FPs under autoblood influence (blood clot) To monitor chronic alterations of the neuronal excitability after prolonged autoblood action up to 360 min the method of slice
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Fig. 2. Neurotrophic effects of L-carnosine. A dose–response data. Note that, low L-carnosine concentrations increased the activity of FP components (conductive fibers— AP LOT, GABABergic inhibitory postsynaptic processes and postsynaptic ionotropic glutamatergic mechanisms—AMPA EPSP). High L-carnosine concentrations stabilized the activity of the FP components.
incubation in whole autoblood without anticoagulant was employed. Such approach enables to study the cell activity under the blood clot formation. We investigated the parameters of evoked FPs in slices incubated in autoblood at 30, 60, 120, and 360 min. Slices were in clot at 37 °C without oxygen delivery. Each time point of experiments had own controls that were handled identically to the treatment conditions. The modifications of the amplitude of FP components are presented in Fig. 3a. The conductive fibers of LOT—the axons of the mitral cells were primarily activated, but response of postsynaptic processes to autoblood (or more exactly clot) action was different at the first time point. Thus, the amplitude of AMPA EPSP and IPSPs decreased compared with the control, whereas the activity of NMDA-dependent processes was already blocked (Fig. 3a). The amplitudes of postsynaptic FP components progressively reduced to 360 min (for AMPA EPSP—8%, U = 9, n = 12, p ≤ 0.05; for NMDA EPSP—1%, U = 1, n = 12, p ≤ 0.05; for IPSPs—3%, U = 3, n = 12, p ≤ 0.05, as compared to control). At this time point the amplitude of AP LOT reduced too (39%, U = 13, n = 8, p ≤ 0.05). Exciting AMPA dependent and inhibitory GABA-ergic mechanisms depressed progressively during the contact with autoblood. NMDA-dependent mechanism was the most vulnerable and its blockade occurred already within the 1st hour of the autoblood action. Evidently the basic mechanisms of electrogenesis in the olfactory cortex slices being in contact with blood clot during 360 min were largely damaged (Figs. 3b and 4). The doubling of a power of electric stimulus failed to retrieve the amplitudes of AP LOT, AMPA and NMDA EPSP components. 3.3. Effects of carnosine pretreatment In the slices pretreated with L-carnosine and then placed in autoblood for 360 min the amplitudes of AP LOT, AMPA and NMDA EPSPs, IPSPs were similar to control values (Figs. 3b and 4). So, activity of presynaptic processes–the amplitude of AP LOT recovered to control level (95%, U = 28, n = 12, p ≥ 0.05, as compared to control). The amplitudes of exciting postsynaptic processes (AMPA and NMDA EPSPs) rose to 90% and 85%, correspondingly, compared with control values (U = 23, n = 12, p ≥ 0.05; U = 21, n = 12, p ≥ 0.05). The amplitude of IPSPs of inhibitory GABA-ergic mechanisms was restored too (85%, U = 25, n = 12, p ≥ 0.05). Therefore, severe damage of the neuronal activity induced by autoblood was overcome by L-carnosine.
Fig. 3. The activities of synaptic glutamatergic and GABABergic mechanisms in olfactory cortex slices (a) and protective effect of the L-carnosine pretreatment (b) under the neurotoxic autoblood (blood clot) action. a—time point of autoblood action: 1—30; 2—60; 3—120; 4—360 min. Isoline is marked by dotted line. All FP components in slices rapidly reduced under the autoblood action. The amplitudes of postsynaptic components (AMPA and NMDA EPSP, and IPSPs) decreased as early as first hour. Activity of conductive fibers was more stable under autoblood influence. b—pretreatment of slices with L-carnosine effectively defended the electrical cell activity, especially the most vulnerable components of FP—NMDA EPSP and IPSPs, from the subsequent injured autoblood action. Calibration: 0.1 mV; 7 ms for panels a and b. During the FPs registration the electronic device for artefact-rejection was used.
