Conditioned Medium Reconditions Hippocampal Neurons against Kainic Acid Induced Excitotoxicity: An In Vitro Study

Abstract

Stem cell therapy is gaining attention as a promising treatment option for neurodegenerative diseases. The functional efficacy of grafted cells is a matter of debate and the recent consensus is that the cellular and functional recoveries might be due to "by-stander" effects of grafted cells. In the present study, we investigated the neuroprotective effect of conditioned medium (CM) derived from human embryonic kidney (HEK) cells in a kainic acid (KA) induced hippocampal degeneration model system in in vitro condition. Hippocampal cell line was exposed to KA (200 µM) for 24 hrs (lesion group) whereas, in the treatment group, hippocampal cell line was exposed to KA in combination with HEK-CM (KA + HEK-CM). We observed that KA exposure to cells resulted in significant neuronal loss. Interestingly, HEK-CM cotreatment completely attenuated the excitotoxic effects of KA. In HEK-CM cotreatment group, the cell viability was ~85-95% as opposed to 47% in KA alone group. Further investigation demonstrated that treatment with HEK-CM stimulated the endogenous cell survival factors like brain derived neurotrophic factors (BDNF) and antiapoptotic factor Bcl-2, revealing the possible mechanism of neuroprotection. Our results suggest that HEK-CM protects hippocampal neurons against excitotoxicity by stimulating the host's endogenous cell survival mechanisms.

Figure 1

Neuromorphological alteration. Representative photomicrographes demonstrating the morphology of H3 cells in different treatment groups. Kainic acid treatment to H3 cells resulted in prominent decrease in cell density and neurites. In contrast, KA + CM group demonstrated healthy cell bodies with dense neurites. Scale Bar 100 μm. Normal control (NC), normal control exposed to HEK conditioned medium (CM), Kainic acid alone treated (KA), and Kainic acid and HEK conditioned medium cotreated (KA + CM).

Figure 2

HEK-CM protects neurons. (a) Photomicrograph of DAPI⁺ cells representing cell density in different treatment groups. (b) Bar diagrams representing the number of viable cells as quantified by DAPI⁺ cells. Scale Bar 200 μm. Normal control (NC), normal control exposed to HEK conditioned medium (CM), Kainic acid alone treated (KA), and Kainic acid and HEK conditioned medium cotreated (KA + CM). Data represented as mean ± SEM. ^* indicates comparison between NC and treatment; ⁺ indicates comparison between KA lesion and treatment; ^*** P < 0.001; ⁺⁺⁺ P < 0.001.

Figure 3

Antiapoptotic effect of HEK-CM. (a) Representative Flow cytometric Scatter Plots demonstrating the apoptotic and antiapoptotic effects of KA and HEK-CM, respectively. (b) Bar diagrams representing the percentage of apoptotic cells in various treatment groups. Normal control (NC), normal control exposed to HEK conditioned medium (CM), Kainic acid alone treated (KA), and Kainic acid and HEK conditioned medium cotreated (KA + CM). Data represented as mean ± SEM. ^* indicates comparison between NC and treatment; ⁺ indicates comparison between KA lesion and treatment; ^*** P < 0.001; ⁺⁺⁺ P < 0.001.

Figure 4

HEK-CM stimulates host's cell survival factors BDNF and Bcl-2. (a) Photomicrograph represents the expression of BDNF in different treatment groups. Scale Bar 200 μm. (b) Bar diagram signifies the fold changes in BDNF fluorescence intensity as compared to NC. (c) MTT Assay. Bar diagram represents the viable cells in different treatment groups. Neutralization of BDNF in HEK-CM diminishes the neuroprotective potential of HEK-CM against excitotoxicity. Nevertheless, significant neuroprotection was observed in KA + CM with anti-BDNF group as compared to KA group suggesting that other growth factors/cytokines in CM might be contributing to the observed neuroprotection. Normal control (NC), normal control exposed to HEK conditioned medium (CM), Kainic acid alone treated (KA), Kainic acid and HEK conditioned medium cotreated (KA + CM), and Kainic acid and BDNF neutralized HEK conditioned medium cotreated (KA + CM with anti-BDNF). Data represented as mean ± SEM. ^* indicates comparison between NC and treatment; ⁺ indicates comparison between KA lesion and treatment; ^* P < 0.05; ^*** P < 0.001; ⁺⁺⁺ P < 0.001.

Figure 5

Messenger RNA expression of antiapoptotic factor Bcl-2 in different treatment groups. Normal control (NC), normal control exposed to HEK conditioned medium (CM), Kainic acid alone treated (KA), and Kainic acid and HEK conditioned medium cotreated (KA + CM).