Neurotoxicity of methotrexate to hippocampal cells in vivo and in vitro
Graphical abstract
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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2022, NeuronCitation Excerpt :Even the beneficial effects of CSF1R inhibitors (Acharya et al., 2016) or anti-inflammatory drugs (Monje et al., 2003) are often evaluated in part by their impact on neurogenesis, which is in turn linked to cognitive function. Enhanced hippocampal neurogenesis has also been found to improve cognition in rodent models of chemotherapy using various chemotherapy agents (e.g., cyclophosphamide [Lyons et al., 2011a], methotrexate [Lyons et al., 2011b; Seigers et al., 2009; Yang et al., 2011], and doxorubicin [El-Agamy et al., 2018; Park et al., 2018]). Oligodendrogenesis, the process by which new oligodendrocytes are generated, is also important in normal brain homeostasis and function (Gibson et al., 2014) and can be disrupted by childhood cancer treatment.