The prime temporal phase of brain edema is supposed to connect with the clot formation in the first hours after hemorrhagic stroke. We attempted to interfere in the process of edema formation having in mind the multiple neuroprotective properties of L-carnosine. The model of hemorrhagic stroke in vitro does very well for it. It is established, that brain slices gain water during incubation in vitro in normal, oxygenated ACSF [18,19,31]. We initially assessed the time course of this water gain in control slices and revealed that water content after 360 min of incubation reached significance of 111–113% (U = 21, p ≥ 0.05) compared with basal water content (100%). We then examined whether the exposure of brain tissue to autoblood in vitro leads to pathological swelling. Swelling increased further during subsequent contact with autoblood. Water content in the slices increased significantly after incubation in autoblood during 360 min (144%, U = 8, n = 12, p ≤ 0.05) (Fig. 5). L-Carnosine applied before the autoblood interfered with swelling of olfactory cortex slices. Water content in pretreated slices was similar to control at all time points of the autoblood contact. Pretreatment of slices with L-carnosine prevented their severe swelling up to 360 min of autoblood action: 144%—autoblood, 108%—L-carnosine (U= 12, n = 12, p ≤ 0.05). Pretreatment of slices with gliatilin—an anti-stroke preparation in concentration of 0.02 μg that corresponds to the doze applied in clinic (1000 mg/70 kg of weight of the person) had no any effect. The water content of slices reached 140% compared with control at the 360 min time point and the difference of water gain in these slices and in slices incubated with autoblood was unreliable (U = 21, n = 8, p ≥ 0.05). Among the antioxidants that showed some efficacy in the models of brain injury, vitamin C (ascorbate) plays an appropriate part [35,36]. Exogenously applied ascorbate was able to compensate for loss of any other single component of the antioxidant network in
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Fig. 4. Carnosine provides complete restoration of the cell activity in the olfactory cortex slices depressed by 360-min autoblood action. Amplitudes of the FPs components (presynaptic—AP LOT; postsynaptic—glutamatergic AMPA and NMDA EPSP and IPSPs as GABAB component) in slices of the control group incubated in ACSF at 37 °C were taken as 100% (empty columns, n = 8). In slices exposed to autoblood during 360 min the amplitudes of AMPA EPSP, NMDA EPSP and IPSPs were inhibited, the amplitude of AP LOT decayed more than half compared to the control (black columns, n = 12). Preincubation of slices with L-carnosine (5 mg/ml in ACSF) during 30 min is able to protect the mechanisms of electrogenesis and to restore the amplitudes of FPs in slices after their subsequent contact with autoblood during 360 min (dashed columns, n = 16) (*p ≤ 0.05 versus control; + p ≤ 0.05 versus autoblood action).
guinea-pig and rat brain slices at the model of H2O2 pathology [37]. Prevention of edema induced by ischemia was achieved by ascorbate application in vitro [19,35]. In the model of hemorrhagic stroke pretreatment of slices with vitamin C resulted in moderate anti edema effects especially after 60 and 120 min of the total incubation time, but water content in such slices was significantly higher versus control for the 360 min time point (135%, U = 21, n = 13, p ≥ 0.05). May be, the reduction of stroke complications induced by ascorbate depends on a specific dose and the severity of stroke [36]. It is known that carnosine could act as natural scavenges from free radical species and by-products of membrane lipids peroxidation [38]. We compared the effect of L-carnosine with the effect of vitamin D3. Vitamin D3 has been shown to play an important role in antioxidative mechanisms. It acutely prevented zinc-induced oxidative injuries acting as a terminator of the lipid peroxidation [39]. In this model of hemorrhagic stroke pretreatment with vitamin D decreased, water content in slices at all time intervals (p b 0.05 for each time point) (Fig. 5). It is obviously that carnosine may offer pluripotent favorable effects by prevention of initial hyperactivation and subsequent total inhibition of glutamate receptor activity induced by contact with autoblood. Our data suggest that L-carnosine involves in synaptic mechanisms as co-mediator at own brain tissue level. Besides, L-carnosine in high exogenous concentration actively modulates glutamatergic and GABAergic synaptic
Fig. 5. Pretreatment of slices with L-carnosine prevented their severe swelling induced by autoblood. Slices contacted with autoblood after their pretreatment with L-carnosine or compared preparations during 30 min. Tissue water content was determined at each time point: ACSF—n = 10; autoblood—n = 12; L-carnosine—n = 15; vitamins C and B—n = 13; gliatilin—n = 8. *p≤ 0.05 for carnosine versus each autoblood points. C—basal water content after slice preparation was taken as 100%.
processes protecting them against injurious blood action. L-Carnosine as antioxidative agent may to secure tissue integrity by decreasing the primary tissue swelling which is a result of the neurotoxic autoblood (blood clot) action.
