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Effect of medroxyprogesterone on development of pentylenetetrazole-induced kindling in mice
Neuroscience 207 (2012) 283–287
EFFECT OF MEDROXYPROGESTERONE ON DEVELOPMENT OF PENTYLENETETRAZOLE-INDUCED KINDLING IN MICE S. A. NASIR,a A. SHARMA,b R. KHANAMa AND D. VOHORAa*
pentylenetetrazole- (PTZ) induced seizure in mice (Frye et al., 2002), maximal electroshock- (MES) induced seizure in rats (White et al., 1995), kainic acid-induced seizure in rats (Nicoletti et al., 1985) and amygdala-kindled seizure in rats (Lonsdale and Burnham, 2003) and so forth. In addition to experimental studies, there are some clinical reports like decreasing catamenial epilepsy in women (Reddy, 2005) suggesting the antiseizure effects of progesterone. The widely accepted mechanism of anticonvulsant action of progesterone is via conversion to a neurosteroid allopregnalonone (3␣, 5␣, tetra hydroprogesterone), which is a potent allosteric modulator of GABAA receptors (Frye, 1995). It is interesting to note that unlike progesterone, medroxyprogesterone (MPA, synthetic progesterone) doesn’t get metabolized to allopregnanolone, which is thought to be responsible for the antiepileptic effects of progesterone via GABAA receptors (Ciriza et al., 2006). However, MPA has interestingly reported efficacy in some experimental and clinical studies including improvement of seizures in catamenial epilepsy in women (Mattson et al., 1984), reduction of kainic acid-induced seizures in male and female rats (Nicoletti et al., 1985) suggesting it has antiepileptic effects. In the present study, the effect of medroxyprogesterone is evaluated for its effect on PTZ-kindling model of epileptogenesis in mice followed by evaluation on kindlinginduced changes in cognitive and motor functions. Because gender can influence the effect of progesterone on seizures, the study was conducted in both male and female mice. To explore whether the effects are mediated via progesterone receptors, an antagonist of progesterone (mifepristone) was also administered.
a Neurobehavioral Pharmacology Laboratory, Department of Pharmacology, Faculty of Pharmacy, Hamdard University, New Delhi 110062, India b
Institute of Genomics & Integrative Biology(IGIB), New Delhi, India
Abstract—In the present study, the effect of medroxyprogesterone (MPA) is evaluated for its effect on pentylenetetrazole (PTZ) kindling model of epileptogenesis in mice followed by evaluation on kindling-induced changes in cognitive and motor functions. To explore whether the effects are mediated via progesterone receptors, a selective antagonist of progesterone (mifepristone, MIF) was also taken. Kindling was induced by once every 2 days treatment with PTZ (25 mg/kg, i.p.) for 5 weeks. The seizure severity during induction of kindling and % incidence of animals kindled at the end of 5 weeks were recorded. The motor function was assessed using a grip strength meter, whereas spatial memory was assessed in a cross maze. MPA (5 and 10 mg/kg, i.p.) significantly reduced the seizure severity scores and produced a significant decrease in the incidence of animals kindled at the end of 5 weeks (P<0.01). A higher efficacy was observed against male mice as compared with females following MPA. MIF neither reduced nor delayed the development of PTZ-induced kindling in mice. Also, it couldn’t reverse the antiepileptogenic effects of MPA. On grip strength test (GST) and spontaneous alternation behavior (SAB), a significant decline in GST and % alternation was observed in kindled mice which was reversed by pre-treatment with MPA. MIF, however, could reverse only the reduced % alternation and not grip strength (GS) in PTZ-kindled animals. The study shows that MPA has antiepileptogenic effects against development of PTZ-induced kindling in mice that may not be mediated via progesterone receptors. © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.
EXPERIMENTAL PROCEDURES
Key words: medroxyprogesterone, mifepristone, diazepam, PTZ kindling, grip strength, spontaneous alternation behavior.
Animals Swiss albino mice weighing between 25 and 35 g and raised at the Central animal house facility of Jamia Hamdard were used. They were housed in polypropylene cages and maintained at 25–30 °C and 50 –55% humidity in a natural light/dark cycle. They were fed on a standard pellet diet (Amrut rat and mice feed; Chakan Oil Mills, Pune, India) and water ad libitum. All procedures involving animals were conducted in accordance with the guidelines of the Animal Ethics Committee, Hamdard University (project # 681). Utmost care was taken to ensure that minimum required animals are used in the study and they are treated in most humane manner.
Literature suggests that progestins can have profound effect on seizure processes (Murialdo et al., 2009; Frye, 2010). Both progesterone and its metabolite have been shown to have antiseizure effect in various animal models (Lonsdale and Burnham, 2007; Singh et al., 2010). Progesterone has shown to be inhibitory to CNS, and it decreases epileptiform activity in various animal models like *Corresponding author. Tel: ⫹91-11-26059688 (ext. 5657). E-mail address: [email protected] or divyavohora@hotmail. com (D. Vohora). Abbreviations: AED, antiepileptic drug; ANOVA, analysis of variance; GS, grip strength; GST, grip strength test; MIF, mifepriston; MPA, medroxyprogesterone; PTZ, pentylenetetrazole; SAB, Spontaneous alternation behavior.
Drugs The studies used the following drugs and chemicals: PTZ and mifepristone (Sigma, USA); MPA acetate (Pfizer, USA); diazepam (Ranbaxy laboratories, India). MPA was administered half an hour before PTZ at doses of 5 mg/kg and 10 mg/kg converted by the
0306-4522/12 $36.00 © 2012 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2012.01.031
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Table 1. Effect of medroxyprogesterone and mifepristone on the incidence of animals kindled following repeated treatment with a subconvulsant dose of pentylenetetrazole in mice S. No.
Groups
% incidence of seizures in male mice (mean⫾SEM)
% incidence of seizures in female mice (mean⫾SEM)
1 2 3 4 5 6 7
Control VEH⫹PTZ MPA1⫹PTZ MPA2⫹PTZ MIF⫹MPA1⫹PTZ MIF⫹PTZ DZP⫹PTZ
0.00$$ 89.29⫾5.05** 71.43⫾6.52**$$ 50.00⫾0.00**,$$ 64.29⫾5.05**,$$ 92.86⫾4.61** 14.29⫾5.05$$
0.00$$ 96.42⫾3.57** 75.00⫾9.44** 82.14⫾7.14** 60.71⫾5.05**,$$ 92.85⫾4.61** 10.71⫾5.05$$
PTZ (25 mg/kg) was administered once every 2 d for 5 wk, whereas MPA1 (5 mg/kg) and MPA2 (10 mg/kg) and MIF (2.5 mg/kg) were administered daily. All treatments were given by i.p. route. The total number of animals in a group was seven; ** P⬍0.01, when compared with normal control (Group 1), $$P⬍0.01, when compared with toxic control (Group 2), significant by analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. Abbreviations: VEH, Vehicle (Sterile water for injection); MPA, medroxyprogesterone; MIF, mifepristone; DZP, diazepam; PTZ, pentylenetetrazole.
method of Freireich et al. (1966) from corresponding rat dose exhibiting protection against kainic acid-induced seizures (Nicoletti et al., 1985). Mifepriston (MIF) was given at a dose of 2.5 mg/kg, 2 h before the administration of PTZ (Borowicz et al., 2002). The solution of MIF was made in dimethylsulfoxide. Diazepam was taken as standard and was given at 3 mg/kg 1.5 h before PTZ (Ali et al., 2006). All drugs were administered intraperitoneally between 9 AM to 12 noon every day.
Induction of kindling Kindling was induced according to the method of Ali et al. (2006). Pentylenetetrazole (PTZ, 25 mg/kg, i.p.) was injected once every 2 days for 5 weeks to induce kindling. The intensity of seizure response was scored as 0⫽no response, 1⫽mouth and facial jerks, 2⫽nodding or myoclonic body jerks, 3⫽forelimb clonus, 4⫽rearing, falling down (loss of postural control), hind limb clonus and forelimb tonus, 5⫽tonic extension of hind limb, status epilecticus, and/or death. Animals were observed for 30 min after PTZ injection. When the animals had a seizure score of 4 on three consecutive administrations, they were defined as being kindled and PTZ treatment was then discontinued. The effect of various treatments on the seizure severity during induction of kindling and % incidence of animals kindled at the end of 5 weeks was recorded.
