Microglia are implicated in the development of paclitaxel chemotherapy-associated cognitive impairment in female mice

Highlights

• Paclitaxel induces microglial inflammation associated with memory recall deficits.

• Machine learning identifies paclitaxel-induced microglial morphological changes.

• Paclitaxel induces transient changes in microglial inflammatory gene expression.

• Microglial depletion aids in restoration of memory recall blunted by paclitaxel.

• Microglial depletion ameliorates hippocampal proinflammatory cytokine expression.

Abstract

Chemotherapy remains a mainstay in the treatment of many types of cancer even though it is associated with debilitating behavioral side effects referred to as “chemobrain,” including difficulty concentrating and memory impairment. The predominant hypothesis in the field is that systemic inflammation drives these cognitive impairments, although the brain mechanisms by which this occurs remain poorly understood. Here, we hypothesized that microglia are activated by chemotherapy and drive chemotherapy-associated cognitive impairments. To test this hypothesis, we treated female C57BL/6 mice with a clinically-relevant regimen of a common chemotherapeutic, paclitaxel (6 i.p. doses at 30 mg/kg), which impairs memory of an aversive stimulus as assessed via a contextual fear conditioning (CFC) paradigm. Paclitaxel increased the percent area of IBA1 staining in the dentate gyrus of the hippocampus. Moreover, using a machine learning random forest classifier we identified immunohistochemical features of reactive microglia in multiple hippocampal subregions that were distinct between vehicle- and paclitaxel-treated mice. Paclitaxel treatment also increased gene expression of inflammatory cytokines in a microglia-enriched population of cells from mice. Lastly, a selective inhibitor of colony stimulating factor 1 receptor, PLX5622, was employed to deplete microglia and then assess CFC performance following paclitaxel treatment. PLX5622 significantly reduced hippocampal gene expression of paclitaxel-induced proinflammatory cytokines and restored memory, suggesting that microglia play a critical role in the development of chemotherapy-associated neuroinflammation and cognitive impairments. This work provides critical evidence that microglia drive paclitaxel-associated cognitive impairments, a key mechanistic detail for determining preventative and intervention strategies for these burdensome side effects.

Introduction

Cancer patients experience many debilitating side effects during and following treatment that impair their quality-of-life. Commonly reported symptoms include fatigue, loss of concentration, and memory loss (Janelsins et al., 2012, Schmidt et al., 2016). In particular, decreased cognitive capacity has been colloquially referred to as “chemobrain,” given that these symptoms frequently arise during cancer treatment. Chemobrain symptoms have been reported to last for months to years following remission (Ahles et al., 2012, Kesler et al., 2013), and chemotherapy-induced central inflammation, likely resulting indirectly via peripheral inflammation, has been posited as an underlying cause of these cognitive deficits (Cheung et al., 2015, Kesler et al., 2013). Chemotherapy leads to peripheral inflammation by inducing cell death in neoplastic and healthy cells, which stimulates proinflammatory cytokine release and attraction of immune cells (Pusztai et al., 2004, Smith et al., 2014). These cytokines are then thought to induce subsequent neuroinflammation by multiple potential routes (reviewed in Cleeland et al., 2003). Additional chemotherapy-induced toxicities (gut permeability, neuropathy) may contribute to or exacerbate peripheral inflammatory signaling to the brain (Obrenovich, 2018). Ultimately, these chemotherapy-induced peripheral cytokines are associated with cognitive deficits (Cheung et al., 2015) and increases in proinflammatory cytokine production in the brain using rodent models (Weymann et al., 2014). Furthermore, chemotherapy-associated disruption of the gut is also associated with the induction of brain-mediated side effects (Grant et al., 2021) Unfortunately, mechanisms that result in brain cytokine production and cognitive deficits during chemotherapy remain poorly understood, resulting in an absence of effective treatment options for these debilitating side effects.