4. Discussion In the present study, we have shown an attenuation of edema by and a recovery of neuronal activity mediated by NMDA and AMPA glutamate and GABA receptors being inhibited by autoblood exposure. It has been shown that synaptic glutamate receptor activation leads to neuronal swelling in substrate deficient human brain and in normoxic brain slices [31,32] and contributes to the initial cell swelling that accompanied anoxic depolarization in neonatal cerebrocortical brain slices [33]. The antagonists of ionotropic glutamate receptor differently affected swelling, a cocktail of these agents was more effective than AP5 or CNQX alone [19], so multiple glutamate receptor types seem to be involved in edema formation during ischemia [32]. Last time L-carnosine is an object of intensive study of many researchers. Protective, antioxidant and antiapoptotic properties of this endogenous dipeptide provide high importance in nervous cell defence in the brain injury. Carnosine compensates deficit in antioxidant protective system of brain damaged by ischemic injury [11–13]. The preventing effect of L-carnosine is accompanied with pronounced protective effect on neurological symptoms and animal mortality in the models of global cerebral ischemia [12,34]. Carnosine appears to influence deleterious pathological processes in focal cerebral ischemia decreasing reactive oxygen species levels and infarct size. It is neuroprotective when administered at time points both before and after the induction of ischemia [15,16]. Carnosine also increased the resistance of neuronal membranes to the in vitro induced oxidation and suppressed the glutamate receptor hyperactivation [7–9,14,15]. In our previous study L-carnosine modulated neuronal function via altered NMDA and AMPA glutamate and GABA receptor activation and promoted the induction and maintenance of long-term potentiation (LTP) in the olfactory cortex slices [30]. The mechanisms by which L-carnosine might act in our model are manifold, from the initial urgent protection of the neuronal activity interfering to decayed receptor activation, especially NMDAmediated, and consequent cell swelling that accompanies ionotropic glutamate-receptor activation. L-carnosine
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Thus, pretreatment of slices with L-carnosine may rescue the mechanisms of synaptic plasticity in olfactory cortex by improving function of glutamate receptors and may offer the significant antiswelling effect in tissue damaged by autoblood (blood clot) contact. The observed disruption of the synaptic receptor activity, appearance of edema in nervous tissue contacting with autoblood (blood clot) suggests that these events may be targets for therapeutic intervention with L-carnosine. It is suggested that carnosine's therapeutic potential should be explored in the nervous tissue in the clinic. References [1] Facchinetti F, Dawson VL, Dawson TM. Free radicals as mediators of neuronal injury. Cell Mol Neurobiol 1998;18:667–82. [2] Qureshi AI, Ali Z, Suri FK, Shuaib A, Baker GA, Todd K, Guterman LR, Hopkins N. Extracellular glutamate and other amino acids in experimental intracerebral hemorrhage: an in vivo microdialysis study. Crit Care Med 2003;31:1482–9. [3] Kasner SE, Demchuk AM, Berrouschot J, Schmutzhard E, Harms L, Verro P, Chalela JA, Abbur R, McGrade H, Christou I, Krieger DW. Predictors of fatal brain edema in massive hemispheric ischemic stroke. Stroke 2001;32:2117–23. [4] Staub CC, Taylor AE. Forward in Edema. New York: Raven Press; 1984. [5] Xi G, Hua Y, Bhasin RR, Ennis SR, Keep RF, Hoff JT. Mechanisms of edema formation after intracerebral hemorrhage: effects of extravasated red blood cells on blood flow and blood-brain barrier integrity. Stroke 2001;32:2932–8. [6] Abe H, Boldyrev A. Metabolic transformation of neuropeptide carnosine modifies its biological activity. Cell Mol Neurobiol 1999;19:163–75. [7] Boldyrev A, Bulygina E, Leinsoo T, Petrushanko I, Tsubone S, Abe H. Protection of neuronal cells against reactive oxygen species by carnosine and related compounds. Comp Biochem Physiol Biochem Mol Biol 2004;137:81–8. [8] Shen Y, Hu WW, Fan YY, Dai HB, Fu QL, Wei EQ, Luo JH, Chen Z. Carnosine protects against NMDA-induced neurotoxicity in differentiated rat PC12 cells through carnosine–histidine–histamine pathway and H(1)/H(3) receptors. Biochem Pharmacol 2007;73:709–17. [9] Tabakman R, Lazarovici P, Kohen R. Neuroprotective effects of carnosine and homocarnosine on pheochromocytoma PC12 cells exposed to ischemia. J Neurosci Res 2002;68:463–9. [10] Horning MS, Blakemore LJ, Trombley PQ. Endogenous mechanisms of neuroprotection: role of zinc, copper, and carnosine. Brain Res 2000;852:56–61. [11] Stvolinsky SL, Kukley ML, Dobrota D, Matejovicova (Vachova) M, Tkac I, Boldyrev AA. Carnosine: an endogenous neuroprotector in the ischemic brain. Cell Mol Neurobiol 1999;19:45–56. [12] Dobrota D, Fedorova T, Stvolinsky S, Babusikova E, Likavcanova K, Drgova A, Strapkova A, Boldyrev A. Carnosine protects the brain of rats and Mongolian gerbils against ischemic injury: after-stroke-effect. Neurochem Res 2005;30: 1283–8. [13] Stvolinsky S, Kukley M, Dobrota D, Mezesova V, Boldyrev A. Carnosine protects rats under global ischemia. Brain Res Bull 2000;53:445–8. [14] Stvolinskii SL, Fedorova TN, Yuneva MO, Boldyrev AA. Protective effect of carnosine on Cu, Zn-superoxide dismutase during impaired oxidative metabolism in the brain in vivo. Bull Exp Biol Med 2003;135:130–2. [15] Min J, Senut MC, Rajanikant K, Greenberg E, Bandagi R, Zemke D, Mousa A, Kassab M, Farooq MU, Gupta R, Majid A. Differential neuroprotective effects of carnosine, anserine, and N-acetyl carnosine against permanent focal ischemia. J Neurosci Res 2008;86(13):2984–91.