Grip strength test The neuromuscular function was determined with the aid of a grip strength meter. The mouse was allowed to hold the grip with its forepaws. The mouse was then pulled back horizontally until it releases its grip. The grip strength reading was directly read from the digital meter (Ali et al., 2004).
Spontaneous alternation behavior (SAB) Spontaneous alternation behavior (SAB) in a cross maze was assessed using the method of Ragozzino and coworkers (1998) suitably modified by us for mice (Vohora et al., 2005). A four-arm wooden cross maze (height: 50 cm, arms: length 23.5 cm, breadth 8 cm, wall height 10 cm) with a central platform (8.8 cm) was used. The dimensions of the maze were similar to the elevated plus
maze for mice as per the method of Itoh et al. (1991). However, unlike the elevated plus maze, the cross maze consisted of four arms with 10-cm walls all of which were open as per the method described for rats (Ragozzino et al., 1998). After being placed in the central platform, mice were allowed to traverse the maze freely for 6 min. The number and sequence of entries were recorded; an alternation is defined as entry into four different arms on an overlapping quintuple set. Five consecutive arm choices within the total set of arm choices constitute a quintuple set. A quintuple set consisting of arm choices B, A, C, B, D comprised an alternation, whereas the set with B, A, D, B, A did not. Percentage alternation was calculated as actual alternations/possible alternations⫻100, where possible alternation was number of arm entries minus four.
Statistical analysis Incidence of seizures, grip strength, and % alteration is expressed as mean⫾SEM and analyzed by ANOVA followed by Dunnett’s t test. Seizure severity is presented as median seizure score with upper and lower quartiles and analyzed by Kruskal–Wallis oneway analysis of ranks test.
RESULTS Effect of MPA on development of PTZ kindling Repeated treatment with PTZ at a subconvulsant dose (25 mg/kg, i.p.), three times a week, induced chemical kindling (Table 1, Figs. 1 and 2). Animals treated with diazepam (3 mg/kg, i.p.) exhibited a significant reduction in incidence (P⬍0.01, Table 1) and severity of seizures (P⬍0.01, Figs. 1–2). MPA (5 and 10 mg/kg, i.p.) effectively delayed the development of PTZ-induced kindling in mice. This was demonstrated by a significant reduction in the seizure severity scores in mice (Figs. 1–2) and a significant decrease in the incidence (Table 1) of seizures at the end of 5 weeks. A dose of 10 mg/kg was more efficacious. A higher efficacy was observed against male mice as compared with females following MPA. Mifepristone (MIF) neither reduced nor delayed the development of PTZ-induced kindling in mice. Pre-treatment with MIF couldn’t reverse the antiepileptogenic effects of MPA. 5
median seizure score
284
4 3
#
2
#
1
#
VEH + PTZ MPA 1 + PTZ MPA 2 + PTZ MIF + MPA 1 + PTZ MIF + PTZ DZP + PTZ
0 7th day 14th day 21st day 28th day 35th day
days of treatment
Fig. 1. Effect of medroxyprogesterone and mifepristone on seizure severity during induction of kindling by PTZ in male mice. PTZ (25 mg/kg) was administered once every 2 d for 5 wk, whereas MPA1 (5 mg/kg) and MPA2 (10 mg/kg) and MIF (2.5 mg/kg) were administered daily. All treatments were given by i.p. route. Data are presented as median seizure score with upper and lower quartiles. # P⬍0.05 vs. VEH⫹PTZ by Kruskal–Wallis one-way analysis by ranks (at the end of 35th day of treatment). The total numbers of animals in a group were seven. VEH: Vehicle (Sterile water for injection); MPA: medroxyprogesterone; MIF: mifepristone; DZP: diazepam; PTZ: pentylenetetrazole.
S. A. Nasir et al. / Neuroscience 207 (2012) 283–287
median seizure score
5 4 3
#
2
#
1
#
VEH + PTZ MPA 1 + PTZ MPA 2 + PTZ MIF + MPA 1 + PTZ MIF + PTZ DZP + PTZ
0 7th day 14th day 21st day 28th day 35th day
days of treatment
Fig. 2. Effect of medroxyprogesterone and mifepristone on seizure severity during induction of kindling by PTZ in female mice. VEH: Vehicle (Sterile water for injection); MPA: medroxyprogesterone; MIF: mifepristone; DZP: diazepam; PTZ: pentylenetetrazole; PTZ (25 mg/kg) was administered once every 2 d for 5 wk, whereas MPA1 (5 mg/kg) and MPA2 (10 mg/kg) and MIF (2.5 mg/kg) were administered daily. All treatments were given by i.p. route. Data are presented as median seizure score with upper and lower quartiles. # P⬍0.05 vs. VEH⫹PTZ by Kruskal–Wallis one-way analysis by ranks (at the end of 35th day of treatment). The total number of animals in a group was seven.
Effect of MPA on grip strength and spontaneous alteration following PTZ kindling On grip strength test (GST) and SAB, a significant decline in GST and percentage alternation was observed in kindled mice (Tables 2–3). Treatment with MPA could reverse these significantly. MIF, however, could reverse only the reduced percentage alternation and not grip strength (GS) in PTZ-kindled animals.
DISCUSSION Current antiepileptic drug (AED) therapy is symptomatic. Treatment strategies that may alleviate seizure-induced neuronal damage can protect against the process leading to epilepsy (epileptogenesis) (Acharya et al., 2008). Steroidal hormones are reported to have neuroprotective poTable 2. Effect of medroxyprogesterone and mifepristone on the grip strength following PTZ-induced kindling in mice S. No.
Groups
Grip strength of male mice (kg)
Grip strength of female mice (kg)
1 2 3 4 5 6 7
Control VEH⫹PTZ MPA1⫹PTZ MPA2⫹PTZ MIF⫹MPA1⫹PTZ MIF⫹PTZ DZP⫹PTZ
0.097⫾0.003$$ 0.065⫾0.002** 0.104⫾0.007$$ 0.072⫾0.007* 0.112⫾0.006$$ 0.053⫾0.003** 0.085⫾0.004
0.086⫾0.002$$ 0.052⫾0.001** 0.103⫾0.004**,$$ 0.090⫾0.005$$ 0.113⫾0.050**,$$ 0.052⫾0.002** 0.084⫾0.002$$
PTZ (25 mg/kg) was administered once every 2 d for5 wk, whereas MPA1 (5 mg/kg) and MPA2 (10 mg/kg) and MIF (2.5 mg/kg) were administered daily. All treatments were given by i.p. route. The total number of animals in a group was seven. Data are represented as mean⫾SEM, ** P⬍0.01, P⬍0.05 when compared with normal control (Group 1), $$P⬍0.01, when compared with PTZ (Group 2), significant by ANOVA followed by Dunnett’s t-test. Abbreviations: VEH, Vehicle (Sterile water for injection); MPA, medroxyprogesterone; MIF, mifepristone; DZP, diazepam; PTZ, pentylenetetrazole.
285
Table 3. Effect of medroxyprogesterone and mifepristone on the spontaneous alternation behavior of mice following PTZ-induced kindling S. No.
