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Palmitone prevents pentylenetetrazole-caused neuronal damage in the CA3 hippocampal region of prepubertal rats
Neuroscience Letters 470 (2010) 111–114
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Palmitone prevents pentylenetetrazole-caused neuronal damage in the CA3 hippocampal region of prepubertal rats E. Cano-Europa a , M.E. González-Trujano b , A. Reyes-Ramírez c , A. Hernández-García a , V. Blas-Valdivia a , R. Ortiz-Butrón a,∗ a
Departamento de Fisiología “Mauricio Russek Berman”, Escuela Nacional de Ciencias Biológicas, I.P.N., Carpio y Plan de Ayala, México, D.F., C.P.11340, Mexico Dirección de Investigaciones en Neurociencias, Instituto Nacional de Psiquiatría Ramón de la Fuente Mu˜ niz, Calzada México-Xochimilco 101, San Lorenzo Huipulco 14370, Mexico Facultad de Estudios Superiores “Zaragoza”, Universidad Nacional Autónoma de México, J.C. Bonilla 66 y Calzada Ignacio Zaragoza, Col. Ejército de Oriente, Iztapalapa 09230, México, D.F., Mexico b c
a r t i c l e
i n f o
Article history: Received 19 August 2009 Received in revised form 16 December 2009 Accepted 24 December 2009 Keywords: Palmitone Anticonvulsant Seizure Neuronal damage
a b s t r a c t Palmitone is a secondary metabolite of polyketide origin extracted from leaves of Annona diversifolia Saff. (Annonaceae). We found that palmitone possesses anticonvulsant properties against penicillin-, 4-AP-, and pentylenetetrazole (PTZ)-caused seizure in adult animals. Some convulsants as PTZ cause neuronal damage in different brain regions such as the CA3 hippocampal region. Our objective was to evaluate if palmitone protects against PTZ-caused seizures and hippocampal neuronal damage in prepubertal rats. We used 32 prepubertal Wistar rats (30–35 days old) divided into four groups of 8 animals; group I was the control group, group II received a single PTZ dose of 50 mg/kg ip, group III received a single palmitone dose of 50 mg/kg ip, and group IV received a palmitone dose of 50 mg/kg ip plus a PTZ dose of 50 mg/kg ip. Ten days after administration, the animals were killed using pentobarbital anesthesia (35 mg/kg). The brains were removed and were embedded in paraffin. Coronal cuts of 7 m were obtained from −2.8 to −3.3 from Bregma. Each section was stained with cresyl violet-eosin. We evaluated the number of normal and abnormal neurons in the CA3 hippocampal region in a 10,000 m2 section. It was observed that palmitone did not prevent the PTZ-caused seizure but palmitone prevents the PTZ-caused neuronal damage in the CA3 hippocampal region. Published by Elsevier Ireland Ltd.
Palmitone (16-hentriacontanone) is an aliphatic ketone isolated from the leaves of Annona diversifolia Saff., a plant used in Mexican traditional medicine to control convulsions. As first described by Gill in 1948, plants belonging to the Annonaceae family have been used to treat epilepsy [8]. There are studies that demonstrated the anticonvulsant effect of Annona muricata and A. diversifolia species on the PTZ-caused seizures in mice [12,20]. As an isolated compound, palmitone has shown an anticonvulsant effect in acute models of epilepsy such as 4-aminopyridine-, bicuculline-, and pentylenetetrazole (PTZ)-caused seizures in mice. In these studies, a delay in the onset of tonic–clonic seizures and reduction in the percentage of mortality were measured [11]. Furthermore, palmitone demonstrated an anticonvulsant effect on the EEG seizures caused by penicillin in rats [9]. Systemic application of PTZ, an antagonist of the GABA-BDZ binding site, excites neurons producing tonic–clonic seizures and mortality, depending on the dose tested. It has also been described that PTZ-caused seizures produce neuronal damage [21]. Moreover,
∗ Corresponding author. Tel.: +525 729 6300/62481/52342; fax: +525 729 62 06. E-mail address: [email protected] (R. Ortiz-Butrón). 0304-3940/$ – see front matter. Published by Elsevier Ireland Ltd. doi:10.1016/j.neulet.2009.12.066
it is known that acute or chronic PTZ administration affects the REDOX environment causing oxidative stress and neuronal damage [6,22,23]. Because the anticonvulsant properties of palmitone are described in adult animals, the objective of this study was to evaluate if palmitone protects against PTZ-caused seizure and hippocampal neuronal damage in prepubertal rats. We used 32 prepubertal Wistar rats (from 30- to 35-days old). The animals were housed at room temperature and maintained on a 12-h light:12-h dark cycle. Food (lab diet) and water were available ad libitum. Experiments were made in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23) and the guidelines of the Laws and Codes of Mexico in The Seventh Title of the Regulations of the General Law of Health Regarding Health Research. Palmitone (16-hentriacontanone) was obtained from a crude extract of A. diversifolia leaves through bio-guided fractionation, as described [11]. A voucher specimen (AN9702) was deposited at the Herbario de Plantas Utiles Efraim Hernandez X, Universidad Autonoma Chapingo, Estado de Mexico, Mexico. Eight animals were randomly assigned into four groups. Group I received dimethyl sulfoxide (DMSO) (Sigma, St. Louis MO, USA) plus
