The Mechanisms By Which AEDs Affect Cognition And The Measures To Prevent The Adverse Effects In Immature Rats

BackgroundEpilepsy is a brain disorder caused by synchronous, rhythmic firing of neurons, displays its highest incidence in the first year of life. There are more and more reports that patients with epilepsy may have impaired cognitive abilities, and antiepileptic drugs (AEDs) therapy may contribute to this impairment. AEDs act via three main mechanisms: (1) limitation of sustained repetitive neuronal firing via blockade of voltage-dependent sodium channels, (2) enhancement ofγ-aminobutyric acid (GABA)-mediated inhibition, and (3) blockade of glutamatergic excitatory neurotransmission.Doctors have noticed that AEDs could cause cognitive impairment in epilepsy for a long time. In recent years multiple new AEDs have been developed and introduced into the clinical practice. And there has been growing interest in the use of new AEDs in the treatment of epilepsy and indications other than epilepsy, including migraine prophylaxis, neuropathic pain syndromes, and neuroprotection. Although there are not many studies about new AEDs, several reports have suggested that newer AEDs may have mild effects on cognition than traditional AEDs. Thus, assessment of the potential adverse cognitive effects of newer AEDs may help us to compare the differences between traditional AEDs and newer AEDs.Except for cognitive impairment AEDs can cause fetal malformation, developmental delay and microcephaly. One of the complicated mechanisms is enhancement of apoptotic neurodegeneration. But it remains open whether other mechanisms such as impairment of migration or proliferation of neuronal progenitors as well as disturbance of synaptogenesis, may also account for neurological deficits seen in humans exposed prenatally or postnatally to AEDs. Thus, the present study investigated the effects of AEDs on apoptotic neurodegeneration, neurotrophins, neurogenesis and mossy fiber sprouting, trying to present some possible mechanisms to explain cognitive impairment associated with AEDs.In recent years a number of laboratories have assessed the effects of enriched environment on the damaged brain. For example, enriched environment can improve memory and motor skills following trauma or destructive lesions, ischemia, stroke, intrauterine alcohol exposure and aging, while the effects of enriched environment on impaired cognition caused by AEDs remain unknown. Thus, the last part of the present study examined the effets of enriched environment on learning and memory following long-term treatment with PB and VPA in developing brain.Furthermore, most previous studies focused on adulthood, there are few studies on immature brain. As we know there are many differences between neonatal brain and adult brain in anatomy and physiology, for example, in immature brain the synaptogenesis and its development is not complete, immature brain is more prone to seizures and has rapid generalization of seizures. All above suggest that the effets of AEDs on immature brain and adult brain may be different. Moreover, patients with epilepsy often receive AEDs treatment for a long time, sometimes for lifetime. Therefore, the effects of AEDs on cognition, its possible mechanisms and the measures to prevent the adverse effects in immature animal, especially the neonate, need to be explored further.Objectives To study the effects of long-term treatment with AEDs on locomotor activity, spatial learning and working memory, and to study the effects of AEDs on apoptosis, neurogenesis and neurotrophins which will provide possible mechanisms from cellular and molecular levels to explain adverse effects of AEDs. We also intended to investigate the effects of enriched environment on impaired cognition caused by AEDs.Methods:1. Neonatal Wistar rats (P7) were randomly assigned to one of five groups: (1) Control group (no drugs, n=12); (2) Phenobarbital (PB, Sigma) group (n=18); (3) Valproate (VPA, Sigma) group (n=18); (4) Lamotrigine (LTG, GlaxoSmithKline group of companies) group (n=18); (5) Topiramate (TPM, Xian-Janssen Pharmaceutical Ltd.) group (n=18). Phenobarbital and valproate were dissolved in distilled water. Lamotrigine and topiramate were dissolved in 1% sodium carboxy methyl cellulose (SCMC). Phenobarbital and valproate were administered intraperitoneally. Lamotrigine and topiramate were administered orally. The doses of the drugs were: (1) PB: 20,40, or 80 mg/kg; (2) VPA: 50, 100, or 200 mg/kg; (3) LTG 20, 40, or 80 mg/kg; (4) TPM 20, 40, or 80 mg/kg. Half of control rats received distilled water injection (i.p.), the rest were administered by SCMC orally. After 3-weeks treatment with AEDs the experimental groups and the controls were ordinally tested for locomotor activity by open field tests on P28, spatial learning by Morris water maze on P30 and working memory by Y maze on P36.2. Neonatal Wistar rats (P7) were randomly assigned to one of five groups: (1) Control group (no drugs, n=24); (2) PB group (n=36); (3) VPA group (n=36); (4) LTG group (n=36); (5) TPM group (n=36). The doses of the drugs were same as the first part. After 3-weeks treatment with AEDs the experimental groups and the controls were divided into two parts. One part was sacrificed by decapitation and the brain was removed on an ice-cold stage. These brains were used in the study of Annexin-V FITC/PI double staining and quantitative real-time detection PCR. The other part that was used in the study of BrdU staining and Timm's staining received an overdose of sodium pentobarbital (60mg/kg, i.p.) and