Groups
% alternation in male mice
% alternation in female mice
1 2 3 4 5 6 7
Control VEH⫹PTZ MPA1⫹PTZ MPA2⫹PTZ MIF⫹MPA1⫹PTZ MIF⫹PTZ DZP⫹PTZ
76.14⫾0.95$$ 44.96⫾1.71** 57.45⫾1.17** 63.48⫾2.02**,$$ 76.08⫾1.14$$ 78.12⫾2.19$$ 60.83⫾1.75**,$$
69.30⫾2.95$$ 42.97⫾1.27** 61.14⫾1.52*,$$ 65.34⫾3.21$$ 79.19⫾1.72**,$$ 72.94⫾1.36$$ 73.07⫾1.59$$
PTZ (25 mg/kg) was administered once every2 d for5 wk, whereas MPA1 (5 mg/kg) and MPA2 (10 mg/kg) and MIF (2.5 mg/kg) were administered daily. All treatments were given by i.p. route. The total number of animals in a group was seven. Data are represented as mean⫾SEM, ** P⬍0.01, * P⬍0.05 when compared with normal control (Group 1), $$P⬍0.01, when compared with PTZ (Group 2), significant by ANOVA followed by Dunnett’s t-test. Abbreviations: VEH, Vehicle (Sterile water for injection); MPA, medroxyprogesterone; MIF, mifepristone; DZP, diazepam; PTZ, pentylenetetrazole.
tential and are increasingly being investigated in seizure disorders. Progesterone is an important hormone in epilepsy research due to its modulating actions in brain (Reddy et al., 2010). It is both a reproductive hormone and a neurosteroid that can modulate neuronal excitability. Progesterone targets multiple molecular and cellular mechanisms that are relevant to epileptogenesis. While it’s cellular actions are mediated via progesterone receptors (Brinton et al., 2008), its antiseizure effects are attributed to its conversion to allopregnanolone, which is a potent positive modulator of GABAA receptor (Frye, 1995). The present study evaluated the effects of an acetylated derivative of progesterone, MPA, which is also the most common progesterone included in hormone therapy on development of PTZ-induced kindling in mice. Unlike progesterone, the MPA’s metabolite does not seem to influence GABAA receptors (McAuley et al., 1993) yet there are reports of its efficacy in catamenial epilepsy in women (Mattson et al., 1984). We found that MPA effectively delayed the development of PTZ-induced kindling in mice. This was demonstrated by a significant reduction in the seizure severity scores and a significant decrease in the incidence of animals kindled at the end of 5 weeks. The protection was, however, lesser as compared with the standard drug diazepam. Although there are several studies available on the anticonvulsant effects of progesterone (Frye, 1995; Mohammad et al., 1998; Reddy et al., 2010), relatively fewer studies have examined the effect of MPA on seizures. There are a few clinical reports where MPA showed improvement in seizure frequency in women with catamenial epilepsy (Mattson et al., 1984; Zimmerman et al., 1973). In our study, MPA exhibited a higher efficacy against male mice as compared with females. This is in contrast with an experimental study against kainic acid induced seizures where MPA was shown to protect female (but not male) animals (Nicoletti et al., 1985). Progesterone, however, was reported to protect against epileptogenesis in male rats as well (Reddy et al., 2010). It is noteworthy that progesterone is considered to be
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an endogenous anticonvulsant (Frye and Scalise, 2000) and is present in brains of both male and female humans and rodents (Hammond et al., 1983) and, thus, could be an effective endogenous anticonvulsant in both. Women with epilepsy are prone to seizures in response to decreased levels of progesterone during perimenstrual periods (Reddy et al., 2010). Jacono and Robertson (1987) reported that progesterone may worsen seizure frequency in epileptic women (Jacono and Robertson, 1987). In addition to this, some women reportedly experienced an increased seizure with MPA (Foldvary-Schaefer et al., 2004). These studies along with the changes related to estrous cycle may explain the lesser efficacy of MPA observed in female mice in our study. In our study, we didn’t take into account different stages of estrous cycle of mouse. The progesterone receptor antagonist MIF neither reduced nor delayed the development of PTZ-induced kindling in mice. This is in agreement with a study on amygdala kindling of rats where MIF didn’t affect the seizure parameters in either sex (Borowicz et al., 2003). Further, its administration before MPA did not reverse the antiepiletogenic effects of MPA on PTZ kindling indicating that progesterone receptors may not mediate such effect of MPA. Contrary to this, progesterone receptor knock out mice were demonstrated to exhibit a marked delay in the rate of kindling pointing toward the involvement of progesterone receptors in kindling progression (Reddy et al., 2010). More recently, the same group also demonstrated that progesterone knock-out, knock-down, and blockade of its receptors had antiepileptogenic effects (Reddy and Mohan, 2011. This shows that activation of progesterone receptors may not mediate the antiepileptic effects of progesterone, and alternative mechanisms may be involved. In line with these findings, if knock-out or inhibition of progesterone receptors is antiepileptogenic, an agonist would be expected to facilitate epileptogenesis. Hence activation of progesterone receptors would counteract the effects mediated by the alternative pathway. Going by this hypothesis, MIF should increase the antiepileptogenic effects of MPA. We too found a mild increase in reduction of % incidence (Table 1) and reduction in seizure severity (Table 3) in female mice when MIF was administered along with MPA. Several mechanisms have been proposed by which progesterone decreases epileptic seizures other than activation of progesterone receptors such as synthesis of a neurosteroid and by promoting neuroprotection (Reddy et al., 2010). Both progesterone and MPA are reported to cause a rapid and transient activation of extracellular receptor kinase, the most common form of mitogen-activated protein kinase (Nilsen and Brinton, 2003), which is thought to be responsible for their proposed neuroprotective effects. It remains to be seen whether the antiepileptogenic effects of MPA in the present study are mediated via their proposed neuroprotective mechanisms. Given the fact that epilepsy and AED therapy are often associated with motor and cognitive dysfunction, the present study also evaluated the effects on motor function and spatial memory following PTZ-induced kindling. We chose to work with spontaneous alternation behavior, as it is
widely used as a model to measure spatial memory in rodents (Ragozzino et al., 1998; Vohora et al., 2005). Further, kindling induced by PTZ is known to impair spatial learning and memory in rodents (Mao et al., 2009; Omrani et al., 2007), whereas progesterone is reported to improve performance in spatial working tasks in rats (Frye et al., 2009) and to enhance cognitive performance in spontaneous alternation tasks (Frye and Walf, 2008). For assessing motor function, GST was used. As expected, a significant decline in the GS and percentage alternation was observed following PTZ kindling indicating a deficit of motor function and spatial memory, respectively. Treatment with MPA could reverse these deficits. In addition to MPA, MIF could also reverse the impairment of the spatial memory in PTZ-kindling mice. The literature reports both impairing and improving effects of progesterone on cognitive functions (Braden et al., 2010; Frye et al., 2009; Frye and Walf, 2008). Although it has been shown to impair memory in radial arm maze and Morris maze (Braden et al., 2010), an improved consolidation of spatial information has been reported in water maze (Frye et al., 2009), Y-maze (Frye and Walf, 2008), and in object recognition test (Lewis et al., 2008). In line with the latter reports on improvement of spatial information by progesterone, MPA (in our study) could reverse the impaired spatial deficits induced by PTZ kindling. It is possible that MPA improves spatial type of memory, and its effects on other types of memory may be differential like progesterone. The latter was reported to improve cognitive performance in the spontaneous alteration and object recognition but not in water maze, contextual and cued conditioned fear test (Frye and Walf, 2008). Though not many studies have evaluated the effects of MIF on memory, recently, it was reported to reverse memory loss induced by spontaneous morphine withdrawal in mice. The effects of MIF were attributed to antagonism of glucocorticoid receptors (Mesripour et al., 2008). In another study, however, MIF did not improve cognitive functions in schizophrenic patients (Gallagher et al., 2005). To summarize, our study is the first to show that not only progesterone but also the synthetic progesterone, MPA, has antiepileptogenic effects against development of PTZ-induced kindling in mice. Mifepristone, a progesterone receptor antagonist, fails to reverse the protective effects of MPA indicating that progesterone receptors may not mediate such effects. Although further studies are absolutely necessary to elucidate the mechanism of antiepileptogenic effects of MPA, our study clearly demonstrates that additional mechanisms (other than conversion to allopregnanolone) may contribute to antiseizure effects of progesterone and/or MPA. Acknowledgments—The work was carried out in Neurobehavioral Pharmacology laboratory funded by Special Assistance Programme of University Grants Commission, New Delhi.
REFERENCES Acharya MM, Hattiangady B, Shetty AK (2008) Progress in neuroprotective strategies for preventing epilepsy. Prog Neurobiol 84:363–404.