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Regulatory Peptides j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / r e g p e p
Neuroprotective properties of L-carnosine in the brain slices exposed to autoblood in the hemorrhagic stroke model in vitro A.Kh. Khama-Murad, A.A. Mokrushin, L.I. Pavlinova ⁎ Pavlov Institute Physiology, Russian Academy Sciences, Saint-Petersburg, Russia
a r t i c l e
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Article history: Received 16 April 2009 Received in revised form 1 December 2009 Accepted 25 November 2010 Available online 9 December 2010 Keywords: Surviving brain slices Edema Focal potentials L-carnosine
a b s t r a c t Neuroprotective properties of L-carnosine have been studied in our in vitro model on olfactory cortex slices of hypertensive rats under a long autoblood (blood clot) influence. Application of L-carnosine (5 mg/ml) on slices before autoblood influence leads to restoration of the activity of glutamatergic and GABA-ergic receptors inhibited in the presence of autoblood and interferes with swelling of slices. L-Carnosine protects a bioelectric activity of nervous cells in case of long influence of autoblood and also renders an anti edema effect. This model of hemorrhagic stroke may provide a perspective for investigating the mechanisms of neuroprotection. Published by Elsevier B.V.
1. Introduction Intracerebral hemorrhage as a result of bleeding leads to neuronal injury through primary factors, including oxidative stress and excitotoxicity [1,2], as well as secondary factors that include edema formation or swelling of a nervous tissue due to increase in the water content [3–5]. Intracerebral hemorrhage has no effective treatment. The neuronal cells of the central nervous system are susceptible to various forms of stroke including hemorrhage. Pharmaceutical pretreatment of stroke may prevent or reduce cellular injury and facilitate subsequent brain recovery. Carnosine (beta-alaninyl-Lhistidine), a specific constituent of excitable tissues of vertebrates and an endogenously synthesized dipeptide, acts as a free radical scavenger [6,7]. Besides it protects nervous cells as was shown in various in vitro models [8–10]. Carnosine exhibits a significant antioxidant protecting effect in case of the brain damaged by ischemic injury and hypobaric hypoxia [11–17]. It has gained great interest because of its potential use as a neuroprotective compound in clinical practice. The elaboration of neuroprotective drugs against insult requires specially developed pharmacological model in vitro. Brain slices are widely used to study ischemia [18,19]. Earlier we proposed a new in vitro model of hemorrhagic stroke which enables to reveal an excitotoxic effect of autoblood and blood clot on bioelectrical activity of neurons mediated by NMDA, AMPA and GABAergic receptors in the surviving olfactory cortex slices [20,21]. Protective effects of carnosine
⁎ Corresponding author. Tel.: +7 812 6842039. E-mail address: [email protected] (L.I. Pavlinova). 0167-0115/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.regpep.2010.11.007
on electrogenesis and edema formation in the injured brain tissue were investigated in this study. 2. Materials and methods 2.1. Tissue preparation and hemorrhagic stroke modeling All animals used in this study were treated with observance of recommendations on ethics of work with the animals offered European Communities Council Direction (86/609 EEC). Olfactory cortex slices were prepared from brain of hypertensive SHR rat males (approximately 200 g body weight) taking into consideration that high blood pressure is known to represent a rather reliable prognostic factor of outcome of intracerebral hemorrhage [22]. After decapitation, slices about 400 μm in thickness prepared within 1 min were placed into artificial cerebrospinal fluid (ACSF), consisted of (in mM): NaCl— 124; KCl—5; CaCl2—2.6; KH2PO4—1.24; MgSO4—1.2; NaHCO3—3; glucose—10; Tris–HCl—23; equilibrated with O2, with osmolarity of 