CSF1R inhibition during cranial radiotherapy reshapes glial dynamics via microglial loss, monocyte engraftment, and accelerated astrocyte reactivity

CSF1R inhibition during cranial radiotherapy reshapes glial dynamics via microglial loss, monocyte engraftment, and accelerated astrocyte reactivity | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results CSF1R inhibition during cranial radiotherapy reshapes glial dynamics via microglial loss, monocyte engraftment, and accelerated astrocyte reactivity Yoran M Leter , Caroline L van Heijningen , Zoey J Tolboom , View ORCID Profile Mark C de Gooijer , Annette C Compter , Olaf van Tellingen , View ORCID Profile Sanne B Schagen , View ORCID Profile Laura E Kuil doi: https://doi.org/10.1101/2025.10.11.681366 Yoran M Leter 1 Division of Pharmacology, the Netherlands Cancer Institute , Amsterdam, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Caroline L van Heijningen 1 Division of Pharmacology, the Netherlands Cancer Institute , Amsterdam, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Zoey J Tolboom 1 Division of Pharmacology, the Netherlands Cancer Institute , Amsterdam, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mark C de Gooijer 1 Division of Pharmacology, the Netherlands Cancer Institute , Amsterdam, the Netherlands 2 Faculty of Biology, Medicine and Health, Division of Cancer Sciences, University of Manchester ; 46 Grafton Street, Manchester, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Mark C de Gooijer Annette C Compter 3 Department of Neuro-Oncology, the Netherlands Cancer Institute , Amsterdam, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Olaf van Tellingen 1 Division of Pharmacology, the Netherlands Cancer Institute , Amsterdam, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sanne B Schagen 4 Division of Psychosocial Research and Epidemiology, the Netherlands Cancer Institute , Amsterdam, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sanne B Schagen Laura E Kuil 1 Division of Pharmacology, the Netherlands Cancer Institute , Amsterdam, the Netherlands 4 Division of Psychosocial Research and Epidemiology, the Netherlands Cancer Institute , Amsterdam, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Laura E Kuil For correspondence: l.kuil{at}nki.nl Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Background Cranial radiotherapy (cRT), a common treatment for central nervous system tumors, induces progressive cognitive problems in over half of patients. In mice, microglial depletion via CSF1R inhibition can mitigate this effect, but the underlying cellular mechanisms remain unclear. We hypothesized that CSF1R inhibition-induced microglial ablation and repopulation improves brain health by modulating microglial reactivity to radiotherapy, which attenuates glial responses to radiotherapy. Methods Nine-week-old male C57BL/6JRj mice received either a CSF1R-inhibitor supplemented diet (pexidartinib, PLX3397) or control diet, followed by fractionated CT-guided cRT (30 Gy) or sham treatment. The pexidartinib diet was discontinued 10 days post-radiotherapy. Animals were sacrificed at three intervals post-radiotherapy, allowing the assessment of temporal changes. Multiple brain regions were assessed by immunohistochemistry for markers of microglia, astrocytes, oligodendrocytes and proliferating cells. Microglial morphological changes were assessed using the semi-automated microglia morphology analysis pipeline mGlia. Results Radiotherapy alone reduced microglial numbers and induced a progressive reactive morphology; mild at 30 days and pronounced at 6 months post cRT. CSF1R inhibition before cRT markedly decreased microglial markers but increased general macrophage markers at 30 days and 6 months after cRT, consistent with monocyte-derived cell engraftment. Morphometric analysis revealed rapid and severe morphological change towards a reactive morphotype at 30 days that persisted until 6 months. Microglial depletion did not prevent loss of neurogenesis or oligodendrocyte progenitor cells (OPCs) and accelerated reactive astrogliosis, though partial OPC recovery in the hippocampus and thalamus was observed at 6 months. Conclusion CSF1R inhibition combined with cRT accelerates reactive gliosis and monocyte-derived macrophage engraftment without protecting vulnerable neural cell populations, though limited long-term OPC recovery occurred. Thus, with this set-up CSF1R inhibition-induced