Microglia play essential roles in maintaining brain homeostasis (Ferro et al., 2021) and regulating learning and memory (Cornell et al., 2022, Parkhurst et al., 2013, Woodburn et al., 2021). As previously reviewed, microglia are essential for experience-driven memory encoding and communication with neurons via multiple signaling pathways, including brain-derived neurotrophic factor (BDNF) (Cornell et al., 2022). In fact, depletion of microglial-derived BDNF worsens performance in memory tasks (Parkhurst et al., 2013). However, in the context of injury, illness, and disease (e.g., chemotherapy treatment), elevated cytokine production and recruitment of peripheral immune cells to the brain by activated microglia (i.e., development of neuroinflammation) can impair cognitive function (Woodburn et al., 2021). A common method of assessing memory impairment in rodent models is the contextual fear conditioning (CFC) paradigm, which measures the time rodents spend freezing when placed in a chamber where they previously received an aversive foot-shock. This task is primarily hippocampal driven (Ji and Maren, 2008) and we have previously demonstrated that paclitaxel causes CFC deficits in BALB/c mice (Loman et al., 2019). Furthermore, previous research has demonstrated that microglia are important mediators of CFC-induced memory consolidation (Yu et al., 2022, Smith et al., 2019, Elmore et al., 2018).

Paclitaxel is a taxane microtubule stabilizing agent that is commonly used to treat ovarian and breast cancers and is classified as an essential drug by the World Health Organization (World Health Organization, 2021). This chemotherapeutic agent causes various brain-mediated side effects in animal models including fatigue (Ray et al., 2011) and cognitive deficits (Huehnchen et al., 2017, Smith et al., 2017) that mimic these symptoms in patients (Ahles et al., 2012). These brain-mediated side effects are associated with inflammatory cytokines in plasma (Pusztai et al., 2004). However, paclitaxel causes limited direct cell death within the brain (Huehnchen et al., 2017, Stage et al., 2020) because it has low absorption capacity across the blood–brain barrier and is rapidly pumped out of the parenchyma by p-glycoprotein (Fellner et al., 2002). Thus, while paclitaxel-induced peripheral inflammation may induce cognitive deficits indirectly, the mechanisms of these deficits are yet unknown. The frequent use of taxane agents for the treatment of cancer, coupled with the devastating side effects they cause, underscores the importance of understanding the underlying brain mechanisms. Chemotherapy-induced neuroinflammation and associated cognitive deficits have been characterized with other chemotherapeutics (reviewed in (Gibson and Monje, 2021)) such as methotrexate (Gibson et al., 2019, Seigers et al., 2010), doxorubicin (Allen et al., 2019), cyclophosphamide (Yang et al., 2010), and 5′ fluorouracil (ElBeltagy et al., 2010). However, the limited studies assessing the role of neuroinflammation in the development of paclitaxel-associated memory impairment do not identify key cellular mechanisms (Chang et al., 2020, Grant et al., 2021, Huehnchen et al., 2017, Li et al., 2018, Loman et al., 2019). Moreover, while astrocytes, oligodendrocytes, and oligodendrocyte precursor cells contribute to methotrexate-induced cognitive impairment (Gibson et al., 2019), the relative contributions of these glial cells have yet to be examined in the context of paclitaxel-induced cognitive impairment.

In the present study, we examined the extent to which paclitaxel-induced microglial and astrocytic activation drives cognitive deficits. We evaluated chemotherapy-induced effects on CFC, a form of hippocampal-dependent memory (Ji and Maren, 2008), and measured hippocampal microglial ionized calcium binding adaptor molecule 1 (IBA1) and astrocytic glial fibrillary acidic protein (GFAP) immunostaining during and following paclitaxel treatment. In addition to traditional immunohistochemical quantification based on percent area of staining, we incorporated signal intensity and morphological characteristics of these signals into machine learning models to detect more subtle paclitaxel-induced cellular differences to reactive microglia and astrocytes. Microglial-specific inflammatory gene expression was assessed and microglial depletion via PLX5622 was investigated as a method of reducing neuroinflammation and paclitaxel-induced CFC deficits. The data indicate a causal role for microglia in the development of paclitaxel chemotherapy-associated cognitive deficits, and distinctive patterns of astrocytic and microglial activation in hippocampal subfields throughout the course of paclitaxel treatment.