295–305 mOsm (OMT-5-01, “Burevestnic”, Russia) for preincubation during 30 min. The concentrations of Ca2+ and Mg2+ were optimized for maximal synaptic activity in olfactory cortex. The temperature was 37 °C, pH 7.2–7.3. After incubation in ACSF at the interval (30, 60, 120, and 360 min) the control slices were placed in perfusion chamber to record the electrophysiological parameters (amplitudes of AP LOT, AMPA and NMDA EPSP, IPSPslow) and were taken for water content determination. To simulate hemorrhagic injury other group of slices was placed in autoblood (3 ml) for electrophysiological parameters and water content study at above indicated terms. The term 360 min was used as maximal, because this interval is corresponded to “time
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window” of the reversibility of the damaged nervous cells when electrogenesis in olfactory slices could be restored [20]. For determination of the L-carnosine protective properties and for comparison its effects with such ones of pharmaceutical preparation the slices were pretreated with L-carnosine (5 mg/ml of the incubation medium) or vitamins C (1.2 mg/ml) and D (0.4 mg/ml), or gliatilin (0.02 μl/ml) during 30 min and then placed in autoblood. Orthodromic stimulation of the lateral olfactory tract (LOT) was conducted using rectangular pulses with a duration of 0.05–0.1 ms and an intensity of 1–3 V with aid of the electrical stimulator (ESU-1, Russia) through platinum bipolar electrodes. Frequency of electric LOT stimulation was 0.003 Hz. Focal potentials (FPs) from slices were recorded using glass microelectrodes filled with 1 M NaCl (resistance 1–5 MΩ) conducted with amplifier (Russia). The recording point was located in the focus of maximal activity at a depth of 270–300 μm. The indifferent silver electrode was located in the chamber. FPs were amplified and then computer-processed using special home-made software. The rate of perfusion of ACSF in perfusion chamber was 2 ml/min. The following individual components of FPs were analyzed: the compound action potential of the lateral olfactory tract (AP LOT, presynaptic component FP), and the non-N-methyl-D-aspartate (nNMDA) and N-methyl-D-aspartate (NMDA) receptor components of the excitatory postsynaptic potentials (EPSPs); the first of these is activated by [alpha]-amino-3-hydroxy-5-methylisoxalol-4-propionic acid (AMPA), the second with NMDA [23–26] referred to as AMPA and NMDA EPSPs; as well as slow (GABABergic) inhibitory postsynaptic potential (IPSPs) which is generated at GABAB receptors activation (Fig. 1). Amplitudes of these components were measured from the isoline to the peak level [23]. The amplitudes of AMPA EPSP were measured over a 1.5–2 ms window centered at the peak of the response. Peak NMDA EPSP was measured as the average potential observed in a 7–8 ms window. Pharmacological evidence of AMPA and NMDA components of focal potential were performed by adding 25 μM CNQX and/or 50 μM APV respectively to the ACSF as previously described [23]. The technique of controlled experiment is used to standardize conditions of individual experiments. To test own neurotrophic influence of L-carnosine on parameters of glutamatergic and GABAergic synaptic cell activity in olfactory cortex slices we tested the following L-carnosine concentrations: 50 μM, 250 μM, 500 μM, 2 mM, 10 mM and 20 mM. To test protective properties of L-carnosine we determined the level of the cell injury and the efficacy of an L-carnosine protection in the neuronal activity recovery by comparing the FP amplitudes in slices pretreated with L-carnosine (5 mg/ml or 20 mM) after 360 min
Fig. 1. Representative response of bioelectrical activity recorded in olfactory cortex slice (FP). AP LOT, action potential of LOT fibers; AMPA EPSP and NMDA EPSP, components of excitatory postsynaptic potential; IPSPs, slow (GABABergic) inhibitory postsynaptic potential; dotted line represents isoline; arrows show the measuring modes of amplitudes of individual FP components.