microglial ablation and repopulation does not improve overall brain health, but is beneficial for OPCs on the long term after cRT. Key points Pexidartinib and cranial radiotherapy have synergistic effects on microglial ablation Infiltrating monocytes repopulate the irradiated brain once the pexidartinib diet is discontinued In the absence of microglia, astrocytes show an accelerated reactivity to radiotherapy Long-term after cranial radiotherapy oligodendrocyte progenitor cell repopulation is enhanced in the pexidartinib treated animals Download figure Open in new tab Introduction Cranial radiotherapy (cRT) is a common treatment for central nervous system (CNS) tumors such as primary brain tumors and brain metastases ( Rahman et al., 2022 ). Brain metastases (BM) occur in 10 to 40% of cancer patients, most frequently stemming from lung, breast, skin, kidney and gastrointestinal cancers ( Rahman et al., 2022 ; Zhang et al., 2012 ). Up to 50% of CNS cancer patients treated with partial or whole-brain radiotherapy develop progressive cognitive impairment ( Cramer et al., 2019 ; Gonzalez-Johnson et al., 2024 ). This impairment, ranging from mild to profound progressive, substantially reduces patients’ quality of life by affecting cognitive performance and daily tasks ( Neo et al., 2017 ). Efforts to reduce cognitive problems upon whole brain radiotherapy are undertaken including treatment with memantine and hippocampal sparing-RT, but in most cases this does not prevent cognitive decline( Brown et al., 2020 ; Gondi et al., 2014 ). There remains a critical need for interventions to prevent, reduce or revert the neurobiological changes to the healthy brain tissue induced by radiation. Cognitive problems following cRT are associated with white matter (myelin), which appears as hyper intensities on MRI in both pediatric and adult brain tumor patients ( Connor et al., 2016 ; Ma et al., 2023 ; Marino et al., 2024 ; Voon et al., 2022 ). Preclinical studies implicate neuroinflammation in radiation-induced cognitive decline ( Cramer et al., 2019 ). We and others showed neurobiological changes upon radiotherapy including the loss of neurogenesis and oligodendrocyte progenitor cells (OPCs), astrocytic hypertrophy, reduced microglial numbers, and altered microglial morphology ( Han et al., 2016 ; Kuil et al., 2025 ; Monje et al., 2003 ; Panagiotakos et al., 2007 ; Voshart et al., 2024 ). OPCs not only generate myelinating oligodendrocytes but also regulate microglia, neuronal circuitry, vascularization, and blood–brain barrier (BBB) integrity ( Kimura et al., 2020 ; Pepper et al., 2018 ). Upon myelin damage, OPCs respond rapidly and play a role in the replenishment of myelination ( Franklin & Ffrench-Constant, 2017 ). Thus, cRT–induced OPC loss may contribute to the late-delayed demyelination observed in treated patients ( Wiggermann et al., 2019 ). Microglia and CNS-associated macrophages, spring from primitive macrophages produced during embryonic yolk sac primitive hematopoiesis and maintain their population by self-renewal throughout life ( Askew et al., 2017 ; Masuda et al., 2022 ). After BBB maturation, microglia remain confined to the CNS, and peripheral macrophages are excluded unless barrier integrity is compromised ( Reddaway et al., 2023 ). Microglia express markers such as IBA1, P2RY12, TMEM119, F4/80, and CX3CR1, with loss of homeostatic markers (P2RY12, CX3CR1, TMEM119) indicating reactive states ( De Simone et al., 2010 ; Paolicelli et al., 2022 ). In their homeostatic state, microglia display a small soma with long, highly branched processes that continuously survey the environment. Upon detecting cell injury or death, they adopt a reactive morphology characterized by an enlarged soma and thick, shortened branches ( Reddaway et al., 2023 ). In severe pathological conditions, including neurodegenerative diseases, infection, stroke, tumors, brain injury, and neuroinflammation, microglia can fully retract their branches, assuming an amoeboid form resembling peripheral macrophages ( Nayak et al., 2014 ; Reddaway et al., 2023 ). Temporary depletion of microglia via colony-stimulating factor 1 receptor (CSF1R) inhibition, followed by rapid repopulation, has been shown in preclinical neurological disease models to reduce inflammation, restore microglial morphology, and improve cognition ( Dagher et al., 2015 ; Henry et al., 2020 ; Iba et al., 2025 ; Thi Lai et al., 2024 ). In cranial irradiation models, persistent microglial activation is linked to cognitive dysfunction, and eliminating microglia during and after radiotherapy using the CSF1R inhibitor PLX5622 prevented these deficits in mice ( Acharya et al., 2016 ; Feng et al., 2021 ; Feng et al., 2016 ; Feng et al., 2018 ). However, these investigations focused primarily on short-term cognitive outcomes, microglial changes, and dendritic complexity. The impact of CSF1R inhibition on other vulnerable populations, such as OPCs and astrocytes, remains largely unknown, with one recent study showing no protection against radiotherapy-induced loss of neurogenesis ( Zhou et al., 2025 ). In this study, we investigate how transient microglial depletion via the CSF1R inhibitor pexidartinib interacts with cranial radiotherapy to influence long-term brain health. We found that combining pexidartinib with radiotherapy synergistically depleted microglia, promoted infiltration of monocyte-derived macrophages, and accelerated astrocyte reactivity, without protecting neural stem cells or oligodendrocyte progenitor cells. Notably, this treatment altered the trajectory of glial responses over months and allowed partial late OPC repopulation, revealing a complex and time-dependent interplay between microglia, astrocytes, and myelinating lineages after cRT. Results Synergistic microglial loss with pexidartinib + cranial radiotherapy To assess microglial sensitivity to cranial radiotherapy (cRT) in the presence or absence of CSF1R inhibition, mice were fed a pexidartinib-supplemented or control diet for three weeks before cRT, continuing until 10 days after treatment ( Fig 1A ). We first assessed the effect of pexidartinib treatment alone and in combination with cRT on microglial abundance. Sham-treated pexidartinib animals displayed reduced IBA1+ cells in cortex, hippocampus, and thalamus (41.3% reduction in cortex, 25.8% reduction in hippocampus, and 85.6% reduction in thalamus, Fig 1B-C ). The magnitude of microglial ablation was lower than the >90% reported previously ( Elmore et al., 2014 ), which might be due to differences in drug formulation in food resulting in 60% lower plasma drug levels. ( Fig. 1D ). Intriguingly, on top of this more modest depletion, combining pexidartinib with cRT produced a near-complete loss of IBA1+ microglia (95.4% cortex, 95.8% hippocampus, 85.6% thalamus; all p<0.05), suggesting enhanced radiosensitivity of pexidartinib-treated microglia as pexidartinib levels in the brain were similar in cRT + pexidartinib and pexidartinib only animals ( Fig 1B-C ). Download figure Open in new tab Figure 1. Experimental set-up of microglial ablation during cRT. A. Schematic representation of the experimental timeline where in yellow the pexidartinib diet (290mg/kg in chow) was supplemented starting 21 days preceding cRT continuously through 10 days post cRT. B. IBA1 IHC in the hippocampus. C. Quantification of IBA1+ cells. D. LC-MS/MS measurement of Pexidartnib levels in plasma and brain. Each dot represents one animal, error bars represent standard deviation. * p<0.05, ** p<0.01, ***p<0.001, ****P<0.0001. Monocyte-derived macrophage infiltration after microglial ablation At 30 days post-treatment, IBA1+ cells repopulated in all groups ( Fig 2A ). However, cRT + pexidartinib animals showed significantly higher IBA1+ cell numbers than cRT alone in cortex, hippocampus, and thalamus (p<0.05)( Fig 2A ; S1-2). IBA1 is known to label microglia, but also CNS-resident macrophages and peripheral macrophages. Therefore, P2RY12 was used as a marker marking homeostatic microglia, not labeling peripheral or CNS-associated macrophages. It is important to note that P2RY12 is downregulated upon microglial reactivity ( Paolicelli et al., 2022 ). P2RY12 staining revealed an almost complete loss of homeostatic microglia in the cRT + pexidartinib group, while cRT alone showed partial loss ( Fig 2A ; S1-2). Lund and colleagues showed that the CSF1R inhibitor-induced empty microglial niche is competitively repopulated by proliferating microglia that are F4/80 low Clec12a − and infiltrating monocytes that are F4/80 high Clec12a + ( Lund et al., 2018 ), which was confirmed by another publication ( Bastos et al., 2025 ). To validate that indeed the availability of the niche and cranial radiotherapy induces widespread monocyte infiltration, F4/80 IHC was performed to distinguish between F4/80 high monocytes and