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Table 1 Duration, latency, and intensity of seizures. Groups
Duration (s) Latency (s) Rate of kindled rats (%)
DMSO + ss
Palmitone + ss
DMSO + PTZ
Palmitone + PTZ
0 0 0 (0/8)
0 0 0 (0/8)
20.6 ± 9.0 131.1 ± 56.7 50 (4/8)
9.9 ± 4.8 139.0 ± 57.9 50 (4/8)
0.9% sterile saline (ss) ip, group II received DMSO plus 50 mg/kg pentylenetetrazol (PTZ, Sigma, St. Louis MO, USA) ip, group III received 50 mg/kg palmitone plus 0.9% ss ip, and group IV received 50 mg/kg palmitone plus 50 mg/kg PTZ ip. All drugs were injected intraperitonially. The injection of PTZ or ss was made 30 min after the injection of DMSO or palmitone. All the rats were monitored during 2 h and the grade of seizures were recorded using the Racine scale [25]. For each rat the latency, duration, and intensity of seizures was recorded. After the experiments the animals were housed at room temperature and maintained in lab conditions. Ten days after the PTZ-injection the rats were killed with an excess of pentobarbital (35 mg/kg). The brains were removed and were fixed with Buoin and embedded in paraffin. Coronal cuts of 7 m were obtained from −2.8 to −3.3 mm from Bregma [24]. Each brain section was stained with cresyl violeteosin, dehydrated, and mounted with resin. We evaluated the number of normal and abnormal neurons in the CA3 hippocampal region within a 10,000 m2 section as previously described [17,3]. Briefly, this evaluation was made using a bidimensional counting method, using ten slices per brain of each CA3 hippocampal region, which were randomly selected from the left and the right hemispheres. The identification of neurons was based on cytoplasmic characteristics including the presence of a readily distinguishable nucleus and a nucleolus and a neuronal shape that is in accordance with the usual rules used in stereological studies to identify neurons [31]. All results are presented as mean ± SE. Statistical analyses for all behavioral tests were evaluated by a one-way ANOVA and a post
hoc Newman–Student–Keuls test. P < 0.05 was considered statistically significant. We did not see differences in latency and duration of any phase according to the Racine scale [25] in all treatments. However, we have in Table 1 the results of the phase 5 (phase which has tonic–clonic seizures). The percentage of kindled rats treated with palmitone was not different from the rats treated only with PTZ. Nor were the latency and the duration of seizures different. For the morphologic study, we found that the use of PTZ produced a marked damage in the CA3 hippocampal region (Fig. 1B) when compared to the control rats (Fig. 1A). The neurons had the characteristic morphological alterations, such as chromatin condensation, nucleolus loss, and cell shrinkage, produced by PTZ (arrows). The group that received PTZ with a preinjection of palmitone had less damage in the CA3 region (Fig. 1D) and the appearance was similar to the control group or to those injected with palmitone only (Fig. 1C). The morphometric study showed that the total number of pyramidal neurons is similar in all the groups (30–35 neurons). We did find that the damage caused by the PTZ injection was prevented by the earlier injection of palmitone. The number of neurons in the PTZ group had a decrease of the number of neurons in 10,000 m2 (Fig. 2A) compared to the group preinjected with palmitone (B). Pediatric epilepsies are devastating neurologic disorders. The developing brain, susceptible to seizures, can cause profound neurologic impairment, enhance subsequent seizure propensity during maturation and in adulthood, and lead to abnormalities in behavioral and cognitive function because of seizure-caused neuronal
Fig. 1. Photomicrographs of the CA3 hippocampal region. (A) DMSO + ss; (B) DMSO + PTZ; (C) palmitone + ss; (D) palmitone + PTZ. Arrows in A, C and D show normal neurons. In B arrows show neuronal damage.
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Fig. 2. (A) The number of pyramidal neurons (PN) in 10,000 m2 of the CA3 hippocampal region. (B) Normal number of pyramidal neurons (NPN) in 10,000 m2 of the CA3 hippocampal region. (*) P < 0.05.
