Hippocampal tissue of patients with refractory temporal lobe epilepsy is associated with astrocyte activation, inflammation, and altered expression of channels and receptors

Abstract

Temporal lobe epilepsy (TLE) is the most common form of focal epilepsy. Previous research has demonstrated several trends in human tissue that, undoubtedly, contribute to the development and progression of TLE. In this study we examined resected human hippocampus tissue for a variety of changes including gliosis that might contribute to the development and presentation of TLE. The study subjects consisted of six TLE patients and three sudden-death controls. Clinicopathological characteristics were evaluated by H&E staining. Immunohistological staining and Western blotting methods were used to analyze the samples. Neuronal hypertrophy was observed in resected epileptic tissue. Immunohistological staining demonstrated that activation of astrocytes was significantly increased in epileptic tissue as compared to corresponding regions of the control group. The Western blot data also showed increased CX43 and AQP4 in the hippocampus and downregulation of Kir4.1, α-syntrophin, and dystrophin, the key constituents of AQP4 multi-molecular complex. These tissues also demonstrated changes in inflammatory factors (COX-2, TGF-β, NF-κB) suggesting that these molecules may play an important role in TLE pathogenesis. In addition we detected increases in metabotropic glutamate receptor (mGluR) 2/3, mGluR5 and kainic acid receptor subunits KA1 (Grik4) and KA2 (Grik5) in patients' hippocampi. We noted increased expression of the α1c subunit comprising class C L-type Ca(2+) channels and calpain expression in these tissues, suggesting that these subunits might have an integral role in TLE pathogenesis. These changes found in the resected tissue suggest that they may contribute to TLE and that the kainic acid receptor (KAR) and deregulation of GluR2 receptor may play an important role in TLE development and disease course. This study identifies alterations in number of commonly studied molecular targets associated with astrogliosis, cellular hypertrophy, water homeostasis, inflammation, and modulation of excitatory neurotransmission in hippocampal tissues from TLE patients.

Figure 1

Sclerosis in the epileptic hippocampus. Characteristic swelling of neurons (white arrows; A) in the chronic epileptic hippocampus (black arrow indicates normal neuron). GFAP staining of representative captures from control and epileptic tissue (GFAP-red; DAPI-blue; B). Quantification of Western blot and immunofluorescent GFAP optical density (C & D, respectively). * P < 0.05.

Figure 2

Aquaporin and associated markers of astrocytic swelling. Representative images taken from 8µm frozen sections stained for AQP4 (green), GFAP (red) and DAPI (blue) from sudden death controls and resected sclerotic hippocampi (A). Elevated levels of AQP4 are apparent in epileptic tissue compared to control. Representative Western blots for AQP4, CX43, and Kir4.1 complex markers are shown (B). Quantification of blot signals relative to β-actin confirmed significantly elevated AQP4 and CX43 concurrent with a significant downregulation of Kir4.1 and associated dystophin and α-syntrophin (C). Data taken from 3 controls, and 6 epileptic hippocampus samples; *P<0.05.

Figure 3

Inflammatory markers upregulation is concurrent with TGF-β increases. Representative Western blots showing inflammatory markers NFκB and COX-2 as well as TGF-β (A). Quantification of 3 control and 6 epileptic hippocampi samples is shown (B). NFκB, COX-2 and TGF-β were significantly elevated in epileptic hippocampi compared to sudden death controls; *P<0.05.

Figure 4

Ionotropic and Metabotropic Glutamate Receptors are elevated in TLE. Representative Western blots showing mGlur2/3 and mGlur5 as well as KA1, and KA2 (A). Quantification of 3 control and 6 epileptic hippocampi samples is shown (B). All receptors were significantly elevated in epileptic hippocampi compared to sudden death controls; *P<0.05.

Figure 5

L-type Ca²⁺ channel α1C expression increases along with m-calpain expression. Representative Western blots from 3 control and 6 epileptic hippocampi samples are shown (A). L-type Ca²⁺ channel and m-calpain were significantly elevated in epileptic hippocampi compared to sudden death controls (B); *P<0.05.