F4/80 low microglia. This revealed widespread monocyte engraftment in cRT + pexidartinib animals ( Fig 2A ; S1-2). Morphometric analysis confirmed a vastly different morphology of IBA1+ cells in the cRT + pexidartinib group ( Fig 2C ). Sholl analysis on hippocampal IBA1+ cells showed increased branching upon radiotherapy alone (p=0.08*), whereas radiotherapy + pexidartinib showed reduced branching (p=0.0003)( Fig 2C ). Download figure Open in new tab Figure 2. Quantification and morphological assessment IBA1 cells A. IHC of IBA1, P2RY12 and F4/80 in the hippocampus at 30 days post cRT. B. IHC of IBA1, P2RY12 and F4/80 in the hippocampus at 6 months post cRT. Each dot represents one animal, error bars represent standard deviation. * p<0.05, ** p<0.01, ***p<0.001, ****P<0.0001. C. Morphometric analysis using mGlia showing a sholl plot and relative cluster distribution plot. Reduced microglial numbers persist half a year post cRT in cortex and thalamus At 6 months post cRT, decreased numbers of IBA1, P2RY12 and F4/80 positive cells were present in the cortex and thalamus in the cRT group (Fig S1-2). In the hippocampus the number of P2RY12+ homeostatic microglia was decreased, but no change in IBA1 and F4/80 was observed ( Fig 2B ). In the cRT + pexidartinib animals the number of P2RY12+ homeostatic microglia was significantly decreased ( Fig 2B ; S1-2), whereas the number of F4/80+ cells was significantly increased ( Fig 2B ; S1-2). The morphology of IBA1+ cells showed differences between groups and over time. Therefore, morphometric analysis was performed on these cells to characterize the effects of cRT alone and the morphological differences induced by cRT + pexidartinib at 30 days and 6 months post cRT. Morphology of IBA1 cells upon cRT is similar regardless of pexidartinib treatment at 6 months Over time, the control groups actually showed almost exclusively cells with a type 2 morphology at 6 months after sham-treatment, whereas at 30 days there were still 33% and 52% of cells presenting a type 1 morphology in sham and pexidartinib groups respectively ( Fig 2C ). This shows that upon aging, the complexity of IBA1+ microglia increases in healthy conditions. At 6 months after cRT, morphological analysis of IBA1+ cells showed morphological adaptation. Decreased branching complexity upon radiotherapy was observed, regardless of pexidartinib treatment (p<0.0001 both groups)( Fig 2C ). Compared to the morphology at 30 days post radiotherapy, the cells in the irradiated brains obtained more often a type 1 morphology (almost 2-fold increase), whereas the pexidartinib treated irradiated animals contained a slight proportional increase in cells with a type 2 morphology (5% at 30 days to 16,67% at 6 months)( Fig 2C ). In the cRT + pexidartinib group the majority of cells belonged to type 1 already at 30 days post cRT, whereas in the cRT only group this only became apparent at 6 months post cRT. This suggests that in the cRT only condition, the IBA1+ cells in the brain might over time also represent some infiltrating monocytes as well as microglia, while at the same time in cRT + pexidartinib animals the IBA1+ cells represent infiltrating monocytes that tend to change their morphology towards microglia-like. Radiotherapy-induced effects on oligodendrocyte progenitor cells (OPCs), myelination, neurogenesis and astrogliosis OPCs (PDGFRA+) were significantly reduced in hippocampus and cortex at 10 days after cRT, unaffected by pexidartinib co-treatment ( Fig 3A ; S3A). This depletion persisted at 30 days, when also the thalamus showed OPC ablation ( Fig 3A ; S3A). By 6 months, partial OPC recovery occurred in cRT + pexidartinib animals in hippocampus and occasionally in thalamus, while cRT alone showed no recovery over time ( Fig 3A ; S3A). Myelin basic protein (MBP) coverage, reflecting myelination, was mildly reduced by cRT in cortex (n.s.) with no rescue by Pexidartinib (Fig S4A). Ki67+ proliferative cells in hippocampal neurogenic regions, reflecting neuronal stem cell proliferation, were nearly absent after cRT at each time point, regardless of pexidartinib treatment ( Fig 3B ; S3B). GFAP+ coverage increased after cRT in multiple regions, reflecting reactive astrocytes ( Fig 3C ; S4B). At early time points (10–30 days), this response was significantly amplified by cRT + pexidartinib in cortex and medial prefrontal cortex, but not hippocampus ( Fig 3C ; S4B). By 6 months, GFAP