of autoblood (blood clot) action with the control values obtained on separate group of slices. 2.2. Determination of total tissue water Percent water and swelling were determined in olfactory cortex slices by wet and dry weight measurements before and after autoblood exposure as described [18,19]. Slices after preincubation in the control medium or after pretreatment with L-carnosine (5 mg/ml of ACSF); vitamin C (1.2 mg/ml); vitamin D (0.4 mg/ml) or gliatilin (0.02 μl/ml) were placed on a filter paper and the excess fluid quickly removed, without extracting water from the tissue. Then slices were weighed on a small piece of preweighed aluminum foil (wet weight, g). The slices were weighed again after incubation with autoblood or in ACSF for 360 min (wet weight, g). After that slices were dried in an oven at 85 °C overnight and re-weighed (dry weight, g). The water content (W) of the slice was calculated as W (wet weight − dry weight) (dry weight−1) and given as g H2O (g dw− 1). The changes in W quantified the swelling of the slice tissue and expressed in Fig. 5 as percentage to control level. All chemical components were from Sigma except gliatilin and vitamins C and D. Gliatilin (choline acetate) commonly used in clinic for treatment of the ischemic and hemorrhagic strokes was from Italpharmeco C. A., Italy; Vitamins C and D were pharmaceutics. The data were processed statistically using nonparametric Wilcoxon– Mann–Whitney U test. The differences were significant at p ≤0.05. 3. Results 3.1. Neurotrophic effects of L-carnosine Carnosine is a dipeptide which is highly concentrated in mammalian olfactory sensory neurons. The biological function of carnosine in the nervous system remains unclear. Besides prevalent evidence concerning carnosine's protective role, it has been suggested that it as co-transmitter or modulator could be implicated in some forms of functional plasticity [27]. Early it was shown that L-carnosine (μM concentrations) evokes inward currents itself in olfactory bulb neurons in cultured slices [28]. At the same time in analogous neuronal culture L-carnosine did not evoke a membrane current or affect currents evoked by glutamate, GABA or glycine even though applied in mM concentrations [29]. In the present study we previously investigated the modifications of the evoked cell activity in control slices in the presence of different L-carnosine concentrations (Fig. 2). L-Carnosine showed dual effect on synaptic activity in neurons of olfactory cortex. In response to incubation with L-carnosine (50 μM) the amplitudes of AP LOT (18%, U = 10, n = 12, p ≤ 0.05); AMPA EPSP (35%, U = 12, n = 12, p ≤ 0.05); IPSPs (34%, U = 9, n = 12, p ≤ 0.05) were increased in control tissue. The change of the NMDA EPSP amplitude was unreliable. The raising of the L-carnosine concentrations (250 μM, 500 μM, 2 mM, 10 mM, and 20 mM) decreased the amplitudes of all FPs components to control level. Under the same conditions L-carnosine induced the modification of synaptic plasticity and facilitated long-term posttetanic potentiation. Electrical tetanization of LOT was accompanied with LTP induction generally prevailed over LTD [30]. Since higher L-carnosine concentrations locate in linear section of dose–response curve and these concentrations correspond to the intrinsic level in structures of the mammalian olfactory sensory analyzer, we used 20 mM of L-carnosine (Fig. 2) in subsequent study. 3.2. Amplitudes of FPs under autoblood influence (blood clot) To monitor chronic alterations of the neuronal excitability after prolonged autoblood action up to 360 min the method of slice
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Fig. 2. Neurotrophic effects of L-carnosine. A dose–response data. Note that, low L-carnosine concentrations increased the activity of FP components (conductive fibers— AP LOT, GABABergic inhibitory postsynaptic processes and postsynaptic ionotropic glutamatergic mechanisms—AMPA EPSP). High L-carnosine concentrations stabilized the activity of the FP components.
