Neurotoxicity of methotrexate to hippocampal cells in vivo and in vitro

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Abstract

This study investigated whether methotrexate (MTX) is neurotoxic to neural progenitor cells in the hippocampus of adult mice and whether it affects hippocampus-dependent behaviors. In addition, the cytotoxicity of MTX was elucidated in rat immature and mature hippocampal cultured cells. The number of Ki-67 (proliferating cell marker)- and doublecortin (immature progenitor neuron marker)-positive cells were significantly time- and dose-dependently changed in the dentate gyrus of adult hippocampi after MTX treatment. A learning and memory task (object recognition memory test) and depression-like behavior test (tail-suspension test) were performed after MTX treatment to assess hippocampal neurogenesis-related behavioral dysfunction. MTX-treated mice showed significant depression-like behaviors and memory defects. The cytotoxicity of MTX in immature hippocampal cells varied in a dose-dependent pattern, but was not changed in the mature cells. MTX induced marked apoptotic changes in immature hippocampal cells, with increase in active caspase-3 and cleaved poly (ADP-ribose) polymerase expressions. Results of this study suggest that the neurotoxic effect of MTX inhibits the proliferation of hippocampal progenitor cells and can cause hippocampal dysfunction, such as depression and cognitive impairment. Additionally, the significantly greater caspase-dependent MTX sensitivity of immature hippocampal cells suggests that the susceptibility of such hippocampal cells depends on their maturation.

Introduction

Cytostatic chemotherapy is often applied in modern oncology; cancer patients live longer and even survive. However, there is increasing clinical interest in its negative side effects on other systems, including cardiological (i.e. arrhythmia) [1], gastrointestinal (i.e. vomiting and mucositis) [2], and psychological disorders (i.e. depression and cognitive impairment) [3], [4], [5].

Methotrexate (MTX) is used in the treatment of many diverse malignancies and in the therapy of diverse autoimmune diseases, such as rheumatoid arthritis and psoriasis, due to its anti-inflammatory and immunosuppressive effects [6], [7]. MTX can be safely administered over a wide dose range in maintenance chemotherapy for acute lymphoblastic leukemia [8]. It exerts antineoplastic effects by competitively inhibiting folate-dependent biochemical processes, thus inhibiting DNA synthesis [9]. However, MTX is a frequently used cytotoxic agent in the clinic and is associated with acute and chronic neurotoxicity [10].

Neurotoxicity is a common and often dose-limiting complication of chemotherapy treatment [11]. The incidence markedly increases when the blood–brain barrier is either overwhelmed or bypassed [12]. Most common neurological complications involve acute alterations in consciousness, seizures, cerebral infarctions, paralysis, neuropathies, leukoencephalopathy, and ototoxicity [13]. Several studies have reported that various chemotherapy-induced neurotoxicities are related to various mechanisms. Anticancer drugs have neurotoxic properties in vitro and in vivo, especially in mature neurons [14]. Excitotoxic mechanisms and caspase-mediated cell death contribute to the neurotoxicity of these compounds in vitro in neuronal cultures and in vivo in the developing rat brain [14]. Additionally, neurotoxicity of mature neuronal cells exposed to anticancer drugs can be alleviated by inhibiting the Rho signaling pathway [15]. Nevertheless, little is known about the precise mechanism of the toxicological effect of chemotherapeutic agents on brain function, especially related to neuronal survival/death.

Adjuvant chemotherapy is frequently applied in the treatment of cancer. The survival rate of patients treated with cytostatics is high; however, the treatment is associated with both short- and long-term side effects. One of the potential long-term effects is cognitive impairment, which primarily manifests as a decrease in attention/concentration, speed of information processing, and memory [16]. For example, some breast cancer survivors experience cognitive defects following chemotherapy [17], [18], [19]. More patients treated with high-dose chemotherapy than patients treated with standard-dose chemotherapy show a defect in cognitive performance compared with healthy control subjects [20], [21]. In addition, several in vivo studies have suggested that MTX has a negative effect on cognitive behavior related to hippocampal cell proliferation [22], [23]. Although several mechanisms have been suggested to explain the cognitive impairment associated with MTX, the precise mechanisms remain poorly understood.

In this study, we examined the time- and dose-dependent changes in the number of Ki-67 (proliferating cell marker)- and doublecortin (DCX; immature progenitor neuron marker)-positive cells in the hippocampal dentate gyrus (DG) of adult C57BL/6 mice after MTX treatment to elucidate the detrimental effect of MTX on neurogenesis in the adult hippocampus. In addition, the effect of MTX on hippocampus-dependent behavioral dysfunction was estimated using a hippocampus-dependent learning paradigm (the object recognition memory test) and depression-like behavior test (the tail-suspension test). Furthermore, this study compared the detrimental effect of MTX treatment on 0-day in vitro (DIV) immature hippocampal cells with that of 14-DIV mature cells to elucidate the differential effect of MTX treatment on immature and mature hippocampal cells in an in vitro system.

Section snippets

Animals

Male C57BL/6 mice aged 8–9 weeks and pregnant Sprague–Dawley rats were obtained from a specific-pathogen-free colony at Oriental, Inc. (Seoul, Korea). The animals were housed in a room which was maintained at 23 ± 2 °C, with relative humidity of 50 ± 5%, artificial lighting from 08:00 to 20:00 h, and 13–18 air changes every hour. The animals were given tap water and commercial rodent chow (Samyang Feed, Seoul, Korea) ad libitum. The Institutional Animal Care and Use Committee at Chonnam National

Histological change in the mouse hippocampus following MTX injection

We first examined histological changes in hippocampal structure using hematoxylin and eosin (H&E) staining. No unusual hippocampal structures were observed in adult mice at 6 h to 14 days after acute MTX exposure (Fig. 1A and B). However, TUNEL staining revealed the presence of apoptotic nuclei in the hippocampal DG of the mice after exposure to MTX, whereas TUNEL-positive apoptotic cells were quite rare in the vehicle-treated controls (Fig. 1C and D). The number of TUNEL-positive apoptotic

Discussion

This study demonstrated the acute administration of MTX induced apoptosis and reduced the number of Ki-67 (proliferating cells)- and DCX (immature progenitor neurons)-positive cells in the adult mouse hippocampus and also induced hippocampal dysfunction, as reflected in cognitive impairment and depression-like behavior. Moreover, our in vitro study revealed that MTX exposure induced apoptotic cell death in immature hippocampal cultured cells through a caspase-dependent pathway, but did not in

Acknowledgments

This work was supported by a National Research Foundation of Korea Grant funded by the Korean Government (NRF-2010-0015393) and a Grant of the Korean Ministry of Education, Science and Technology (The Regional Core Research Program/Biohousing Research Institute). This work was supported by the Biohousing Research Center.

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