damage [30]. Consequently, it is important to develop strategies that prevent seizure and neuronal damage. For that reason, we pretreated the prepubertal rat with palmitone before the PTZ-caused seizure. We demonstrated that palmitone prevented PTZ-caused hippocampal damage though palmitone did not prevent PTZcaused seizure. It has been described that the immature brain (between P0 and P10) is more susceptible to the development of seizures than the mature brain [19]. However, it has not been studied the susceptibility between pre-puber animals and adults. We did not found palmitone prevent PTZ-causes seizure because prepuber rats have lesser serum concentration of 17-estradiol and testosterone than adults rats [13]. It was observed that high concentration of these hormones promotes seizures. It could be explained because hippocampal glial cells converted testosterone in 17-estradiol or dihydrotestosterone by the aromatase and 5␣reductase enzymes, respectively. Besides, dihydrotestosterone is converted to 3␣-androstanediol by 3␣-hydroxysteroid oxidoreductase which modulates GABAA receptor [27]. The PTZ-causes myoclonic and clonic seizures result from activation of forebrain structures, whereas the tonic extension is mediated by diencephalon and brainstem structures [4,2]. But the hippocampus is actually one of the brain areas that show the most vulnerability in epilepsy [14]. It is generally believed that PTZ blocks the benzodiazepine receptor site of GABAA , but also PTZ exerts its effects by binding other allosteric sites like the picrotoxin-binding site of this post-synaptic GABAA receptor [18]. The inhibition of GABAergic neurons results in the activation of glutamatergic and motor neurons [16,28]. Moreover, the PTZ enhanced glutamate binding in the cerebral cortex [5] and its receptors in the hippocampus [29]. Additionally, the PTZ-caused neuronal hyperactivity caused hippocampal oxidative stress and cellular damage [26,15]. The neuroprotector effect of palmitone is unknown; however it could be that hippocampal neurons metabolize it and used the palmitone as palmitic acid. As a result, palmitone could promote post-translational reversible changes in some proteins that modulate the glutamatergic system such as postsynaptic density protein 95 (PSD 95). It has been demonstrated that palmitoylation of PSD95 reduced activation of n-nitric oxide synthase (EC 1.14.13.39) preventing NO formation [7]. Thus, if NO concentration is reduced, the gluatamate release and NMDA receptor activation-mediated excitotoxicity is decreased. This hypothesis is probable because González-Trujano [10] demonstrated by using a microdialysis technique that palmitone reduced the extracellular concentration of glutamate. There are many peptidases derived from neurons and glia in the brain, such as prolyl oligopeptidase (POP) (prolyl endopeptidase, post-proline cleaving enzyme; EC 3.4.21.26) and endopeptidase 24.15 (EP 24.15) (PZ-peptidase; EC 3.4.24.15), which are associated with PTZ-caused hippocampal damage. Alterations of these peptidases are prevented by MK-801 (a potent noncompetitive
antagonist of the NMDA receptor) pretreatment [1]. Thus, palmitone reduces the glutamate-activated NMDA receptor and prevents the decrease in peptidase activity and hippocampal damage. This was a preliminary study about the potential of palmitone as neuroprotector in a model of temporal lobe epilepsy in prepubertal rats and it opens the field for studying the molecular mechanism for future experiments.
References [1] M.M. Ahmed, M. Arif, T. Chikuma, T. Kato, Pentylenetetrazol-induced seizures affect the levels of prolyl oligopeptidase, thimet oligopeptidase and glial proteins in rat brain regions, and attenuation by MK-801 pretreatment, Neurochem. Int. 47 (2005) 248–259. [2] Y. Ben-Ari, E. Tremblay, D. Riche, G. Ghilini, R. Naquet, Electrographic, clinical and pathological alterations following systemic administration of kainic acid, bicuculline or pentetrazole: metabolic mapping using the deoxyglucose method with special reference to the pathology of epilepsy, Neuroscience 6 (1981) 1361–1391. [3] V. Blas-Valdivia, E. Cano-Europa, A. Hernandez-Garcia, R. Ortiz-Butron, Hippocampus and amygdala neurotoxicity produced by systemic lidocaine in adult rats, Life Sci. 81 (2007) 691–694. [4] R.A. Browning, Role of the brain-stem reticular formation in tonic–clonic seizures: lesion and pharmacological studies, Fed. Proc. 44 (1985) 2425–2431. [5] L.F. da Silva, P. Pereira, E. Elisabetsky, A neuropharmacological analysis of PTZinduced kindling in mice, Gen. Pharmacol. 