coverage was similarly elevated in both irradiated groups, reflecting accelerated gliosis in cRT + pexidartinib compared to cRT alone ( Fig 3C ; S4B). Download figure Open in new tab Figure 3. IHC analysis of OPCs, neurogenesis and astrocytes A. IHC of PDGFRA labelling OPCs in the hippocampus at 10 days post cRT and quantification of PDGFRA+ cells on all three time points B. IHC of Ki67 labelling proliferation in the sub-ventricular zone (neurogenic site) at 10 days post cRT and quantification of Ki67+ cells normalized to total cells on all three time points. C. IHC of GFAP labelling astrocytes in the cortex at 10 days post cRT and quantification of GFAP+ pixel coverage normalized to total region area on all three time points. Each dot represents one animal, error bars represent standard deviation. * p<0.05, ** p<0.01, ***p<0.001, ****P<0.0001. Discussion Cranial radiotherapy (cRT) triggers complex, long-lasting changes in the brain’s glial landscape. Here, we show that transient CSF1R inhibition with pexidartinib during cRT synergistically depletes microglia, drives rapid and widespread engraftment of monocyte-derived macrophages, and accelerates astrocytic reactivity yet fails to protect neural stem cells or oligodendrocyte progenitor cells (OPCs). Over time, infiltrating monocytes adopt microglia-like morphologies and support partial OPC repopulation, but neither myelin integrity nor neurogenesis recover, underscoring the limited neuroprotective potential of this strategy. Synergistic microglial depletion upon cRT + pexidartinib The current study demonstrates that the mild microglia depletion upon CSF1R inhibition is aggravated by radiotherapy treatment in a synergistic manner. Others have shown that another CSF1R inhibitor GW2580 does not affect microglial numbers, but do sensitize them to reactive oxygen species, which might make microglia more sensitive to radiotherapy ( Soto-Diaz et al., 2021 ). Another possibility explaining the increased microglial radio sensitivity is that pexidartinib-induced reduction of microglia induced proliferation in the remaining microglia, increasing the number of microglia in mitosis. It is well known that cells in mitosis are more sensitive to DNA-damaging radiotherapy ( Cosper et al., 2023 ; Stobbe et al., 2002 ). Empty microglial niche allows for infiltration of the brain by monocyte-derived cells Our findings challenge the prevailing assumption that microglial ablation during cRT is broadly beneficial. Previous rodent studies reported improved cognition after CSF1R inhibition combined with cRT ( Acharya et al., 2016 ; Feng et al., 2018 ), but largely examined short-term outcomes and focused on microglial reactivity and dendritic complexity. By extending the observation window to six months and interrogating multiple glial lineages, we reveal that while pexidartinib intensifies early microglial loss, this also creates an empty niche permissive to monocyte infiltration. Consistent with lineage-tracing studies ( Bastos et al., 2025 ; Du et al., 2024 ; Feng et al., 2021 ; Hohsfield et al., 2020 ; Morganti et al., 2014 ; Xu et al., 2020 ), the infiltrating cells remain abundant long-term, suggesting stable engraftment. Zhou and colleagues (2025) showed that depletion of microglia delayed dead neuronal progenitor removal and strongly increased CCL2 levels resulting in a pro-inflammatory environment ( Zhou et al., 2025 ). A pro-inflammatory environment could have persistent negative effects as pro-inflammatory conditions have been found to negatively influence microglia migration towards the ADP gradient, a process modulated by the P2RY12 receptor and needed for microglia immune response ( De Simone et al., 2010 ). These elevated CCL2 levels likely induced the infiltration of monocytes in these conditions as it is known that monocytes can be attracted to the brain by CCL2-CCR2 signaling ( Howe et al., 2017 ; Mondini et al., 2019 ; Murugan et al., 2020 ; Varvel et al., 2016 ). Thus, this is likely happening in our model as well, shown by the infiltration of F4/80 high cells and absence of P2RY12+ cells in the cRT pexidartinib animals 30 days post cRT. Brain-engrafted monocyte-derived cells become microglia-like over time Assessing the morphology at 30 days after treatment showed increased branching complexity upon cRT, as well as in the sham-treated animals over time. These findings are in line with what we previously observed in early-aging (77 weeks) in female animals and with the previously reported morphological changes upon cRT in male animals ( Kuil et al., 2025 ). Based on the change in morphology of IBA1+ cells in the cRT + pexidartinib animals over time between 30 days and 6 months and the mild increase in P2RY12+ homeostatic microglial cells numbers, one might speculate that the infiltrating monocyte-derived microglia acquire a microglial-like identity. This has been suggested by Feng and colleagues upon radiotherapy, but also in other conditions, e.g. stroke, lesions, cuprizone treatment ( Chen et al., 2020 ; Feng et al., 2021 ; Mildner et al., 2007 ). However, the debate on whether these cells truly can become microglia in terms of transcriptome and identity remains inconclusive ( Bastos et al., 2025 ; Cronk et al., 2018 ). Microglial ablation during and after cRT did not prevent NSC and OPC loss Microglial ablation during radiotherapy did not prevent the loss of neurogenesis, which is an early and persistent effect of cRT. In line with our observations, Zhou et al showed that loss of neurogenesis was induced by radiotherapy regardless of the presence or absence of microglia, accomplished using a genetic microglial ablation model in juvenile mice ( Zhou et al., 2025 ). This challenges the general perception that upon radiotherapy microglia are detrimental for neurogenesis due to their radiotherapy-induced reactivity leading to the loss of neurogenesis ( Gibson & Monje, 2021 ; Monje et al., 2003 ; Voshart et al., 2024 ). cRT-induced loss of OPCs was also not prevented by pexidartinib treatment. This is unexpected, since Gibson et al showed that chemotherapy-induced OPC loss could be prevented by CSF1R inhibitor treatment ( Gibson et al., 2019 ). Interestingly, long-term post cRT OPCs showed enhanced repopulation in pexidartinib + cRT animals compared to cRT animals. This suggests that the microenvironment induced by pexidartinib treatment in combination with cRT is, in the long-term, beneficial for OPC repopulation. This could be the result of having monocyte-derived macrophages grafted in the brain instead of endogenous (reactive) microglia, or due to the accelerated immune response consisting of reactive macrophages and reactive astrocytes, which is initiated already early on upon cRT. CSF1R inhibitor treatment during cRT induced an accelerated astrocyte reactivity A seminal in vitro study by Liddelow et al. showed that activated microglia release IL-1α, TNF, and C1q, which are necessary to induce the formation of neurotoxic astrocytes ( Liddelow et al., 2017 ). Along the same lines, Gibson et al showed that chemotherapy treatment did not induce reactivity of astrocytes in vitro using monocultures (2019). However in the presence of medium of chemotherapy-induced reactive microglia, reactive astrocytes were induced ( Gibson et al., 2019 ). Unfortunately, they did not assess astrocytes in their in vivo CSF1R inhibitor + chemotherapy model. Our data shows that microglial presence is not required for astrocyte reactivity upon cRT since the absence of microglia, or their death, accelerated the astrocytic reactivity to cRT. Rapid response of both microglia and astrocytes upon LPS (triggering immune reactions) or EAE (myelin damage) models have been found in previous in vivo studies ( Chanaday & Roth, 2016 ; Xingi et al., 2023 ). However, studies that tried to induce reactive astrogliosis in the absence of microglia are limited. One study showed that minocycline did not prevent astrocytic reactivity in response to stroke ( Bourget et al., 2022 ). However, in their additional experiment using CSF1R inhibitor treatment, stroke itself did induce astrocytes, but did not reach significance, hampering the assessment of a possible improvement upon CSF1R inhibitor treatment ( Bourget et al., 2022 ). Thus, the complex interaction between microglia and astrocytes remains elusive, but in the absence of microglia, our cRT model induces a major astrocytic response that is sustained over time. Timing and extend of microglial ablation and repopulation might be critical In future research, it might be valuable to alter the timing of microglia depletion in relation to the moment of cRT. For example Henry et al. (2020) depleted microglia 1 month after traumatic brain injury followed by quick repopulation, allowing microglia to continue their homeostatic function shortly after injury ( Henry et al., 2020 ). In doing