On different mechanisms of axitinib and diazepam antiseizure action in pentylenetetrazol-induced kindling model
Description
The present work aimed to evaluate the histomorphology characteristics of hippocampal structures and determine the severity of seizures after treatment with the tyrosine kinase B axitinib inhibitor in fully developed and postponed periods in PTZ-kindled rats. Considering that benzodiazepines cause antiseizure effects via GABAA receptors (Nicholson et al., 2018), while inhibitors of Tyr-kinase inhibit them (Dunne et al., 1998) we have also studied antiseizure effectiveness of diazepam at both stages of PTZ-kindling. Axitinib treatment (10.0 mg/kg, p.o.) performed at the early phase of kindling resulted in complete protection from generalized tonic-clonic seizures and a significant reduction of seizures compared to the control (H=10.988, P=0.001). PTZ testing (35.0 mg/kg, i.p.) in a postponed period caused generalized seizures in all animals and with repeated character in most (6 out of 7) of them. Seizure severity was higher when compared with fully developed kindling (H=4.116, P=0.042). Treatment with axitinib prevented generalized tonic-clonic seizures in most rats (5 out of six) and significantly reduced seizure severity (H=5.989, P=0.014) . At the same time, seizure severity in the postponed period exceeded such one in the early phase of kindling in axitinib-treated rats (H=4.275, P=0.039). Diazepam (1.0 mg/kg, i.p.) prevented generalized tonic-clonic seizures in 5 out of 6 rats and reduced seizure severity in fully developed kindling in comparison with the control (H=7.773, P<0.005) (Fig. 3, C). Testing PTZ administration caused tonic-clonic seizures in all rats with repeated fits in 5 out of 6 animals in postponed periods. Diazepam administration (1.0 mg/kg, p.o.) did not prevent generalized seizures in 2 out of 7 rats but reduced seizure severity compared with the control (H=6.647, P=0.01). Seizure severity was not significantly different from those registered after diazepam treatment in the early period of kindling (H=1.114, P=0.291). Immunohistochemical, light and elctron micropscopy data revealed neurodegeneration in CA1 hippocampal zone along with the accumulation collagen IV - marker of angiogenesis.
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The PTZ kindled seizures were modelled via daily PTZ (35.0 mg/kr, i.p.) administrations during three weeks. Groups of observations and design of experimental protocols are presented in Fig. 1 and Fig. 2.(File: DESIGN of TREATMENT) Diazepam ("Calmpose," "Ranbaxy Diagnostics," India) was injected at a dose of 1.0 mg/kg, i.p., and axitinib ("Sigma Aldrich") was given orally (p.o.) in doses of 10.0 mg/kg (Fig. 1, DESIGN). Axitinib was dissolved in 5.0% methylcellulose (Methocel, “Sigma Aldrich”) and diazepam – in 0.9% saline and 5.0% dextrose. During the final ten days of kindled PTZ injections, axitinib was given 60 minutes prior to PTZ, and convulsions were calculated following a 21-t PTZ injection during the initial kindling period. Also, axitinib was administered daily from the 5th till the 14th day of the two weeks free from PTZ in a postponed period of kindling. Diazepam was administered 30 min before PTZ was tested in early and postponed periods (Fig. 2). Control animals were treated with Methocel and with 0.9% saline and 5.0% dextrose. Histopathological analyses Brain tissues were removed for the histopathological evaluation. Ten days, the samples were fixed with 10% formaldehyde. Then, the tissues were processed by alcohol series (70-80-96-100 %) and xylene series and embedded in paraffin for light microscope analyses (LM). The sections were cut at 0.5μm and stained with cresyl violet. A camera attachment and a microscope were used to capture the tissue photographs. The analyses were performed using cellSens Entry software with the aid of a light microscope with camera attachment (Olympus, BX43, Center Valley, PA, USA). For the transmission electron microscopic analysis (TEM), samples were processed with graded acetone, propylene oxide, osmium tetroxide, acetone, propylene oxide, and Araldite and embedded in resin blocks. Following tissue processing, 70 nm sections were taken on 200 mesh copper grids and contrasted using uranyl acetate–lead citrate solutions for assessments under an electron microscope (JEOL JSM-7001F, JEOL Ltd., Tokyo, Japan). For histopathological assessments, consecutive 2 µm-thick sections were also put onto glass slides and stained with a 1% toluidine blue and sodium borate mixture on a hot plate. Immunohistochemical analysis For immunohistochemical analyses, 5μm thickness sections were incubated in 3% H2O2, washed with BSA, and the anti-Collagen IV antibodies were applied for 2 hours. After that, the section was washed with PBS and incubated with biotinylated anti-rabbit IgG. Immunoreactivity was confirmed with a streptavidin-biotin complex package and visualized with diaminobenzidine tetrahydrochloride (DAPI). Areas were counter-stained with Mayer's hematoxylin.