31 (1998) 47–50. [6] H. Franke, H. Kittner, Morphological alterations of neurons and astrocytes and changes in emotional behavior in pentylenetetrazol-kindled rats, Pharmacol. Biochem. Behav. 70 (2001) 291–303. [7] M. Fukata, Y. Fukata, H. Adesnik, R. Nicoll, D. Bredt, Identification of PSD-95 palmitoylating enzymes, Neuron 44 (2004) 987–996. [8] R.C. Gill, Curariform drug, Brit (2009) 619–627. [9] E. Gonzalez, E. Tapia, L. Lopez-Meraz, A. Navarrete, A. Reyes-Ramirez, A. Martinez, Anticonvulsant effect of Annona diversifolia Saff. and palmitone on penicillin-induced convulsive activity. A behavioral and EEG study in rats, Epilepsia 47 (2006) 1810–1817. [10] M. González-Trujano, Estudio del efecto de la palmitona: participación de los opioides endógenos, del GABA y de los aminoácidos excitadores, CINVESTAV (2002). [11] M.E. Gonzalez-Trujano, A. Navarrete, B. Reyes, E. Cedillo-Portugal, E. Hong, Anticonvulsant properties and bio-guided isolation of palmitone from leaves of Annona diversifolia, Planta Med. 67 (2001) 136–141. [12] M.E. González-Trujano, C.A. Navarrete, B. Reyes, E. Hong, Some pharmacological effects of the ethanol extract of leaves of Annona diversifolia on the central nervous system in Mice, Phytol. Res. 12 (1998) 600–602. [13] C. Guerra-Araiza, A. Reyna-Neyra, A.M. Salazar, M.A. Cerbon, S. Morimoto, I. Camacho-Arroyo, Progesterone receptor isoforms expression in the prepuberal and adult male rat brain, Brain Res. Bull. 54 (2001) 13–17. [14] K.Z. Haas, E.F. Sperber, L.A. Opanashuk, P.K. Stanton, S.L. Moshe, Resistance of immature hippocampus to morphologic and physiologic alterations following status epilepticus or kindling, Hippocampus 11 (2001) 615–625. [15] R. Huang, C. Bell-Horner, M. Dibas, D. Covey, J. Drewe, G. Dillon, Pentylenetetrazole-induced inhibition of recombinant gamma—aminobutyric acid type A (GABAA) receptors: mechanism and site of action, J. Pharmacol. Exp. Ther. 298 (2001) 986–995. [16] D. Kondziella, J. Hammer, O. Sletvold, U. Sonnewald, The pentylenetetrazolekindling model of epilepsy in SAMP8 mice: glial-neuronal metabolic interactions, Neurochem. Int. 43 (2003) 629–637. [17] G.E. Lopez-Galindo, E. Cano-Europa, R. Ortiz-Butron, Ketamine prevents lidocaine-caused neurotoxicity in the CA3 hippocampal and basolateral amygdala regions of the brain in adult rats, J. Anesth. 22 (2008) 471–474. [18] R.L. Macdonald, J.L. Barker, Different actions of anticonvulsant and anesthetic barbiturates revealed by use of cultured mammalian neurons, Science 200 (1978) 775–777.
114
E. Cano-Europa et al. / Neuroscience Letters 470 (2010) 111–114
[19] S.L. Moshe, B.J. Albala, R.F. Ackermann, J. Engel, Increased seizure susceptibility of the immature brain, Brain Res. 283 (1983) 81–85. [20] P. NˇıGouemo, B. Koudogbo, H.P. Tchivounda, A. Nguema, M.M. Etoua, Effects of ethanol extract of Annona muricata on pentylenetetrazol induced convulsive seizures in mice, Phytol. Res. 11 (1997) 243– 245. [21] J.H. Park, H. Cho, H. Kim, K. Kim, Repeated brief epileptic seizures by pentylenetetrazole cause neurodegeneration and promote neurogenesis in discrete brain regions of freely moving adult rats, Neuroscience 140 (2006) 673–684. [22] N. Patsoukis, G. Zervoudakis, N.T. Panagopoulos, C.D. Georgiou, F. Angelatou, N.A. Matsokis, Thiol redox state (TRS) and oxidative stress in the mouse hippocampus after pentylenetetrazol-induced epileptic seizure, Neurosci. Lett. 357 (2004) 83–86. [23] N. Patsoukis, G. Zervoudakis, C.D. Georgiou, F. Angelatou, N.A. Matsokis, N.T. Panagopoulos, Effect of pentylenetetrazol-induced epileptic seizure on thiol redox state in the mouse cerebral cortex, Epilepsy Res. 62 (2004) 65– 74. [24] G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1999.