S. A. Nasir et al. / Neuroscience 207 (2012) 283–287 Ali A, Pillai KK, Ahmad FJ, Dua Y, Vohora D (2004) Evidence of the antiepileptic potential of amiloride with neuropharmacological benefits in rodent models of epilepsy and behaviour. Epilepsy Behav 5:322–328. Ali A, Pillai KK, Ahmad FJ, Dua Y, Vohora D (2006) Anticonvulsant effect of amiloride in pentylenetetrazole-induced status epilepticus in mice. Pharmacol Rep 58:242–245. Borowicz KK, Kleinrok Z, Czuczwar SJ (2003) Influence of sex hormone antagonists on the anticonvulsant action of conventional antiepileptic drugs against amygdala-kindled seizures in male and female rats. Eur Neuropsychopharmacol 13:257–265. Borowicz KK, Luszczki J, Matuszek M, Kleinrok Z, Czuczwar SJ (2002) Effects of tamoxifen, mifepristone and cyproterone on the electroconvulsive threshold and pentylenetetrazole-induced convulsion in mice. Pol J Pharmacol 54:103–109. Braden BB, Talboom JS, Crain ID, Simard AR, Lukas RJ, Prokai P, Scheldrup MR, Bowman BL, Bimonte-Nelson BA (2010) Medroxyprogesterone acetate impairs memory and alters the GABAergic system in aged surgically menopausal rats. Neurobiol Learn Mem 93:444 – 453. Brinton RD, Thompson RF, Foy MR, Baudry M, Wang J, Finch CE, Morgan TE, Pike CJ, Mack WJ, Stanczyk FZ, Nilsen J (2008) Progesterone receptors: form and function in brain. Front Neuroendocrinol 29:313–339. Ciriza I, Carrero P, Frye CA, Garcia-segura LM (2006) Reduced metabolites mediate neuroprotective effects of progesterone in the adult rat hippocampus. The synthetic progestin medroxyprogesterone acetate (Provera) is not neuroprotective. J Neurobiol 66:916 –928. Foldvary-Schaefer N, Harden C, Herzog A, Falcone T (2004) Hormones and seizures. Cleve Clin J Med 71:S11–S18. Freireich EJ, Gehan EA, Rall DP (1966) Quantitative comparison of toxicity of anticancer agents in mouse, rat, hamster, dog, monkey and man. Cancer Chemother Rep 50:219 –244. Frye CA (1995) The neurosteroid 3c, 5c THP has anti seizure and possible neuroprotective effects in an animal model of epileptogenesis. Brain Res 696:113–120. Frye CA (2010) Effects and mechanisms of progestogens and androgens in ictal activity. Epilepsia 51(3):135–140. Frye CA, Llaneza DC, Walf AA (2009) Progesterone can enhance consolidation and/or performance in spatial, object and working memory tasks in Long–Evans rats. Anim Behav 78:279 –286. Frye CA, Rhodes ME, Walf A, Harney J (2002) Progesterone reduces pentylenetetrazole-induced ictal activity of wild-type mice but not those deficient in type I 5␣-reductase. Epilepsia 43:14 –17. Frye CA, Scalise TJ (2000) Anti-seizure effects of progesterone and 3␣, 5␣-THP in kainic acid and perforant pathway models of epilepsy. Psychoneuroendocrinology 25:407– 420. Frye CA, Walf AA (2008) Effects of progesterone administration and APPswe ⫹ PSEN1De9 mutation for cognitive performance of midaged mice. Neurobiol Learn Mem 89:17–26. Gallagher P, Watson S, Smith MS, Ferrier IN, Young AH (2005) Effects of adjunctive mifepristone (RU-486) administration on neurocognitive function and symptoms in schizophrenia. Biol Psychiatry 57:155–161. Hammond GL, Hirvonen J, Vihko R (1983) Progesterone, androstenedione, testosterone, 5␣-dihydrotestosterone and androsterone concentrations in specific regions of the human brain. J Steroid Biochem 18:185–189. Itoh J, Nabeshima T, Kameyama T (1991) Utility of an elevated plusmaze for dissociation of amnesic and behavioral effects of drugs in mice. Eur J Pharmacol 194:71–76. Jacono JJ, Robertson JMD (1987) The effects of estrogen, progesterone, and ionized calcium on seizures during the menstrual cycle of epileptic women. Epilepsia 28:571–577.
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Lewis MC, Orr PT, Frick KM (2008) Differential effects of acute progesterone administration on spatial and object memory in middleaged and aged female C57BL/6 mice. Horm Behav 54:455– 462. Lonsdale D, Burnham WM (2003) The anticonvulsant effects of progesterone and 5␣-dihydroprogesterone on amygdala-kindled seizures in rats. Epilepsia 44:1494 –1499. Lonsdale D, Burnham WM (2007) The anticonvulsant effects of allopregnanolone against amygdala-kindled seizures in female rats. Neurosci Lett 411:147–151. Mao RR, Tian M, Yue-Xiong YX, Qi-Xin Z, Lin X, Jun C (2009) Effects of pentylenetetrazol-induced brief convulsive seizures on spatial memory and fear memory. Epilepsy Behav 15:441– 444. Mattson RH, Cramer JA, Caldwell BV, Siconolfi BC (1984) Treatment of seizures with medroxyprogesterone acetate: preliminary report. Neurology 34:1255–1258. McAuley JW, Kroboth PD, Stiff DD, Reynolds IJ (1993) Modulation of [3H] flunitrazepam binding by natural and synthetic progestational agents. Pharmacol Biochem Behav 45:77– 83. Mesripour A, Hajhashemi V, Rabbani M (2008) Metyrapone and mifepristone reverse memory loss induced by spontaneous morphine withdrawal in mice. Eur Psychiatry 23 (Suppl 2):S292. Mohammad S, Abolhassan A, Pourgholami MH (1998) Evaluation of the anticonvulsant profile of progesterone in male amygdala-kindled rats. Epilepsy Res 30:195–202. Murialdo G, Magri F, Tamagno G, Ameri P, Camera A, Colnaghi S, Perucca P, Ravera G, Galimberti CA (2009) Seizure frequency and sex steroids in women with partial epilepsy on antiepileptic therapy. Epilepsia 50:1920 –1926. Nicoletti F, Speciale C, Sortino MA, Summa G, Caruso G, Patti F, Canonico PL (1985) Comparative effects of estradiol benzoate, the antiestrogen clomiphene citrate, and the progestin medroxyprogesterone acetate on kainic acid-induced seizures in male and female rats. Epilepsia 26:252–257. Nilsen J, Brinton RD (2003) Divergent impact of progesterone and medroxyprogesterone acetate (Provera) on nuclear mitogen-activated protein kinase signaling. Proc Natl Acad Sci U S A 100:10506 –10511. Omrani A, Ghadami MR, Fathi N, Tahmasian M, Fathollahi Y, Touhidi A (2007) Naloxone improves impairment of spatial performance induced by pentylenetetrazol kindling in rats. Neuroscience 145:824 – 831. Ragozzino ME, Pal SN, Unick K, Stefani MR, Gold PE (1998) Modulation of hippocampal acetylcholine release and spontaneous alternation scores by intrahippocampal glucose injections. J Neurosci 18:1595–1601. Reddy DS (2005) Pharmacotherapy of catamenial epilepsy. Indian J Pharmacol 37:288 –293. Reddy DS, Gangisetty O, Briyal S (2010) Disease-modifying activity of progesterone in the hippocampus kindling model of epileptogenesis. Neuropharmacology 59:573–581. Singh S, Hota D, Prakash A, Khanduja KL, Arora SK, Chakrabarti A (2010) Allopregnanolone, the active metabolite of progesterone protects against neuronal damage in picrotoxin-induced seizure model in mice. Pharmacol Biochem Behav 94:416 – 422. Vohora D, Pal SN, Pillai KK (2005) Modulation of spontaneous alternation behavior of mice treated with thioperamide and tacrine in a cross maze task. Fundam Clin Pharmacol 19:531–532. White HS, Woodhead JH, Franklin MR, Swinyard EA, Wolf HA (1995) General principles, experimental selection, quantification, and evaluation of antiepileptic drugs. Antiepilep. Drugs 4:99 –110. Zimmerman AW, Holden KR, Reiter EO, Dekaban AS (1973) Medroxyprogesterone acetate in the treatment of seizures associated with menstruation. J Paediatr 83:959 –963.
