The neuropsychological symptoms related to chemotherapy treatment remain a major, unresolved challenge with prevention hampered by insufficient understanding of their pathophysiology. Long-term neuroimmune changes have been identified as a hallmark feature shared by these symptoms , although, the exact timeline of mechanistic events preceding neuroinflammation, and the relationship between different glial cells driving this neuroinflammatory response, remain unclear. Here we provide a longitudinal study of region-specific, neuroimmune changes occurring in response to systemically administered 5-FU, and position impaired neurogenesis along with microglial activation as key initiating events in the resulting neuroinflammatory sequelae, perpetuated by astrocytic reactivity. We have shown for the first time in vivo that, after 5-FU, impaired neurogenesis may be related to reduced BCL2 expression and, following the acute activation of microglia, MMP activity and IL-6 signalling share a clear relationship with astrocyte reactivity (Fig. 7). These data underscore the acute origins of chronic neuropsychological symptoms highlighting the importance of neuroprotective strategies in acute supportive care.
We have shown that DCX staining was significantly reduced within 24 and 48 hours of systemic administration of 5-FU, establishing that newly born neuronal progenitor cells have been targeted, suggestive of direct cytotoxicity. To confirm whether there is active cell death occurring, additional markers of apoptosis and autophagy could be investigated in future studies. This supports the hypothesis that the inhibition of adult hippocampal neurogenesis by chemotherapy drives the development of the neuropsychological symptoms associated with these treatments . Furthermore, activation of microglia, but not astrocytes, was seen at acute timepoints following 5-FU. Our observation indicates that chemotherapy agents act directly on microglia, driving and initiating a neuroinflammatory response consistent with the findings of a sophisticated in vitro experiment that demonstrated significant reactivity of microglial, but not astrocyte, cell cultures when exposed to the chemotherapy agent methotrexate . Instead, the treatment of astrocytes with conditioned media from methotrexate-exposed microglia initiated broad astrocytic reactivity, establishing that microglia may respond directly to chemotherapeutics, but astrocytes require reactive microglia to drive their activation . In the current study, increased Iba1 staining was observed at acute timepoints, and a global increase to GFAP staining was seen at subacute and chronic timepoints and is to the best of our knowledge, the first to confirm the in vivo temporal relationship between glial cell activation, following chemotherapy treatment. These findings, along with the observed decrease in levels of DCX following 5-FU, support the possibility of 5-FU acting directly within the CNS to initiate the neuroimmune changes seen at acute timepoints following treatment.
The presence of cytotoxic injury implies 5-FU has the capacity to access the brain to induce damage. Unlike many other chemotherapy drugs, 5-FU has a small molecular weight and therefore is capable of crossing the BBB . However, despite 5-FU being routinely utilised for the treatment of CNS malignancies, its efficacy is somewhat limited by the agent’s high polarity . As such, the concentration of 5-FU that truly accesses the brain and the capacity for this dose to induce neurotoxicity remains unclear. A further consideration in the ability of 5-FU to induce direct cytotoxicity is its short half-life of only 8 to 20 minutes in vivo, and its rapid catabolism in the liver . As such, identifying the presence and distribution of 5-FU within the brain, using emerging techniques such as spatial metabolomics with mass spectrometry imaging, which provides greater sensitivity to low yield, small molecules compared to traditional techniques , is critical to understanding the role of direct cytotoxicity in the neuroimmune changes occurring post 5-FU treatment, as well as its distribution among critical brain regions such as the hippocampus.