[25] R.J. Racine, Modification of seizure activity by electrical stimulation. I. After-discharge threshold, Electroencephalogr. Clin. Neurophysiol. 32 (1972) 269–279. [26] R. Ramanjaneyulu, M. Ticku, Interactions of pentamethylenetetrazole and tetrazole analogues with the picrotoxinin site of the benzodiazepine-GABA receptor-ionophore complex, Eur. J. Pharmacol. 98 (1984) 337–345. [27] D.S. Reddy, Testosterone modulation of seizure susceptibility is mediated by neurosteroids 3alpha-androstanediol and 17beta-estradiol, Neuroscience 129 (2004) 195–207. [28] L. Rocha, M. Briones, R.F. Ackermann, B. Anton, N.T. Maidment, C.J. Evans, J. Engel, Pentylenetetrazol-induced kindling: early involvement of excitatory and inhibitory systems, Epilepsy Res. 26 (1996) 105–113. [29] H. Schroeder, A. Becker, V. Hoellt, Sensitivity and density of glutamate receptor subtypes in the hippocampal formation are altered in pentylenetetrazolekindled rats, Exp. Brain Res. 120 (1998) 527–530. [30] C.E. Stafstrom, S.L. Moshe, J.W. Swann, A. Nehlig, M.P. Jacobs, P.A. Schwartzkroin, Models of pediatric epilepsies: strategies and opportunities, Epilepsia 47 (2006) 1407–1414. [31] M.J. West, Regionally specific loss of neurons in the aging human hippocampus, Neurobiol. Aging 14 (1993) 287–293.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Palmitone prevents pentylenetetrazole-caused neuronal damage in the CA3 hippocampal region of prepubertal rats E. Cano-Europa a , M.E. González-Trujano b , A. Reyes-Ramírez c , A. Hernández-García a , V. Blas-Valdivia a , R. Ortiz-Butrón a,∗ a
Departamento de Fisiología “Mauricio Russek Berman”, Escuela Nacional de Ciencias Biológicas, I.P.N., Carpio y Plan de Ayala, México, D.F., C.P.11340, Mexico Dirección de Investigaciones en Neurociencias, Instituto Nacional de Psiquiatría Ramón de la Fuente Mu˜ niz, Calzada México-Xochimilco 101, San Lorenzo Huipulco 14370, Mexico Facultad de Estudios Superiores “Zaragoza”, Universidad Nacional Autónoma de México, J.C. Bonilla 66 y Calzada Ignacio Zaragoza, Col. Ejército de Oriente, Iztapalapa 09230, México, D.F., Mexico b c
a r t i c l e
i n f o
Article history: Received 19 August 2009 Received in revised form 16 December 2009 Accepted 24 December 2009 Keywords: Palmitone Anticonvulsant Seizure Neuronal damage
a b s t r a c t Palmitone is a secondary metabolite of polyketide origin extracted from leaves of Annona diversifolia Saff. (Annonaceae). We found that palmitone possesses anticonvulsant properties against penicillin-, 4-AP-, and pentylenetetrazole (PTZ)-caused seizure in adult animals. Some convulsants as PTZ cause neuronal damage in different brain regions such as the CA3 hippocampal region. Our objective was to evaluate if palmitone protects against PTZ-caused seizures and hippocampal neuronal damage in prepubertal rats. We used 32 prepubertal Wistar rats (30–35 days old) divided into four groups of 8 animals; group I was the control group, group II received a single PTZ dose of 50 mg/kg ip, group III received a single palmitone dose of 50 mg/kg ip, and group IV received a palmitone dose of 50 mg/kg ip plus a PTZ dose of 50 mg/kg ip. Ten days after administration, the animals were killed using pentobarbital anesthesia (35 mg/kg). The brains were removed and were embedded in paraffin. Coronal cuts of 7 m were obtained from −2.8 to −3.3 from Bregma. Each section was stained with cresyl violet-eosin. We evaluated the number of normal and abnormal neurons in the CA3 hippocampal region in a 10,000 m2 section. It was observed that palmitone did not prevent the PTZ-caused seizure but palmitone prevents the PTZ-caused neuronal damage in the CA3 hippocampal region. Published by Elsevier Ireland Ltd.
Palmitone (16-hentriacontanone) is an aliphatic ketone isolated from the leaves of Annona diversifolia Saff., a plant used in Mexican traditional medicine to control convulsions. As first described by Gill in 1948, plants belonging to the Annonaceae family have been used to treat epilepsy [8]. There are studies that demonstrated the anticonvulsant effect of Annona muricata and A. diversifolia species on the PTZ-caused seizures in mice [12,20]. As an isolated compound, palmitone has shown an anticonvulsant effect in acute models of epilepsy such as 4-aminopyridine-, bicuculline-, and pentylenetetrazole (PTZ)-caused seizures in mice. In these studies, a delay in the onset of tonic–clonic seizures and reduction in the percentage of mortality were measured [11]. Furthermore, palmitone demonstrated an anticonvulsant effect on the EEG seizures caused by penicillin in rats [9]. Systemic application of PTZ, an antagonist of the GABA-BDZ binding site, excites neurons producing tonic–clonic seizures and mortality, depending on the dose tested. It has also been described that PTZ-caused seizures produce neuronal damage [21]. Moreover,
∗ Corresponding author. Tel.: +525 729 6300/62481/52342; fax: +525 729 62 06. E-mail address: [email protected] (R. Ortiz-Butrón). 0304-3940/$ – see front matter. Published by Elsevier Ireland Ltd. doi:10.1016/j.neulet.2009.12.066
