Lamotrigine decreased hippocampal damage and improved vascular risk markers in a rat model of pentylenetetrazole induced kindling seizure

Abstract

Various antiepileptic drugs (AEDs) especially enzyme-inducing AEDs might be associated with increased vascular risk, through impairment of the endogenous antioxidative ability which may trigger oxygen-dependent tissue injury. Lamotrigine (LTG) a non-enzyme-inducing AED has scarce information regarding its effects on oxidative stress. The present study aimed to study the possible modulation of vascular risk factors of epileptogenesis by LTG, in a rat model of kindling seizure induced by pentylenetetrazole (PTZ). Four groups of male Wister rats were used; vehicle control group, PTZ group (alternate day PTZ, 30 mg/kg, i.p), LTG/PTZ group (LTG 20 mg/kg/day p.o and alternate day PTZ) and LTG group. The study period was 5 weeks. Lipoproteins and total homocysteine (tHcy), malondialdehyde (MDA) and reduced glutathione (GSH) were measured. Aortic endothelial function study and histopathological examination of the rats' brains, aortas and coronaries were conducted. Serum total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C), tHcy, MDA, GSH levels were significantly higher in epileptic rats than normal controls rats. A decrease in HDL-cholesterol with high atherosclerotic index was also demonstrated. The administration of LTG improved the PTZ-kindled seizures. It produced a significant decrease in TC, TG and LDL-cholesterol, MDA, aortic GSH and increase in HDL-cholesterol with no significant effect on serum GSH and tHcy levels. LTG improved endothelium-dependent relaxation, decreased hippocampal neurodegenerative changes and atherosclerotic changes of aortas and coronaries. LTG decreased seizures severity, hippocampal damage and improved vascular risk markers in this rat model of kindling seizures.

Keywords: Antiepileptic drugs; Epilepsy; Homocysteine; Lamotrigine; Pentylenetetrazole.

Fig. 1

(A-C) The effect of Pentylenetetrazole and lamotrigine on (A) average Racine score. (B) latency to GTCS (generalized tonic clonic seizures). (C) seizure score. PTZ (Pentylenetetrazole, 30 mg/kg), LTG (lamotrigine, 20 mg/kg). Data are expressed as mean±SD, number of animals=7. For average Racine's score and latency to GTCS, ^*p<0.05 compared to PTZ were determined by unpaired t-test. For seizure score; ^#p<0.05 compared to control group, ^*p<0.05 compared to PTZ group by analysis of variance (ANOVA) followed by Newman-Keuls multiple comparison test.

Fig. 2

Serum homocysteine level in control rats, PTZ treated rats, PTZ+LTG and LTG treated rats. PTZ (pentylenetetrazole, 30 mg/kg), LTG (lamotrigine, 20 mg/kg). Data are expressed as mean±SD, number of animals=7. ^#p<0.05 compared to control group by analysis of variance (ANOVA), followed by Newman-Keuls multiple comparison test.

Fig. 3

(A, B) Serum and aortic malondialdehyde levels in control rats, PTZ treated rats, PTZ+LTG and LTG treated rats. PTZ (pentylenetetrazole, 30 mg/kg), LTG (lamotrigine, 20 mg/kg). Data are expressed as mean±SD, number of animals=7. ^#p<0.05 compared to control group, ^*p<0.05 compared to PTZ group by analysis of variance (ANOVA), followed by Newman-Keuls multiple comparison test.

Fig. 4

(A, B) Serum and aortic reduced glutathione (GSH) levels in control rats, PTZ treated rats, PTZ+LTG and LTG treated rats. PTZ (pentylenetetrazole, 30 mg/kg), LTG (lamotrigine, 20 mg/kg). Data are expressed as mean±SD, number of animals=7. ^#p<0.05 compared to control group by analysis of variance (ANOVA), followed by Newman-Keuls multiple comparison test.

Fig. 5

(A-H) Pearson's correlation coefficients between serum total homocysteine (tHcy) concentration and both serum lipids and oxidative stress parameters in control, PTZ (Pentylenetetrazole) treated, PTZ+LTG (lamotrigine) and LTG treated rats. LDL, low density lipoprotein; HDL, high density lipoprotein.