While it is plausible that 5-FU enters the CNS to directly damage newly born neuronal progenitor cells and activate microglia, other triggers cannot be ignored, particularly given that we used only a single dose of 5-FU. Inflammatory processes are documented to induce neurodegeneration and cell death, with neuroinflammation being a hallmark feature shared by numerous neurodegenerative disorders . Importantly, intense peripheral inflammation and the generation of associated danger signals (e.g. DAMP’s, PAMP’s and MAMP’s) are commonly induced by a range of chemotherapy agents. These inflammatory responses are triggered by cytotoxic injury of chemotherapy drugs against tumor cells and the collateral damage they cause to healthy tissue, in particular the highly proliferative intestinal mucosa . For example, gastrointestinal mucositis and breakdown of the mucosal barrier, permits translocation of gut-derived inflammatory mediators and bacterial endotoxins such as lipopolysaccharide (LPS), into systemic circulation where they are able to impact BBB integrity . Apoptosis within the intestinal mucosa and resulting peaks in peripheral endotoxin levels have been observed at 6 and 24 hours following chemotherapy treatment respectively in mice. In that same study, damage to BBB integrity was identified at 24, 48 and 72 hours post treatment, highlighting a clear time course of events facilitating peripheral to central communication . With increases to BBB permeability and the movement of peripheral inflammatory mediators into the CNS, it is possible that these peripherally-derived mediators act to induce the acute damage to neuronal progenitor cells and activation of microglia seen following 5-FU treatment, or may work in conjunction with 5-FU to induce neuronal cell death [39–41].
After the transient increase to microglial activation observed at acute timepoints following 5-FU, the downstream neuroinflammatory response characterised in our study involved increases to TIMP-1 and associated MMP expression as well as IL-6R, with long term astrocyte reactivity. A compensatory increase to TIMP-1 expression, reflective of increases to the overall levels of MMPs present within the CNS, is seen in astrocytes when exposed to conditioned media from activated microglial cell cultures . We identified a positive correlation between TIMP-1 expression and astrocyte activation within the prefrontal cortex and CA3 region of the hippocampus, with peaks in expression and activation observed at subacute timepoints. Given that microglia are known to produce MMPs , it is likely that the acute activation of microglia induces increased TIMP-1 expression, to compensate for increased MMP activity. In the chronic setting following 5-FU treatment, TIMP-1 expression returned to levels similar to those seen at baseline. However, IL-6R expression peaked in the chronic setting and this was positively correlated with the level of astrocyte activation observed in the hypothalamus. Trans signalling via the IL-6R is critical in mediating the pro-inflammatory action of IL-6  and as such IL-6 signalling may underlie the chronic activation of astrocytes and resulting neuroinflammation following 5-FU. Therefore, our data imply that acute microglial activation and resulting MMP activity are initiating factors in downstream astrocyte activation and TIMP-1 expression within the subacute setting and IL-6 signalling as a critical mediator perpetuating chronic astrocyte reactivity following 5-FU.
Our evidence suggests that chronic neuropsychological symptoms have acute cytotoxic origins. This new knowledge challenges the current management guidelines which are fundamentally reactive. In addition to being variably implemented, neuropsychological symptoms are largely rehabilitated through group training, which entails repetitive completion of tasks posing a mental challenge, as well as psychological intervention and pharmacotherapy when symptoms develop . If our data are indeed translatable to humans, we suggest that ongoing work should be focussed on protecting the brain in the acute phases of chemotherapy treatment by either reinforcing the BBB or mitigating peripheral signals that drive neurological changes.
Our study is the first to longitudinally characterise the neuroimmune changes occurring post 5-FU treatment, however it has some limitations. Notably, we did not undertake behavioural phenotyping in these animals as the main aim of this study was to investigate the timeline of molecular events preceding the chronic neuroinflammation seen following chemotherapy. While this neuroinflammatory response is proposed to underlie the neuropsychological side effects of chemotherapy treatment, the inclusion of behavioural analyses probing cognitive and psychological function would have introduced significant confounding factors without providing additional data relevant to our fundamental research question. As established by Mandillo et al. stress related to the repetitive handling required by a battery of behavioural assessments, is the primary confounding variable affecting reliability and reproducibility of these tests [45, 46]. Given that stress can drive the development of neuronal damage, including both neuroinflammation and deficits in neurogenesis [23, 47], the inclusion of behavioural analyses and the resulting stress introduced by repeated handling may have masked the true effects of 5-FU on the outcome measures reported in this study. A further limitation is that all transcriptomic analyses were performed using the frontal cortex of the brain and as such no conclusions can be made regarding regional changes in the expression of the genes investigated. These analyses could, however, provide further clarification to the physiological changes occurring in critical brain regions, such as the hippocampus, following chemotherapy and thus is an avenue for future research to build on the time course of neurological events defined by the current study.