it is known that acute or chronic PTZ administration affects the REDOX environment causing oxidative stress and neuronal damage [6,22,23]. Because the anticonvulsant properties of palmitone are described in adult animals, the objective of this study was to evaluate if palmitone protects against PTZ-caused seizure and hippocampal neuronal damage in prepubertal rats. We used 32 prepubertal Wistar rats (from 30- to 35-days old). The animals were housed at room temperature and maintained on a 12-h light:12-h dark cycle. Food (lab diet) and water were available ad libitum. Experiments were made in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23) and the guidelines of the Laws and Codes of Mexico in The Seventh Title of the Regulations of the General Law of Health Regarding Health Research. Palmitone (16-hentriacontanone) was obtained from a crude extract of A. diversifolia leaves through bio-guided fractionation, as described [11]. A voucher specimen (AN9702) was deposited at the Herbario de Plantas Utiles Efraim Hernandez X, Universidad Autonoma Chapingo, Estado de Mexico, Mexico. Eight animals were randomly assigned into four groups. Group I received dimethyl sulfoxide (DMSO) (Sigma, St. Louis MO, USA) plus
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Table 1 Duration, latency, and intensity of seizures. Groups
Duration (s) Latency (s) Rate of kindled rats (%)
DMSO + ss
Palmitone + ss
DMSO + PTZ
Palmitone + PTZ
0 0 0 (0/8)
0 0 0 (0/8)
20.6 ± 9.0 131.1 ± 56.7 50 (4/8)
9.9 ± 4.8 139.0 ± 57.9 50 (4/8)
0.9% sterile saline (ss) ip, group II received DMSO plus 50 mg/kg pentylenetetrazol (PTZ, Sigma, St. Louis MO, USA) ip, group III received 50 mg/kg palmitone plus 0.9% ss ip, and group IV received 50 mg/kg palmitone plus 50 mg/kg PTZ ip. All drugs were injected intraperitonially. The injection of PTZ or ss was made 30 min after the injection of DMSO or palmitone. All the rats were monitored during 2 h and the grade of seizures were recorded using the Racine scale [25]. For each rat the latency, duration, and intensity of seizures was recorded. After the experiments the animals were housed at room temperature and maintained in lab conditions. Ten days after the PTZ-injection the rats were killed with an excess of pentobarbital (35 mg/kg). The brains were removed and were fixed with Buoin and embedded in paraffin. Coronal cuts of 7 m were obtained from −2.8 to −3.3 mm from Bregma [24]. Each brain section was stained with cresyl violeteosin, dehydrated, and mounted with resin. We evaluated the number of normal and abnormal neurons in the CA3 hippocampal region within a 10,000 m2 section as previously described [17,3]. Briefly, this evaluation was made using a bidimensional counting method, using ten slices per brain of each CA3 hippocampal region, which were randomly selected from the left and the right hemispheres. The identification of neurons was based on cytoplasmic characteristics including the presence of a readily distinguishable nucleus and a nucleolus and a neuronal shape that is in accordance with the usual rules used in stereological studies to identify neurons [31]. All results are presented as mean ± SE. Statistical analyses for all behavioral tests were evaluated by a one-way ANOVA and a post
hoc Newman–Student–Keuls test. P < 0.05 was considered statistically significant. We did not see differences in latency and duration of any phase according to the Racine scale [25] in all treatments. However, we have in Table 1 the results of the phase 5 (phase which has tonic–clonic seizures). The percentage of kindled rats treated with palmitone was not different from the rats treated only with PTZ. Nor were the latency and the duration of seizures different. For the morphologic study, we found that the use of PTZ produced a marked damage in the CA3 hippocampal region (Fig. 1B) when compared to the control rats (Fig. 1A). The neurons had the characteristic morphological alterations, such as chromatin condensation, nucleolus loss, and cell shrinkage, produced by PTZ (arrows). The group that received PTZ with a preinjection of palmitone had less damage in the CA3 region (Fig. 1D) and the appearance was similar to the control group or to those injected with palmitone only (Fig. 1C). The morphometric study showed that the total number of pyramidal neurons is similar in all the groups (30–35 neurons). We did find that the damage caused by the PTZ injection was prevented by the earlier injection of palmitone. The number of neurons in the PTZ group had a decrease of the number of neurons in 10,000 m2 (Fig. 2A) compared to the group preinjected with palmitone (B). Pediatric epilepsies are devastating neurologic disorders. The developing brain, susceptible to seizures, can cause profound neurologic impairment, enhance subsequent seizure propensity during maturation and in adulthood, and lead to abnormalities in behavioral and cognitive function because of seizure-caused neuronal
Fig. 1. Photomicrographs of the CA3 hippocampal region. (A) DMSO + ss; (B) DMSO + PTZ; (C) palmitone + ss; (D) palmitone + PTZ. Arrows in A, C and D show normal neurons. In B arrows show neuronal damage.
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Fig. 2. (A) The number of pyramidal neurons (PN) in 10,000 m2 of the CA3 hippocampal region. (B) Normal number of pyramidal neurons (NPN) in 10,000 m2 of the CA3 hippocampal region. (*) P < 0.05.