was perfused with different solution. And the rats that were used for BrdU staining received BrdU injection (100 mg/kg, i.p.) 36 hr before anaesthesia.3. Neonatal Wistar rats (P7) were randomly assigned to one of four groups: (1) PB group (n=18); (2) PB +EE group (n=18); (3) VPA group (n=18); (4) VPA+EE group (n=18). The doses of the drugs were: (1) PB: 20, 40, or 80 mg/kg; (2) VPA: 50, 100, or 200 mg/kg. Rats in PB+EE and VPA+EE groups were raised in an enriched environment. An enriched environment was created in a plastic rectangular container measuring 115 cm (length) X 40 cm (width) X 40 cm (height). The bottom of the environment was covered with the cage bedding that was also used for the plastic cages in our animal housing facility. Several tactile and visual stimuli were provided in the form of animal toys. There was a treadmill and several places where the animals could hide. There also was a moving object in the form of a clock with a colorful pendulum and a mirror. Auditory stimuli were provided in the form of Mozart's piano concerts. Every 3rd day, the arrangement of objects in the environment was changed. When not in the enriched environment, rats were treated in the same manner as the controls. After 3-weeks treatment with AEDs and EE, rats were ordinally tested for locomotor activity by open field tests on P28, spatial learning by Morris water maze on P30 and working memory by Y maze on P36.Results:1. All rats were alive during experimental period. VPA-treated rats gained increased weight than normal rats while LTG -treated rats lost weight during the study (P<0.05). In open field test different dose of PB-treated rats showed increased locomotor activity while high dose of LTG (40, 80 mg/kg)-treated rats exhibited decreased activity. In morris water maze, the PB group spent more time in finding the platform but less time in the target quadrant at all dose levels compared with control rats(P<0.05); the VPA group spent more time in finding the platform but less time in the target quadrant at dose of 100 and 200 mg/kg ( P<0.05); only high-dose LTG group spent more time in finding the platform ( PO.05) but no differences were noted in the probe test (P>0.05); TPM group also spent more time in finding the platform but less time in the target quadrant at dose of 40 and 80 mg/kg. In Y maze test, control rats learned the left-right alternative memory model. PB and TPM group had deficits in working memory and no differences were found in total errors between the former ten trials and the latter ten trials in these rats. Total errors in PB and TPM group significantly increased compared to control rats in both training sessions and retention sessions (PB group, training session F=4.72, P<0.05; retention session F=23.9, P<0.01; TPM group, training session F=4.35, P<0.05; retention session F=15.1, P<0.01).While significant decrease was found in TE between the former ten trials and the latter ten trials in VPA and LTG-treated group (P<0.05). And no differences were found between VPA or LTG-treated group and control group in both training sessions and retention sessions (P>0.05).2. Long-term treatment with AEDs caused significant reduction in brain weight: PB (80mg/kg) led to a significant decrease in mean brain weight of 12% (2.03±0.16 vs 2.32±0.24 in control rats, P<0.05), VPA (200mg/kg) resulted in a significant decrease in weight of rain of 15% (1.95±0.26, P< 0.01), LTG (80mg/kg) resulted in a decrease of brain weight of 6% (2.16±0.12 vs 2.34±0.21 in control rats, P>0.05), TPM (80mg/kg) led to a significant decrease in brain weight of 8% (2.11±0.17, P<0.05). Traditional AEDs phenobarbital (20 mg/kg) and valproate (50 mg/kg) caused apoptotic neurodegeneration and suppressed the expression of BDNF and NT-3 in the developing brain. While new AEDs such as topiramate (40 mg/kg) and lamotrigine (80 mg/kg) caused apoptotic neurodegeneration and decreased expression of BDNF and NT-3 only at high dose levels. Neurogenesis increased in the rats treated with valproate (50 mg/kg, 212±43, t=3.39 P<0.01) and lamotrigine (20 mg/kg, 195±18, t=4.23 P<0.01) but their effect on mossy fiber sprouting was not obvious in all rats (P>0.05).3. PB+EE group performed significantly better than the PB group in morris water maze and Y maze (P<0.05) , but no differences were noted between 80 mg/kg PB+EE group and PB group (P>0.05). The improvement was also observed in VPA+EE group. The results of the probe test paralleled the water maze testing. But no differences were found in locomotor activity between the enriched group and nonenriched group (P>0.05).Conclusions:1. Traditional AEDs affects multiple cognitive domains in the range of therapeutic dose. Although reports have suggested that newer AEDs may have fewer effects on cognition this study finds that newer AEDs such as LTG and TPM also impair cognition at high dose level. However, the degree and domain of impairment caused by different AEDs are various.2. PB, VPA, LTG and TPM cause apoptotic neurodegeneration in the developing brain at different dose levels. Neuronal death is associated with reduced expression of BDNF and NT-3. Interestingly, VPA and LTG cause increased neurogenesis in dentate gyrus with an absence of mossy fiber sprouting. These findings present one possible mechanism to explain cognitive impairment associated with exposure of humans to antiepileptic therapy.3. Enriched environment can improve cognitive outcome of rats treated with AEDs. But the extent of improvement is related to the dose of AEDs. The improvement effect of EE is not obvious when the PB is at high level.