(Accepted 14 January 2012) (Available online 25 January 2012)
EFFECT OF MEDROXYPROGESTERONE ON DEVELOPMENT OF PENTYLENETETRAZOLE-INDUCED KINDLING IN MICE S. A. NASIR,a A. SHARMA,b R. KHANAMa AND D. VOHORAa*
pentylenetetrazole- (PTZ) induced seizure in mice (Frye et al., 2002), maximal electroshock- (MES) induced seizure in rats (White et al., 1995), kainic acid-induced seizure in rats (Nicoletti et al., 1985) and amygdala-kindled seizure in rats (Lonsdale and Burnham, 2003) and so forth. In addition to experimental studies, there are some clinical reports like decreasing catamenial epilepsy in women (Reddy, 2005) suggesting the antiseizure effects of progesterone. The widely accepted mechanism of anticonvulsant action of progesterone is via conversion to a neurosteroid allopregnalonone (3␣, 5␣, tetra hydroprogesterone), which is a potent allosteric modulator of GABAA receptors (Frye, 1995). It is interesting to note that unlike progesterone, medroxyprogesterone (MPA, synthetic progesterone) doesn’t get metabolized to allopregnanolone, which is thought to be responsible for the antiepileptic effects of progesterone via GABAA receptors (Ciriza et al., 2006). However, MPA has interestingly reported efficacy in some experimental and clinical studies including improvement of seizures in catamenial epilepsy in women (Mattson et al., 1984), reduction of kainic acid-induced seizures in male and female rats (Nicoletti et al., 1985) suggesting it has antiepileptic effects. In the present study, the effect of medroxyprogesterone is evaluated for its effect on PTZ-kindling model of epileptogenesis in mice followed by evaluation on kindlinginduced changes in cognitive and motor functions. Because gender can influence the effect of progesterone on seizures, the study was conducted in both male and female mice. To explore whether the effects are mediated via progesterone receptors, an antagonist of progesterone (mifepristone) was also administered.
a Neurobehavioral Pharmacology Laboratory, Department of Pharmacology, Faculty of Pharmacy, Hamdard University, New Delhi 110062, India b
Institute of Genomics & Integrative Biology(IGIB), New Delhi, India
Abstract—In the present study, the effect of medroxyprogesterone (MPA) is evaluated for its effect on pentylenetetrazole (PTZ) kindling model of epileptogenesis in mice followed by evaluation on kindling-induced changes in cognitive and motor functions. To explore whether the effects are mediated via progesterone receptors, a selective antagonist of progesterone (mifepristone, MIF) was also taken. Kindling was induced by once every 2 days treatment with PTZ (25 mg/kg, i.p.) for 5 weeks. The seizure severity during induction of kindling and % incidence of animals kindled at the end of 5 weeks were recorded. The motor function was assessed using a grip strength meter, whereas spatial memory was assessed in a cross maze. MPA (5 and 10 mg/kg, i.p.) significantly reduced the seizure severity scores and produced a significant decrease in the incidence of animals kindled at the end of 5 weeks (P<0.01). A higher efficacy was observed against male mice as compared with females following MPA. MIF neither reduced nor delayed the development of PTZ-induced kindling in mice. Also, it couldn’t reverse the antiepileptogenic effects of MPA. On grip strength test (GST) and spontaneous alternation behavior (SAB), a significant decline in GST and % alternation was observed in kindled mice which was reversed by pre-treatment with MPA. MIF, however, could reverse only the reduced % alternation and not grip strength (GS) in PTZ-kindled animals. The study shows that MPA has antiepileptogenic effects against development of PTZ-induced kindling in mice that may not be mediated via progesterone receptors. © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.
EXPERIMENTAL PROCEDURES
Key words: medroxyprogesterone, mifepristone, diazepam, PTZ kindling, grip strength, spontaneous alternation behavior.
Animals Swiss albino mice weighing between 25 and 35 g and raised at the Central animal house facility of Jamia Hamdard were used. They were housed in polypropylene cages and maintained at 25–30 °C and 50 –55% humidity in a natural light/dark cycle. They were fed on a standard pellet diet (Amrut rat and mice feed; Chakan Oil Mills, Pune, India) and water ad libitum. All procedures involving animals were conducted in accordance with the guidelines of the Animal Ethics Committee, Hamdard University (project # 681). Utmost care was taken to ensure that minimum required animals are used in the study and they are treated in most humane manner.
Literature suggests that progestins can have profound effect on seizure processes (Murialdo et al., 2009; Frye, 2010). Both progesterone and its metabolite have been shown to have antiseizure effect in various animal models (Lonsdale and Burnham, 2007; Singh et al., 2010). Progesterone has shown to be inhibitory to CNS, and it decreases epileptiform activity in various animal models like *Corresponding author. Tel: ⫹91-11-26059688 (ext. 5657). E-mail address: [email protected] or divyavohora@hotmail. com (D. Vohora). Abbreviations: AED, antiepileptic drug; ANOVA, analysis of variance; GS, grip strength; GST, grip strength test; MIF, mifepriston; MPA, medroxyprogesterone; PTZ, pentylenetetrazole; SAB, Spontaneous alternation behavior.
Drugs The studies used the following drugs and chemicals: PTZ and mifepristone (Sigma, USA); MPA acetate (Pfizer, USA); diazepam (Ranbaxy laboratories, India). MPA was administered half an hour before PTZ at doses of 5 mg/kg and 10 mg/kg converted by the
0306-4522/12 $36.00 © 2012 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2012.01.031
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Table 1. Effect of medroxyprogesterone and mifepristone on the incidence of animals kindled following repeated treatment with a subconvulsant dose of pentylenetetrazole in mice S. No.
Groups
% incidence of seizures in male mice (mean⫾SEM)
% incidence of seizures in female mice (mean⫾SEM)
1 2 3 4 5 6 7
Control VEH⫹PTZ MPA1⫹PTZ MPA2⫹PTZ MIF⫹MPA1⫹PTZ MIF⫹PTZ DZP⫹PTZ
0.00$$ 89.29⫾5.05** 71.43⫾6.52**$$ 50.00⫾0.00**,$$ 64.29⫾5.05**,$$ 92.86⫾4.61** 14.29⫾5.05$$
0.00$$ 96.42⫾3.57** 75.00⫾9.44** 82.14⫾7.14** 60.71⫾5.05**,$$ 92.85⫾4.61** 10.71⫾5.05$$
PTZ (25 mg/kg) was administered once every 2 d for 5 wk, whereas MPA1 (5 mg/kg) and MPA2 (10 mg/kg) and MIF (2.5 mg/kg) were administered daily. All treatments were given by i.p. route. The total number of animals in a group was seven; ** P⬍0.01, when compared with normal control (Group 1), $$P⬍0.01, when compared with toxic control (Group 2), significant by analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. Abbreviations: VEH, Vehicle (Sterile water for injection); MPA, medroxyprogesterone; MIF, mifepristone; DZP, diazepam; PTZ, pentylenetetrazole.
method of Freireich et al. (1966) from corresponding rat dose exhibiting protection against kainic acid-induced seizures (Nicoletti et al., 1985). Mifepriston (MIF) was given at a dose of 2.5 mg/kg, 2 h before the administration of PTZ (Borowicz et al., 2002). The solution of MIF was made in dimethylsulfoxide. Diazepam was taken as standard and was given at 3 mg/kg 1.5 h before PTZ (Ali et al., 2006). All drugs were administered intraperitoneally between 9 AM to 12 noon every day.
Induction of kindling Kindling was induced according to the method of Ali et al. (2006). Pentylenetetrazole (PTZ, 25 mg/kg, i.p.) was injected once every 2 days for 5 weeks to induce kindling. The intensity of seizure response was scored as 0⫽no response, 1⫽mouth and facial jerks, 2⫽nodding or myoclonic body jerks, 3⫽forelimb clonus, 4⫽rearing, falling down (loss of postural control), hind limb clonus and forelimb tonus, 5⫽tonic extension of hind limb, status epilecticus, and/or death. Animals were observed for 30 min after PTZ injection. When the animals had a seizure score of 4 on three consecutive administrations, they were defined as being kindled and PTZ treatment was then discontinued. The effect of various treatments on the seizure severity during induction of kindling and % incidence of animals kindled at the end of 5 weeks was recorded.