damage [30]. Consequently, it is important to develop strategies that prevent seizure and neuronal damage. For that reason, we pretreated the prepubertal rat with palmitone before the PTZ-caused seizure. We demonstrated that palmitone prevented PTZ-caused hippocampal damage though palmitone did not prevent PTZcaused seizure. It has been described that the immature brain (between P0 and P10) is more susceptible to the development of seizures than the mature brain [19]. However, it has not been studied the susceptibility between pre-puber animals and adults. We did not found palmitone prevent PTZ-causes seizure because prepuber rats have lesser serum concentration of 17-estradiol and testosterone than adults rats [13]. It was observed that high concentration of these hormones promotes seizures. It could be explained because hippocampal glial cells converted testosterone in 17-estradiol or dihydrotestosterone by the aromatase and 5␣reductase enzymes, respectively. Besides, dihydrotestosterone is converted to 3␣-androstanediol by 3␣-hydroxysteroid oxidoreductase which modulates GABAA receptor [27]. The PTZ-causes myoclonic and clonic seizures result from activation of forebrain structures, whereas the tonic extension is mediated by diencephalon and brainstem structures [4,2]. But the hippocampus is actually one of the brain areas that show the most vulnerability in epilepsy [14]. It is generally believed that PTZ blocks the benzodiazepine receptor site of GABAA , but also PTZ exerts its effects by binding other allosteric sites like the picrotoxin-binding site of this post-synaptic GABAA receptor [18]. The inhibition of GABAergic neurons results in the activation of glutamatergic and motor neurons [16,28]. Moreover, the PTZ enhanced glutamate binding in the cerebral cortex [5] and its receptors in the hippocampus [29]. Additionally, the PTZ-caused neuronal hyperactivity caused hippocampal oxidative stress and cellular damage [26,15]. The neuroprotector effect of palmitone is unknown; however it could be that hippocampal neurons metabolize it and used the palmitone as palmitic acid. As a result, palmitone could promote post-translational reversible changes in some proteins that modulate the glutamatergic system such as postsynaptic density protein 95 (PSD 95). It has been demonstrated that palmitoylation of PSD95 reduced activation of n-nitric oxide synthase (EC 1.14.13.39) preventing NO formation [7]. Thus, if NO concentration is reduced, the gluatamate release and NMDA receptor activation-mediated excitotoxicity is decreased. This hypothesis is probable because González-Trujano [10] demonstrated by using a microdialysis technique that palmitone reduced the extracellular concentration of glutamate. There are many peptidases derived from neurons and glia in the brain, such as prolyl oligopeptidase (POP) (prolyl endopeptidase, post-proline cleaving enzyme; EC 3.4.21.26) and endopeptidase 24.15 (EP 24.15) (PZ-peptidase; EC 3.4.24.15), which are associated with PTZ-caused hippocampal damage. Alterations of these peptidases are prevented by MK-801 (a potent noncompetitive
antagonist of the NMDA receptor) pretreatment [1]. Thus, palmitone reduces the glutamate-activated NMDA receptor and prevents the decrease in peptidase activity and hippocampal damage. This was a preliminary study about the potential of palmitone as neuroprotector in a model of temporal lobe epilepsy in prepubertal rats and it opens the field for studying the molecular mechanism for future experiments.
References [1] M.M. Ahmed, M. Arif, T. Chikuma, T. Kato, Pentylenetetrazol-induced seizures affect the levels of prolyl oligopeptidase, thimet oligopeptidase and glial proteins in rat brain regions, and attenuation by MK-801 pretreatment, Neurochem. Int. 47 (2005) 248–259. [2] Y. Ben-Ari, E. Tremblay, D. Riche, G. Ghilini, R. Naquet, Electrographic, clinical and pathological alterations following systemic administration of kainic acid, bicuculline or pentetrazole: metabolic mapping using the deoxyglucose method with special reference to the pathology of epilepsy, Neuroscience 6 (1981) 1361–1391. [3] V. Blas-Valdivia, E. Cano-Europa, A. Hernandez-Garcia, R. Ortiz-Butron, Hippocampus and amygdala neurotoxicity produced by systemic lidocaine in adult rats, Life Sci. 81 (2007) 691–694. [4] R.A. Browning, Role of the brain-stem reticular formation in tonic–clonic seizures: lesion and pharmacological studies, Fed. Proc. 44 (1985) 2425–2431. [5] L.F. da Silva, P. Pereira, E. Elisabetsky, A neuropharmacological analysis of PTZinduced kindling in mice, Gen. Pharmacol. 31 (1998) 47–50. [6] H. Franke, H. Kittner, Morphological alterations of neurons and astrocytes and changes in emotional behavior in pentylenetetrazol-kindled rats, Pharmacol. Biochem. Behav. 