incubation in whole autoblood without anticoagulant was employed. Such approach enables to study the cell activity under the blood clot formation. We investigated the parameters of evoked FPs in slices incubated in autoblood at 30, 60, 120, and 360 min. Slices were in clot at 37 °C without oxygen delivery. Each time point of experiments had own controls that were handled identically to the treatment conditions. The modifications of the amplitude of FP components are presented in Fig. 3a. The conductive fibers of LOT—the axons of the mitral cells were primarily activated, but response of postsynaptic processes to autoblood (or more exactly clot) action was different at the first time point. Thus, the amplitude of AMPA EPSP and IPSPs decreased compared with the control, whereas the activity of NMDA-dependent processes was already blocked (Fig. 3a). The amplitudes of postsynaptic FP components progressively reduced to 360 min (for AMPA EPSP—8%, U = 9, n = 12, p ≤ 0.05; for NMDA EPSP—1%, U = 1, n = 12, p ≤ 0.05; for IPSPs—3%, U = 3, n = 12, p ≤ 0.05, as compared to control). At this time point the amplitude of AP LOT reduced too (39%, U = 13, n = 8, p ≤ 0.05). Exciting AMPA dependent and inhibitory GABA-ergic mechanisms depressed progressively during the contact with autoblood. NMDA-dependent mechanism was the most vulnerable and its blockade occurred already within the 1st hour of the autoblood action. Evidently the basic mechanisms of electrogenesis in the olfactory cortex slices being in contact with blood clot during 360 min were largely damaged (Figs. 3b and 4). The doubling of a power of electric stimulus failed to retrieve the amplitudes of AP LOT, AMPA and NMDA EPSP components. 3.3. Effects of carnosine pretreatment In the slices pretreated with L-carnosine and then placed in autoblood for 360 min the amplitudes of AP LOT, AMPA and NMDA EPSPs, IPSPs were similar to control values (Figs. 3b and 4). So, activity of presynaptic processes–the amplitude of AP LOT recovered to control level (95%, U = 28, n = 12, p ≥ 0.05, as compared to control). The amplitudes of exciting postsynaptic processes (AMPA and NMDA EPSPs) rose to 90% and 85%, correspondingly, compared with control values (U = 23, n = 12, p ≥ 0.05; U = 21, n = 12, p ≥ 0.05). The amplitude of IPSPs of inhibitory GABA-ergic mechanisms was restored too (85%, U = 25, n = 12, p ≥ 0.05). Therefore, severe damage of the neuronal activity induced by autoblood was overcome by L-carnosine.
Fig. 3. The activities of synaptic glutamatergic and GABABergic mechanisms in olfactory cortex slices (a) and protective effect of the L-carnosine pretreatment (b) under the neurotoxic autoblood (blood clot) action. a—time point of autoblood action: 1—30; 2—60; 3—120; 4—360 min. Isoline is marked by dotted line. All FP components in slices rapidly reduced under the autoblood action. The amplitudes of postsynaptic components (AMPA and NMDA EPSP, and IPSPs) decreased as early as first hour. Activity of conductive fibers was more stable under autoblood influence. b—pretreatment of slices with L-carnosine effectively defended the electrical cell activity, especially the most vulnerable components of FP—NMDA EPSP and IPSPs, from the subsequent injured autoblood action. Calibration: 0.1 mV; 7 ms for panels a and b. During the FPs registration the electronic device for artefact-rejection was used.
The prime temporal phase of brain edema is supposed to connect with the clot formation in the first hours after hemorrhagic stroke. We attempted to interfere in the process of edema formation having in mind the multiple neuroprotective properties of L-carnosine. The model of hemorrhagic stroke in vitro does very well for it. It is established, that brain slices gain water during incubation in vitro in normal, oxygenated ACSF [18,19,31]. We initially assessed the time course of this water gain in control slices and revealed that water content after 360 min of incubation reached significance of 111–113% (U = 21, p ≥ 0.05) compared with basal water content (100%). We then examined whether the exposure of brain tissue to autoblood in vitro leads to pathological swelling. Swelling increased further during subsequent contact with autoblood. Water content in the slices increased significantly after incubation in autoblood during 360 min (144%, U = 8, n = 12, p ≤ 0.05) (Fig. 5). L-Carnosine applied before the autoblood interfered with swelling of olfactory cortex slices. Water content in pretreated slices was similar to control at all time points of the autoblood contact. Pretreatment of slices with L-carnosine prevented their severe swelling up to 360 min of autoblood action: 144%—autoblood, 108%—L-carnosine (U= 12, n = 12, p ≤ 0.05). Pretreatment of slices with gliatilin—an anti-stroke preparation in concentration of 0.02 μg that corresponds to the doze applied in clinic (1000 mg/70 kg of weight of the person) had no any effect. The water content of slices reached 140% compared with control at the 360 min time point and the difference of water gain in these slices and in slices incubated with autoblood was unreliable (U = 21, n = 8, p ≥ 0.05). Among the antioxidants that showed some efficacy in the models of brain injury, vitamin C (ascorbate) plays an appropriate part [35,36]. Exogenously applied ascorbate was able to compensate for loss of any other single component of the antioxidant network in
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Fig. 4. Carnosine provides complete restoration of the cell activity in the olfactory cortex slices depressed by 360-min autoblood action. Amplitudes of the FPs components (presynaptic—AP LOT; postsynaptic—glutamatergic AMPA and NMDA EPSP and IPSPs as GABAB component) in slices of the control group incubated in ACSF at 37 °C were taken as 100% (empty columns, n = 8). In slices exposed to autoblood during 360 min the amplitudes of AMPA EPSP, NMDA EPSP and IPSPs were inhibited, the amplitude of AP LOT decayed more than half compared to the control (black columns, n = 12). Preincubation of slices with L-carnosine (5 mg/ml in ACSF) during 30 min is able to protect the mechanisms of electrogenesis and to restore the amplitudes of FPs in slices after their subsequent contact with autoblood during 360 min (dashed columns, n = 16) (*p ≤ 0.05 versus control; + p ≤ 0.05 versus autoblood action).