so, one can investigate whether pro-inflammatory conditions can be prevented by microglia depletion after cRT and the effect this has on the cognitive outcome long term. However, the study from Hohsfield et al (2020) suggests that even after BBB recovery for 3.5 months after radiotherapy, CSF1R inhibitor-induced microglial ablation still results in repopulation from peripheral monocytes instead of microglial expansion ( Hohsfield et al., 2020 ). To prevent infiltration of monocytes one might adjust the CSF1R dosing to induce only partial ablation, resulting in higher microglial numbers in the brain that are able to repopulate the niche. This strategy has induced positive effects on synaptic connectivity and cognitive function in aged mice ( Strackeljan et al., 2025 ). Implications While microglia depletion strategies have been considered for mitigating radiation-induced cognitive decline, our data indicate that timing, extent of ablation, and the nature of repopulating cells are critical variables that can dramatically alter the glial response. Future work should test delayed or partial microglial ablation and assess the functional properties of engrafted monocytes Limitations The less-than-anticipated microglial depletion in sham + pexidartinib animals, likely due to formulation differences, complicates direct comparison with earlier reports. Cognitive outcomes were not assessed here, so functional implications of the observed glial remodeling remain speculative. Furthermore, it has not been established whether monocyte-derived cells are beneficial, detrimental, or neutral for long-term brain health in healthy conditions nor after cRT. Conclusion This work reveals that pexidartinib induced CSF1R inhibition during cranial radiotherapy reshapes glial dynamics by driving microglial loss, monocyte infiltration, and accelerated astrocyte reactivity without safeguarding vulnerable neural populations. These results call for a reassessment of microglia-targeted interventions in cRT, emphasizing the need for precision in manipulating the neuro-immune environment to promote recovery. Funding This research was funded by the Dutch Cancer Society; grant number NKI 2015-7937 acquired by SS. This research was supported by an institutional grant of the Dutch Cancer Society and of the Dutch ministry of Health, Welfare and Sports to the Netherlands Cancer Institute. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements We would like to thank the animal pathology core facility and animal facility of the NKI for assistance. Funder Information Declared Dutch Cancer Society, https://ror.org/0368jnd28 , NKI 2015-7937 References ↵ Acharya , M. M. , Green , K. N. , Allen , B. D. , Najafi , A. R. , Syage , A. , Minasyan , H. , Le , M. T. , Kawashita , T. , Giedzinski , E. , Parihar , V. K. , West , B. L. , Baulch , J. E. , & Limoli , C. L. ( 2016 ). 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Share CSF1R inhibition during cranial radiotherapy reshapes glial dynamics via microglial loss, monocyte engraftment, and accelerated astrocyte reactivity Yoran M Leter , Caroline L van Heijningen , Zoey J Tolboom , Mark C de Gooijer , Annette C Compter , Olaf van Tellingen , Sanne B Schagen , Laura E Kuil bioRxiv 2025.10.11.681366; doi: https://doi.org/10.1101/2025.10.11.681366 Share This Article: Copy Citation Tools CSF1R inhibition during cranial radiotherapy reshapes glial dynamics via microglial loss, monocyte engraftment, and accelerated astrocyte reactivity Yoran M Leter , Caroline L van Heijningen , Zoey J Tolboom , Mark C de Gooijer , Annette C Compter , Olaf van Tellingen , Sanne B Schagen , Laura E Kuil bioRxiv 2025.10.11.681366; doi: https://doi.org/10.1101/2025.10.11.681366 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Neuroscience Subject Areas All Articles Animal Behavior and Cognition (7635) Biochemistry (17697) Bioengineering (13894) Bioinformatics (41951) Biophysics (21455) Cancer Biology (18592) Cell Biology (25507) Clinical Trials (138) Developmental Biology (13380) Ecology (19903) Epidemiology (2067) Evolutionary Biology (24321) Genetics (15610) Genomics (22509) Immunology (17737) Microbiology (40398) Molecular Biology (17182) Neuroscience (88618) Paleontology (667) Pathology (2833) Pharmacology and Toxicology (4825) Physiology (7641) Plant Biology (15158) Scientific Communication and Education (2046) Synthetic Biology (4296) Systems Biology (9825) Zoology (2271)