Grip strength test The neuromuscular function was determined with the aid of a grip strength meter. The mouse was allowed to hold the grip with its forepaws. The mouse was then pulled back horizontally until it releases its grip. The grip strength reading was directly read from the digital meter (Ali et al., 2004).
Spontaneous alternation behavior (SAB) Spontaneous alternation behavior (SAB) in a cross maze was assessed using the method of Ragozzino and coworkers (1998) suitably modified by us for mice (Vohora et al., 2005). A four-arm wooden cross maze (height: 50 cm, arms: length 23.5 cm, breadth 8 cm, wall height 10 cm) with a central platform (8.8 cm) was used. The dimensions of the maze were similar to the elevated plus
maze for mice as per the method of Itoh et al. (1991). However, unlike the elevated plus maze, the cross maze consisted of four arms with 10-cm walls all of which were open as per the method described for rats (Ragozzino et al., 1998). After being placed in the central platform, mice were allowed to traverse the maze freely for 6 min. The number and sequence of entries were recorded; an alternation is defined as entry into four different arms on an overlapping quintuple set. Five consecutive arm choices within the total set of arm choices constitute a quintuple set. A quintuple set consisting of arm choices B, A, C, B, D comprised an alternation, whereas the set with B, A, D, B, A did not. Percentage alternation was calculated as actual alternations/possible alternations⫻100, where possible alternation was number of arm entries minus four.
Statistical analysis Incidence of seizures, grip strength, and % alteration is expressed as mean⫾SEM and analyzed by ANOVA followed by Dunnett’s t test. Seizure severity is presented as median seizure score with upper and lower quartiles and analyzed by Kruskal–Wallis oneway analysis of ranks test.
RESULTS Effect of MPA on development of PTZ kindling Repeated treatment with PTZ at a subconvulsant dose (25 mg/kg, i.p.), three times a week, induced chemical kindling (Table 1, Figs. 1 and 2). Animals treated with diazepam (3 mg/kg, i.p.) exhibited a significant reduction in incidence (P⬍0.01, Table 1) and severity of seizures (P⬍0.01, Figs. 1–2). MPA (5 and 10 mg/kg, i.p.) effectively delayed the development of PTZ-induced kindling in mice. This was demonstrated by a significant reduction in the seizure severity scores in mice (Figs. 1–2) and a significant decrease in the incidence (Table 1) of seizures at the end of 5 weeks. A dose of 10 mg/kg was more efficacious. A higher efficacy was observed against male mice as compared with females following MPA. Mifepristone (MIF) neither reduced nor delayed the development of PTZ-induced kindling in mice. Pre-treatment with MIF couldn’t reverse the antiepileptogenic effects of MPA. 5
median seizure score
284
4 3
#
2
#
1
#
VEH + PTZ MPA 1 + PTZ MPA 2 + PTZ MIF + MPA 1 + PTZ MIF + PTZ DZP + PTZ
0 7th day 14th day 21st day 28th day 35th day
days of treatment
Fig. 1. Effect of medroxyprogesterone and mifepristone on seizure severity during induction of kindling by PTZ in male mice. PTZ (25 mg/kg) was administered once every 2 d for 5 wk, whereas MPA1 (5 mg/kg) and MPA2 (10 mg/kg) and MIF (2.5 mg/kg) were administered daily. All treatments were given by i.p. route. Data are presented as median seizure score with upper and lower quartiles. # P⬍0.05 vs. VEH⫹PTZ by Kruskal–Wallis one-way analysis by ranks (at the end of 35th day of treatment). The total numbers of animals in a group were seven. VEH: Vehicle (Sterile water for injection); MPA: medroxyprogesterone; MIF: mifepristone; DZP: diazepam; PTZ: pentylenetetrazole.
S. A. Nasir et al. / Neuroscience 207 (2012) 283–287
median seizure score
5 4 3
#
2
#
1
#
VEH + PTZ MPA 1 + PTZ MPA 2 + PTZ MIF + MPA 1 + PTZ MIF + PTZ DZP + PTZ
0 7th day 14th day 21st day 28th day 35th day
days of treatment
Fig. 2. Effect of medroxyprogesterone and mifepristone on seizure severity during induction of kindling by PTZ in female mice. VEH: Vehicle (Sterile water for injection); MPA: medroxyprogesterone; MIF: mifepristone; DZP: diazepam; PTZ: pentylenetetrazole; PTZ (25 mg/kg) was administered once every 2 d for 5 wk, whereas MPA1 (5 mg/kg) and MPA2 (10 mg/kg) and MIF (2.5 mg/kg) were administered daily. All treatments were given by i.p. route. Data are presented as median seizure score with upper and lower quartiles. # P⬍0.05 vs. VEH⫹PTZ by Kruskal–Wallis one-way analysis by ranks (at the end of 35th day of treatment). The total number of animals in a group was seven.
Effect of MPA on grip strength and spontaneous alteration following PTZ kindling On grip strength test (GST) and SAB, a significant decline in GST and percentage alternation was observed in kindled mice (Tables 2–3). Treatment with MPA could reverse these significantly. MIF, however, could reverse only the reduced percentage alternation and not grip strength (GS) in PTZ-kindled animals.
DISCUSSION Current antiepileptic drug (AED) therapy is symptomatic. Treatment strategies that may alleviate seizure-induced neuronal damage can protect against the process leading to epilepsy (epileptogenesis) (Acharya et al., 2008). Steroidal hormones are reported to have neuroprotective poTable 2. Effect of medroxyprogesterone and mifepristone on the grip strength following PTZ-induced kindling in mice S. No.
Groups
Grip strength of male mice (kg)
Grip strength of female mice (kg)
1 2 3 4 5 6 7
Control VEH⫹PTZ MPA1⫹PTZ MPA2⫹PTZ MIF⫹MPA1⫹PTZ MIF⫹PTZ DZP⫹PTZ
0.097⫾0.003$$ 0.065⫾0.002** 0.104⫾0.007$$ 0.072⫾0.007* 0.112⫾0.006$$ 0.053⫾0.003** 0.085⫾0.004
0.086⫾0.002$$ 0.052⫾0.001** 0.103⫾0.004**,$$ 0.090⫾0.005$$ 0.113⫾0.050**,$$ 0.052⫾0.002** 0.084⫾0.002$$
PTZ (25 mg/kg) was administered once every 2 d for5 wk, whereas MPA1 (5 mg/kg) and MPA2 (10 mg/kg) and MIF (2.5 mg/kg) were administered daily. All treatments were given by i.p. route. The total number of animals in a group was seven. Data are represented as mean⫾SEM, ** P⬍0.01, P⬍0.05 when compared with normal control (Group 1), $$P⬍0.01, when compared with PTZ (Group 2), significant by ANOVA followed by Dunnett’s t-test. Abbreviations: VEH, Vehicle (Sterile water for injection); MPA, medroxyprogesterone; MIF, mifepristone; DZP, diazepam; PTZ, pentylenetetrazole.
285
Table 3. Effect of medroxyprogesterone and mifepristone on the spontaneous alternation behavior of mice following PTZ-induced kindling S. No.