guinea-pig and rat brain slices at the model of H2O2 pathology [37]. Prevention of edema induced by ischemia was achieved by ascorbate application in vitro [19,35]. In the model of hemorrhagic stroke pretreatment of slices with vitamin C resulted in moderate anti edema effects especially after 60 and 120 min of the total incubation time, but water content in such slices was significantly higher versus control for the 360 min time point (135%, U = 21, n = 13, p ≥ 0.05). May be, the reduction of stroke complications induced by ascorbate depends on a specific dose and the severity of stroke [36]. It is known that carnosine could act as natural scavenges from free radical species and by-products of membrane lipids peroxidation [38]. We compared the effect of L-carnosine with the effect of vitamin D3. Vitamin D3 has been shown to play an important role in antioxidative mechanisms. It acutely prevented zinc-induced oxidative injuries acting as a terminator of the lipid peroxidation [39]. In this model of hemorrhagic stroke pretreatment with vitamin D decreased, water content in slices at all time intervals (p b 0.05 for each time point) (Fig. 5). It is obviously that carnosine may offer pluripotent favorable effects by prevention of initial hyperactivation and subsequent total inhibition of glutamate receptor activity induced by contact with autoblood. Our data suggest that L-carnosine involves in synaptic mechanisms as co-mediator at own brain tissue level. Besides, L-carnosine in high exogenous concentration actively modulates glutamatergic and GABAergic synaptic
Fig. 5. Pretreatment of slices with L-carnosine prevented their severe swelling induced by autoblood. Slices contacted with autoblood after their pretreatment with L-carnosine or compared preparations during 30 min. Tissue water content was determined at each time point: ACSF—n = 10; autoblood—n = 12; L-carnosine—n = 15; vitamins C and B—n = 13; gliatilin—n = 8. *p≤ 0.05 for carnosine versus each autoblood points. C—basal water content after slice preparation was taken as 100%.
processes protecting them against injurious blood action. L-Carnosine as antioxidative agent may to secure tissue integrity by decreasing the primary tissue swelling which is a result of the neurotoxic autoblood (blood clot) action.
4. Discussion In the present study, we have shown an attenuation of edema by and a recovery of neuronal activity mediated by NMDA and AMPA glutamate and GABA receptors being inhibited by autoblood exposure. It has been shown that synaptic glutamate receptor activation leads to neuronal swelling in substrate deficient human brain and in normoxic brain slices [31,32] and contributes to the initial cell swelling that accompanied anoxic depolarization in neonatal cerebrocortical brain slices [33]. The antagonists of ionotropic glutamate receptor differently affected swelling, a cocktail of these agents was more effective than AP5 or CNQX alone [19], so multiple glutamate receptor types seem to be involved in edema formation during ischemia [32]. Last time L-carnosine is an object of intensive study of many researchers. Protective, antioxidant and antiapoptotic properties of this endogenous dipeptide provide high importance in nervous cell defence in the brain injury. Carnosine compensates deficit in antioxidant protective system of brain damaged by ischemic injury [11–13]. The preventing effect of L-carnosine is accompanied with pronounced protective effect on neurological symptoms and animal mortality in the models of global cerebral ischemia [12,34]. Carnosine appears to influence deleterious pathological processes in focal cerebral ischemia decreasing reactive oxygen species levels and infarct size. It is neuroprotective when administered at time points both before and after the induction of ischemia [15,16]. Carnosine also increased the resistance of neuronal membranes to the in vitro induced oxidation and suppressed the glutamate receptor hyperactivation [7–9,14,15]. In our previous study L-carnosine modulated neuronal function via altered NMDA and AMPA glutamate and GABA receptor activation and promoted the induction and maintenance of long-term potentiation (LTP) in the olfactory cortex slices [30]. The mechanisms by which L-carnosine might act in our model are manifold, from the initial urgent protection of the neuronal activity interfering to decayed receptor activation, especially NMDAmediated, and consequent cell swelling that accompanies ionotropic glutamate-receptor activation. L-carnosine
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