The Novel Effect of Immunomodulator-Glatiramer Acetate on Epileptogenesis and Epileptic Seizures

Abstract

Background/Aims: Immunological mechanisms can be triggered as a response to central nervous system insults and can lead to seizures. In this study an investigation was made to determine if glatiramer acetate (GA), an immunomodulator currently used in the treatment of multiple sclerosis, could protect rats from pilocarpine-induced seizures and chronic epilepsy. Methods: Two groups of adult male Sprague-Dawley rats, experimental (GA) and control, were used in the study. The systemic IL-1α and IL-1β levels at baseline were checked as well as status epilepticus (SE), and the spontaneous recurrent seizure (SRS) stage by enzyme-linked immunosorbent assay. The GA group was given GA (150 μg/kg, ip) and the control group was given a saline injection prior to pilocarpine-induced seizures. Seizure susceptibility, severity and mortality were evaluated, using Racine seizure classification and hippocampal damage was evaluated by Nissl staining. The GA group received GA (150 μg/kg/day, ip) daily after SE, and the chronic spontaneous seizures were evaluated by long-term video recording, and mossy fiber sprouting was evaluated by Timm staining. The IL-1α and IL-1β levels were correlated with seizure activities. The TNF-α level in the hippocampus was determined at the SRS stage by immunohistochemistry. The effect of GA on ionic currents and action potentials (APs) in NG108-15 differentiated neurons was investigated using patch-clamp technology. Results: It was found that latency to severe seizures was significantly longer in the GA (p < 0.01) group, which also had SE of shorter duration and less frequent SRS (p < 0.01). GA attenuated acute hippocampal neuron loss and chronic mossy fiber sprouting in the CA3 and the SRS-reduction correlated with the reduction of IL-1α, but not with IL-1β or TNF-α levels. Mechanistically, GA reduced the peak amplitude of voltage-gated Na⁺ current (I_Na), with a negative shift in the inactivation curve of I_Na and reduced the amplitude of APs along with decreased firing of APs. Conclusion: GA might serve as a neuroexcitability modulator which attenuates pilocarpine-induced acute and chronic excitotoxicity. Sodium channel attenuation was partially independent of the immunomodulatory effect.

Telmisartan, an Antagonist of Angiotensin II Receptors, Accentuates Voltage-Gated Na+ Currents and Hippocampal Neuronal Excitability

“…On day 1, the rats were injected with lithium chloride (3 meq/kg; ip) and methylscopolamine (25 mg/kg; sc) and then subjected to pilocarpine (60 mg/kg; sc)-induced seizures. The behavioral characteristics of the rats during epileptic seizures were similar to those reported elsewhere ( Lai et al, 2018a , b ; Hung et al, 2019 ). During pilocarpine-induced status epilepticus, the rats were given zoletil (50 mg/kg, ip) and xylazine (20 mg, ip) and atropine (0.2 mg/kg, sc) if the status epilepticus lasted for 20 min ( Mello et al, 1993 ).…”

“…Paraformaldehyde was then used for perfusion. Coronal sections (20 μm thick) of the hippocampus were fixed in formaldehyde, as previously described ( Chang et al, 2003 ; Lai et al, 2018a , b ). The cresyl violet-stained sections were examined for gross indications of damage to the hippocampus.…”

“…The severity of neuron loss in different subfields of the hippocampus was scored semiquantitatively as follows: 0 = no neuron loss, 1 ≤ 10% neuron loss, 2 = between 11 and 50% neuron loss, and 3 ≥ 50% neuron loss ( Pitkänen et al, 2006 ; Lai et al, 2018a , b ; Hung et al, 2019 ). Counts were made at 400 × using the Image Plus 2.0 computer image analysis system (Motic, Richmond, BC, Canada), and the hippocampal subfields were defined by an imaginary line connecting the tips of the granule cell layer blades, which separated the Cornu Ammonis3c (CA3c) (medially) from the CA3b (laterally) and the CA2 from the CA1 ( Chang et al, 2003 ; Lai et al, 2018a , b ; Hung et al, 2019 ). Values from the different groups were determined by an investigator blind to the study design, after which they were averaged in each group.…”

“…On day 18, the rats’ brains were removed, and coronal sections (20 μm thick) through the entire hippocampus were cut on a Leica CM1900 freezing microtome. Every sixth section was stained with Timm’s stain ( Chang et al, 2003 ; Lai et al, 2018a , b ; Hung et al, 2019 ). The sections were developed in the dark for 10–45 min in a 200-ml solution containing 5.1 g of citric acid, 4.7 g of sodium citrate, 3.47 g of hydroquinone, 212.25 mg of AgNO 3 , and 120 ml of 50% gum arabic.…”

“…Timm’s staining was assessed from the septal area to the temporal hippocampus (the region between 2.8 and 3.8 mm posterior to the bregma). We used a semiquantitative scale to evaluate the degree of mossy fiber sprouting in the pyramidal and infrapyramidal areas of the hippocampus CA3 region and that in the granular cell and inner molecular layers of the dentate gyrus ( Chang et al, 2003 ; Lai et al, 2018a , b ; Hung et al, 2019 ). The score criteria were as follows: 0, no granules; 1, occasional discrete granule bundles; 2, occasional-to-moderate granules; 3, prominent granules; 4, a prominent nearly continuous granule band; and 5, a continuous or nearly continuous dense granule band.…”