Groups
% alternation in male mice
% alternation in female mice
1 2 3 4 5 6 7
Control VEH⫹PTZ MPA1⫹PTZ MPA2⫹PTZ MIF⫹MPA1⫹PTZ MIF⫹PTZ DZP⫹PTZ
76.14⫾0.95$$ 44.96⫾1.71** 57.45⫾1.17** 63.48⫾2.02**,$$ 76.08⫾1.14$$ 78.12⫾2.19$$ 60.83⫾1.75**,$$
69.30⫾2.95$$ 42.97⫾1.27** 61.14⫾1.52*,$$ 65.34⫾3.21$$ 79.19⫾1.72**,$$ 72.94⫾1.36$$ 73.07⫾1.59$$
PTZ (25 mg/kg) was administered once every2 d for5 wk, whereas MPA1 (5 mg/kg) and MPA2 (10 mg/kg) and MIF (2.5 mg/kg) were administered daily. All treatments were given by i.p. route. The total number of animals in a group was seven. Data are represented as mean⫾SEM, ** P⬍0.01, * P⬍0.05 when compared with normal control (Group 1), $$P⬍0.01, when compared with PTZ (Group 2), significant by ANOVA followed by Dunnett’s t-test. Abbreviations: VEH, Vehicle (Sterile water for injection); MPA, medroxyprogesterone; MIF, mifepristone; DZP, diazepam; PTZ, pentylenetetrazole.
tential and are increasingly being investigated in seizure disorders. Progesterone is an important hormone in epilepsy research due to its modulating actions in brain (Reddy et al., 2010). It is both a reproductive hormone and a neurosteroid that can modulate neuronal excitability. Progesterone targets multiple molecular and cellular mechanisms that are relevant to epileptogenesis. While it’s cellular actions are mediated via progesterone receptors (Brinton et al., 2008), its antiseizure effects are attributed to its conversion to allopregnanolone, which is a potent positive modulator of GABAA receptor (Frye, 1995). The present study evaluated the effects of an acetylated derivative of progesterone, MPA, which is also the most common progesterone included in hormone therapy on development of PTZ-induced kindling in mice. Unlike progesterone, the MPA’s metabolite does not seem to influence GABAA receptors (McAuley et al., 1993) yet there are reports of its efficacy in catamenial epilepsy in women (Mattson et al., 1984). We found that MPA effectively delayed the development of PTZ-induced kindling in mice. This was demonstrated by a significant reduction in the seizure severity scores and a significant decrease in the incidence of animals kindled at the end of 5 weeks. The protection was, however, lesser as compared with the standard drug diazepam. Although there are several studies available on the anticonvulsant effects of progesterone (Frye, 1995; Mohammad et al., 1998; Reddy et al., 2010), relatively fewer studies have examined the effect of MPA on seizures. There are a few clinical reports where MPA showed improvement in seizure frequency in women with catamenial epilepsy (Mattson et al., 1984; Zimmerman et al., 1973). In our study, MPA exhibited a higher efficacy against male mice as compared with females. This is in contrast with an experimental study against kainic acid induced seizures where MPA was shown to protect female (but not male) animals (Nicoletti et al., 1985). Progesterone, however, was reported to protect against epileptogenesis in male rats as well (Reddy et al., 2010). It is noteworthy that progesterone is considered to be
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an endogenous anticonvulsant (Frye and Scalise, 2000) and is present in brains of both male and female humans and rodents (Hammond et al., 1983) and, thus, could be an effective endogenous anticonvulsant in both. Women with epilepsy are prone to seizures in response to decreased levels of progesterone during perimenstrual periods (Reddy et al., 2010). Jacono and Robertson (1987) reported that progesterone may worsen seizure frequency in epileptic women (Jacono and Robertson, 1987). In addition to this, some women reportedly experienced an increased seizure with MPA (Foldvary-Schaefer et al., 2004). These studies along with the changes related to estrous cycle may explain the lesser efficacy of MPA observed in female mice in our study. In our study, we didn’t take into account different stages of estrous cycle of mouse. The progesterone receptor antagonist MIF neither reduced nor delayed the development of PTZ-induced kindling in mice. This is in agreement with a study on amygdala kindling of rats where MIF didn’t affect the seizure parameters in either sex (Borowicz et al., 2003). Further, its administration before MPA did not reverse the antiepiletogenic effects of MPA on PTZ kindling indicating that progesterone receptors may not mediate such effect of MPA. Contrary to this, progesterone receptor knock out mice were demonstrated to exhibit a marked delay in the rate of kindling pointing toward the involvement of progesterone receptors in kindling progression (Reddy et al., 2010). More recently, the same group also demonstrated that progesterone knock-out, knock-down, and blockade of its receptors had antiepileptogenic effects (Reddy and Mohan, 2011. This shows that activation of progesterone receptors may not mediate the antiepileptic effects of progesterone, and alternative mechanisms may be involved. In line with these findings, if knock-out or inhibition of progesterone receptors is antiepileptogenic, an agonist would be expected to facilitate epileptogenesis. Hence activation of progesterone receptors would counteract the effects mediated by the alternative pathway. Going by this hypothesis, MIF should increase the antiepileptogenic effects of MPA. We too found a mild increase in reduction of % incidence (Table 1) and reduction in seizure severity (Table 3) in female mice when MIF was administered along with MPA. Several mechanisms have been proposed by which progesterone decreases epileptic seizures other than activation of progesterone receptors such as synthesis of a neurosteroid and by promoting neuroprotection (Reddy et al., 2010). Both progesterone and MPA are reported to cause a rapid and transient activation of extracellular receptor kinase, the most common form of mitogen-activated protein kinase (Nilsen and Brinton, 2003), which is thought to be responsible for their proposed neuroprotective effects. It remains to be seen whether the antiepileptogenic effects of MPA in the present study are mediated via their proposed neuroprotective mechanisms. Given the fact that epilepsy and AED therapy are often associated with motor and cognitive dysfunction, the present study also evaluated the effects on motor function and spatial memory following PTZ-induced kindling. We chose to work with spontaneous alternation behavior, as it is
widely used as a model to measure spatial memory in rodents (Ragozzino et al., 1998; Vohora et al., 2005). Further, kindling induced by PTZ is known to impair spatial learning and memory in rodents (Mao et al., 2009; Omrani et al., 2007), whereas progesterone is reported to improve performance in spatial working tasks in rats (Frye et al., 2009) and to enhance cognitive performance in spontaneous alternation tasks (Frye and Walf, 2008). For assessing motor function, GST was used. As expected, a significant decline in the GS and percentage alternation was observed following PTZ kindling indicating a deficit of motor function and spatial memory, respectively. Treatment with MPA could reverse these deficits. In addition to MPA, MIF could also reverse the impairment of the spatial memory in PTZ-kindling mice. The literature reports both impairing and improving effects of progesterone on cognitive functions (Braden et al., 2010; Frye et al., 2009; Frye and Walf, 2008). Although it has been shown to impair memory in radial arm maze and Morris maze (Braden et al., 2010), an improved consolidation of spatial information has been reported in water maze (Frye et al., 2009), Y-maze (Frye and Walf, 2008), and in object recognition test (Lewis et al., 2008). In line with the latter reports on improvement of spatial information by progesterone, MPA (in our study) could reverse the impaired spatial deficits induced by PTZ kindling. It is possible that MPA improves spatial type of memory, and its effects on other types of memory may be differential like progesterone. The latter was reported to improve cognitive performance in the spontaneous alteration and object recognition but not in water maze, contextual and cued conditioned fear test (Frye and Walf, 2008). Though not many studies have evaluated the effects of MIF on memory, recently, it was reported to reverse memory loss induced by spontaneous morphine withdrawal in mice. The effects of MIF were attributed to antagonism of glucocorticoid receptors (Mesripour et al., 2008). In another study, however, MIF did not improve cognitive functions in schizophrenic patients (Gallagher et al., 2005). To summarize, our study is the first to show that not only progesterone but also the synthetic progesterone, MPA, has antiepileptogenic effects against development of PTZ-induced kindling in mice. Mifepristone, a progesterone receptor antagonist, fails to reverse the protective effects of MPA indicating that progesterone receptors may not mediate such effects. Although further studies are absolutely necessary to elucidate the mechanism of antiepileptogenic effects of MPA, our study clearly demonstrates that additional mechanisms (other than conversion to allopregnanolone) may contribute to antiseizure effects of progesterone and/or MPA. Acknowledgments—The work was carried out in Neurobehavioral Pharmacology laboratory funded by Special Assistance Programme of University Grants Commission, New Delhi.
REFERENCES Acharya MM, Hattiangady B, Shetty AK (2008) Progress in neuroprotective strategies for preventing epilepsy. Prog Neurobiol 84:363–404.