70 (2001) 291–303. [7] M. Fukata, Y. Fukata, H. Adesnik, R. Nicoll, D. Bredt, Identification of PSD-95 palmitoylating enzymes, Neuron 44 (2004) 987–996. [8] R.C. Gill, Curariform drug, Brit (2009) 619–627. [9] E. Gonzalez, E. Tapia, L. Lopez-Meraz, A. Navarrete, A. Reyes-Ramirez, A. Martinez, Anticonvulsant effect of Annona diversifolia Saff. and palmitone on penicillin-induced convulsive activity. A behavioral and EEG study in rats, Epilepsia 47 (2006) 1810–1817. [10] M. González-Trujano, Estudio del efecto de la palmitona: participación de los opioides endógenos, del GABA y de los aminoácidos excitadores, CINVESTAV (2002). [11] M.E. Gonzalez-Trujano, A. Navarrete, B. Reyes, E. Cedillo-Portugal, E. Hong, Anticonvulsant properties and bio-guided isolation of palmitone from leaves of Annona diversifolia, Planta Med. 67 (2001) 136–141. [12] M.E. González-Trujano, C.A. Navarrete, B. Reyes, E. Hong, Some pharmacological effects of the ethanol extract of leaves of Annona diversifolia on the central nervous system in Mice, Phytol. Res. 12 (1998) 600–602. [13] C. Guerra-Araiza, A. Reyna-Neyra, A.M. Salazar, M.A. Cerbon, S. Morimoto, I. Camacho-Arroyo, Progesterone receptor isoforms expression in the prepuberal and adult male rat brain, Brain Res. Bull. 54 (2001) 13–17. [14] K.Z. Haas, E.F. Sperber, L.A. Opanashuk, P.K. Stanton, S.L. Moshe, Resistance of immature hippocampus to morphologic and physiologic alterations following status epilepticus or kindling, Hippocampus 11 (2001) 615–625. [15] R. Huang, C. Bell-Horner, M. Dibas, D. Covey, J. Drewe, G. Dillon, Pentylenetetrazole-induced inhibition of recombinant gamma—aminobutyric acid type A (GABAA) receptors: mechanism and site of action, J. Pharmacol. Exp. Ther. 298 (2001) 986–995. [16] D. Kondziella, J. Hammer, O. Sletvold, U. Sonnewald, The pentylenetetrazolekindling model of epilepsy in SAMP8 mice: glial-neuronal metabolic interactions, Neurochem. Int. 43 (2003) 629–637. [17] G.E. Lopez-Galindo, E. Cano-Europa, R. Ortiz-Butron, Ketamine prevents lidocaine-caused neurotoxicity in the CA3 hippocampal and basolateral amygdala regions of the brain in adult rats, J. Anesth. 22 (2008) 471–474. [18] R.L. Macdonald, J.L. Barker, Different actions of anticonvulsant and anesthetic barbiturates revealed by use of cultured mammalian neurons, Science 200 (1978) 775–777.
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E. Cano-Europa et al. / Neuroscience Letters 470 (2010) 111–114
[19] S.L. Moshe, B.J. Albala, R.F. Ackermann, J. Engel, Increased seizure susceptibility of the immature brain, Brain Res. 283 (1983) 81–85. [20] P. NˇıGouemo, B. Koudogbo, H.P. Tchivounda, A. Nguema, M.M. Etoua, Effects of ethanol extract of Annona muricata on pentylenetetrazol induced convulsive seizures in mice, Phytol. Res. 11 (1997) 243– 245. [21] J.H. Park, H. Cho, H. Kim, K. Kim, Repeated brief epileptic seizures by pentylenetetrazole cause neurodegeneration and promote neurogenesis in discrete brain regions of freely moving adult rats, Neuroscience 140 (2006) 673–684. [22] N. Patsoukis, G. Zervoudakis, N.T. Panagopoulos, C.D. Georgiou, F. Angelatou, N.A. Matsokis, Thiol redox state (TRS) and oxidative stress in the mouse hippocampus after pentylenetetrazol-induced epileptic seizure, Neurosci. Lett. 357 (2004) 83–86. [23] N. Patsoukis, G. Zervoudakis, C.D. Georgiou, F. Angelatou, N.A. Matsokis, N.T. Panagopoulos, Effect of pentylenetetrazol-induced epileptic seizure on thiol redox state in the mouse cerebral cortex, Epilepsy Res. 62 (2004) 65– 74. [24] G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1999.
[25] R.J. Racine, Modification of seizure activity by electrical stimulation. I. After-discharge threshold, Electroencephalogr. Clin. Neurophysiol. 32 (1972) 269–279. [26] R. Ramanjaneyulu, M. Ticku, Interactions of pentamethylenetetrazole and tetrazole analogues with the picrotoxinin site of the benzodiazepine-GABA receptor-ionophore complex, Eur. J. Pharmacol. 98 (1984) 337–345. [27] D.S. Reddy, Testosterone modulation of seizure susceptibility is mediated by neurosteroids 3alpha-androstanediol and 17beta-estradiol, Neuroscience 129 (2004) 195–207. [28] L. Rocha, M. Briones, R.F. Ackermann, B. Anton, N.T. Maidment, C.J. Evans, J. Engel, Pentylenetetrazol-induced kindling: early involvement of excitatory and inhibitory systems, Epilepsy Res. 26 (1996) 105–113. [29] H. Schroeder, A. Becker, V. Hoellt, Sensitivity and density of glutamate receptor subtypes in the hippocampal formation are altered in pentylenetetrazolekindled rats, Exp. Brain Res. 120 (1998) 527–530. [30] C.E. Stafstrom, S.L. Moshe, J.W. Swann, A. Nehlig, M.P. Jacobs, P.A. Schwartzkroin, Models of pediatric epilepsies: strategies and opportunities, Epilepsia 47 (2006) 1407–1414. [31] M.J. West, Regionally specific loss of neurons in the aging human hippocampus, Neurobiol. Aging 14 (1993) 287–293.