Fig. 6

(A) Maximal contractile response (E-max) induced by phenylephrine (PE). (B) Effective concentration 50 (EC50) of PE. (C) Relaxant responses to acetylcholine (Ach) in the endothelium-intact aortic rings of control rats, PTZ treated rats, PTZ+LTG and LTG treated rats. PTZ (pentylenetetrazole, 30mg/kg), LTG (lamotrigine, 20 mg/kg).Data are expressed as mean±SD, number of animals=7. ^#p<0.05 compared to control group, ^*p<0.05 compared to PTZ group by analysis of variance (ANOVA), followed by Newman-Keuls multiple comparison test.

Fig. 7

(rows A-D): A photomicrograph of CA3 region of the hippocampus: Row A, - Stained with H&E, ×540; (1) Control rat: notice that the pyramidal layer is formed of closely packed pyramidal cells with vesicular nuclei (↑). (2) Pentylenetetrazole (PTZ) treated rat: showing the presence of many deeply stained cells having pyknotic nuclei and perineuronal spaces (▸) an apparent decrease in the number of the pyramidal cells. (3) PTZ-Lamotrigine (LTG) treated rat: Pyramidal cells having vesicular nuclei and basophilic cytoplasm (↑). Few shrunken, darkly stained neurons are seen (▸) within the pyramidal layer. (4) LTG treated rat: nearly similar to control group (↑). Row B, Stained with Toluidine blue ×540: (1) Control rat: cytoplasm of the pyramidal cells appears studded with Nissl granules (↑). (2) PTZ treated rat: an apparent decrease in the Nissl granules of the pyramidal cells (↑). (3) LTG-PTZ treated rat: an apparent increase in the Nissl granules content of the pyramidal cells (↑) compared to that of PTZ treated rats. (4) LTG treated rat: picture nearly similar to control group. Row C, stained with Caspase-3 ×540; (1) Control rat: negative caspse-3 immuno- reaction in pyramidal cells. (2) PTZ treated rat: positive brownish caspase-3 immuno- reaction in pyramidal cells (↑). (3) PTZ-LTG treated rat: positive brownish caspase-3 immuno- reaction in few pyramidal cells (↑) when compared to PTZ group. (4): LTG treated rat: negative caspase-3 immuno- reaction in pyramidal cells. Row D, stained with Glial fibrillary acidic protein (GFAP) ×540. (1) Control rat: positive reaction for GFAP immuno-staining in few astrocytes (↑). (2) PTZ treated rat: an apparent increase in astrocytes (↑) size with longer cytoplasmic processes, number and GFAP immunoreactivity compared to control rats. (3) PTZ-LTG treated rat: an apparent decrease in the number of GFAP immunoreactive astrocytes (↑) compared to PTZ group. (4) LTG treated rat: few GFAP immunoreactive astrocytes (↑).

Fig. 8

(row A, B): Row A, A photomicrograph of a transverse section in a rat thoracic aorta (H&E, ×540); (1) Control rat: the tunica intima lined with endothelial cells (↑), tunica media (M) is relatively thick and formed mainly of wavy acidophilic elastic laminae () and smooth muscle cells (▲). Notice tunica adventitia is thin. (2) PTZ treated rat: clumps of cells with darkly stained nuclei (↑) attached to the endothelium of tunica intima which is apparently irregular. Few of the smooth muscle cells of tunica media show foamy acidophilic cytoplasm with karyolitic nucleus (▲) or clearly vacuolated cytoplasm with peripheral flat pyknotic nucleus (↑↑). (3) PTZ-LTG treated rat: apparently regular tunica intima (↑). Most of the smooth muscle cells are nearly similar to control group, others show foamy acidophilic cytoplasm (↑↑) and few number shows vacuolated cytoplasm with deeply stained nucleus (▲). (4) LTG treated rat: tunicae of the aorta are nearly similar to the control group. Row B, A photomicrograph of a section a coronary artery of the heart (H&E, ×540); (1) Control rat: an intact endothelial lining (↑). The tunica media (M) is formed of smooth muscle fibres with rod shaped vesicular nuclei (▲). The adventitia (A) is formed of loose connective tissue. (2) PTZ treated rat: multiple vacuoles in the tunica intima and tunica media (↑). Notice adherence of blood elements to the endothelium (▲). Exudates are also seen (). (3) PTZ-LTG treated rat: few vacuolated cells are seen in tunica media (↑). (4) LTG treated rat: nearly intact tunicae.