S. A. Nasir et al. / Neuroscience 207 (2012) 283–287 Ali A, Pillai KK, Ahmad FJ, Dua Y, Vohora D (2004) Evidence of the antiepileptic potential of amiloride with neuropharmacological benefits in rodent models of epilepsy and behaviour. Epilepsy Behav 5:322–328. Ali A, Pillai KK, Ahmad FJ, Dua Y, Vohora D (2006) Anticonvulsant effect of amiloride in pentylenetetrazole-induced status epilepticus in mice. Pharmacol Rep 58:242–245. Borowicz KK, Kleinrok Z, Czuczwar SJ (2003) Influence of sex hormone antagonists on the anticonvulsant action of conventional antiepileptic drugs against amygdala-kindled seizures in male and female rats. Eur Neuropsychopharmacol 13:257–265. Borowicz KK, Luszczki J, Matuszek M, Kleinrok Z, Czuczwar SJ (2002) Effects of tamoxifen, mifepristone and cyproterone on the electroconvulsive threshold and pentylenetetrazole-induced convulsion in mice. Pol J Pharmacol 54:103–109. Braden BB, Talboom JS, Crain ID, Simard AR, Lukas RJ, Prokai P, Scheldrup MR, Bowman BL, Bimonte-Nelson BA (2010) Medroxyprogesterone acetate impairs memory and alters the GABAergic system in aged surgically menopausal rats. Neurobiol Learn Mem 93:444 – 453. Brinton RD, Thompson RF, Foy MR, Baudry M, Wang J, Finch CE, Morgan TE, Pike CJ, Mack WJ, Stanczyk FZ, Nilsen J (2008) Progesterone receptors: form and function in brain. Front Neuroendocrinol 29:313–339. Ciriza I, Carrero P, Frye CA, Garcia-segura LM (2006) Reduced metabolites mediate neuroprotective effects of progesterone in the adult rat hippocampus. The synthetic progestin medroxyprogesterone acetate (Provera) is not neuroprotective. J Neurobiol 66:916 –928. Foldvary-Schaefer N, Harden C, Herzog A, Falcone T (2004) Hormones and seizures. Cleve Clin J Med 71:S11–S18. Freireich EJ, Gehan EA, Rall DP (1966) Quantitative comparison of toxicity of anticancer agents in mouse, rat, hamster, dog, monkey and man. Cancer Chemother Rep 50:219 –244. Frye CA (1995) The neurosteroid 3c, 5c THP has anti seizure and possible neuroprotective effects in an animal model of epileptogenesis. Brain Res 696:113–120. Frye CA (2010) Effects and mechanisms of progestogens and androgens in ictal activity. Epilepsia 51(3):135–140. Frye CA, Llaneza DC, Walf AA (2009) Progesterone can enhance consolidation and/or performance in spatial, object and working memory tasks in Long–Evans rats. Anim Behav 78:279 –286. Frye CA, Rhodes ME, Walf A, Harney J (2002) Progesterone reduces pentylenetetrazole-induced ictal activity of wild-type mice but not those deficient in type I 5␣-reductase. Epilepsia 43:14 –17. Frye CA, Scalise TJ (2000) Anti-seizure effects of progesterone and 3␣, 5␣-THP in kainic acid and perforant pathway models of epilepsy. Psychoneuroendocrinology 25:407– 420. Frye CA, Walf AA (2008) Effects of progesterone administration and APPswe ⫹ PSEN1De9 mutation for cognitive performance of midaged mice. Neurobiol Learn Mem 89:17–26. Gallagher P, Watson S, Smith MS, Ferrier IN, Young AH (2005) Effects of adjunctive mifepristone (RU-486) administration on neurocognitive function and symptoms in schizophrenia. Biol Psychiatry 57:155–161. Hammond GL, Hirvonen J, Vihko R (1983) Progesterone, androstenedione, testosterone, 5␣-dihydrotestosterone and androsterone concentrations in specific regions of the human brain. J Steroid Biochem 18:185–189. Itoh J, Nabeshima T, Kameyama T (1991) Utility of an elevated plusmaze for dissociation of amnesic and behavioral effects of drugs in mice. Eur J Pharmacol 194:71–76. Jacono JJ, Robertson JMD (1987) The effects of estrogen, progesterone, and ionized calcium on seizures during the menstrual cycle of epileptic women. Epilepsia 28:571–577.
287
Lewis MC, Orr PT, Frick KM (2008) Differential effects of acute progesterone administration on spatial and object memory in middleaged and aged female C57BL/6 mice. Horm Behav 54:455– 462. Lonsdale D, Burnham WM (2003) The anticonvulsant effects of progesterone and 5␣-dihydroprogesterone on amygdala-kindled seizures in rats. Epilepsia 44:1494 –1499. Lonsdale D, Burnham WM (2007) The anticonvulsant effects of allopregnanolone against amygdala-kindled seizures in female rats. Neurosci Lett 411:147–151. Mao RR, Tian M, Yue-Xiong YX, Qi-Xin Z, Lin X, Jun C (2009) Effects of pentylenetetrazol-induced brief convulsive seizures on spatial memory and fear memory. Epilepsy Behav 15:441– 444. Mattson RH, Cramer JA, Caldwell BV, Siconolfi BC (1984) Treatment of seizures with medroxyprogesterone acetate: preliminary report. Neurology 34:1255–1258. McAuley JW, Kroboth PD, Stiff DD, Reynolds IJ (1993) Modulation of [3H] flunitrazepam binding by natural and synthetic progestational agents. Pharmacol Biochem Behav 45:77– 83. Mesripour A, Hajhashemi V, Rabbani M (2008) Metyrapone and mifepristone reverse memory loss induced by spontaneous morphine withdrawal in mice. Eur Psychiatry 23 (Suppl 2):S292. Mohammad S, Abolhassan A, Pourgholami MH (1998) Evaluation of the anticonvulsant profile of progesterone in male amygdala-kindled rats. Epilepsy Res 30:195–202. Murialdo G, Magri F, Tamagno G, Ameri P, Camera A, Colnaghi S, Perucca P, Ravera G, Galimberti CA (2009) Seizure frequency and sex steroids in women with partial epilepsy on antiepileptic therapy. Epilepsia 50:1920 –1926. Nicoletti F, Speciale C, Sortino MA, Summa G, Caruso G, Patti F, Canonico PL (1985) Comparative effects of estradiol benzoate, the antiestrogen clomiphene citrate, and the progestin medroxyprogesterone acetate on kainic acid-induced seizures in male and female rats. Epilepsia 26:252–257. Nilsen J, Brinton RD (2003) Divergent impact of progesterone and medroxyprogesterone acetate (Provera) on nuclear mitogen-activated protein kinase signaling. Proc Natl Acad Sci U S A 100:10506 –10511. Omrani A, Ghadami MR, Fathi N, Tahmasian M, Fathollahi Y, Touhidi A (2007) Naloxone improves impairment of spatial performance induced by pentylenetetrazol kindling in rats. Neuroscience 145:824 – 831. Ragozzino ME, Pal SN, Unick K, Stefani MR, Gold PE (1998) Modulation of hippocampal acetylcholine release and spontaneous alternation scores by intrahippocampal glucose injections. J Neurosci 18:1595–1601. Reddy DS (2005) Pharmacotherapy of catamenial epilepsy. Indian J Pharmacol 37:288 –293. Reddy DS, Gangisetty O, Briyal S (2010) Disease-modifying activity of progesterone in the hippocampus kindling model of epileptogenesis. Neuropharmacology 59:573–581. Singh S, Hota D, Prakash A, Khanduja KL, Arora SK, Chakrabarti A (2010) Allopregnanolone, the active metabolite of progesterone protects against neuronal damage in picrotoxin-induced seizure model in mice. Pharmacol Biochem Behav 94:416 – 422. Vohora D, Pal SN, Pillai KK (2005) Modulation of spontaneous alternation behavior of mice treated with thioperamide and tacrine in a cross maze task. Fundam Clin Pharmacol 19:531–532. White HS, Woodhead JH, Franklin MR, Swinyard EA, Wolf HA (1995) General principles, experimental selection, quantification, and evaluation of antiepileptic drugs. Antiepilep. Drugs 4:99 –110. Zimmerman AW, Holden KR, Reiter EO, Dekaban AS (1973) Medroxyprogesterone acetate in the treatment of seizures associated with menstruation. J Paediatr 83:959 –963.
(Accepted 14 January 2012) (Available online 25 January 2012)