With the goal of understanding the role of microglia under physiological and various pathological conditions, two novel microglia ablation models have recently been reported, either genetically targeting microglia [
15‐
17] or through pharmacologically targeting the CSF1 receptor (CSF1R) with PLX3397 or 5622 [
13,
14,
18‐
20]. Although both models effectively ablate microglia and have been demonstrated to be valuable tools in the field, a major difference between the two systems is that whereas the genetic ablation model can lead to the upregulation of select cytokines and induces astrocyte activation [
15], the pharmacological model, in general, appears not to cause such actions [
18,
57]. The upregulation of cytokines and astrocyte activation in the genetic ablation model may cause additional deficits in the brain that are not specifically related to microglia function. In this study, we describe a consistent loss of ventricle pathology in both the Cx3cr1-iDTR and iDTR mouse models following Dtx administration that has not been reported previously.
Given the wide utilization of the microglial ablation models and the subsequent large number of high impact publications using these models [
13‐
20,
22‐
25], it is important to carefully characterize these models utilizing multiple analyses and methods. The genetic model of microglia depletion has been used in over 700 publications since its conception. It has been used to investigate microglia repopulation in age-dependent studies to better understand mechanisms behind adult microglia replenishing [
15]. This model has also been used to investigate how microglia play a role in learning and memory [
16]. Additionally, it has also been used in a variety of neurological disorder-focused studies, including Alzheimer’s disease, seizures, and stroke [
58‐
60]. The PLX model of microglia ablation has similarly been used to examine stroke, Alzheimer’s disease, and Parkinson’s disease [
13,
14,
21,
61]. In our characterization of the genetic microglia ablation model, we discovered that Cx3cr1-iDTR mice develop a robust, unpredicted neuropathology, distinguished by a significant loss of ventricular spaces (Fig.
2), which was not observed in the pharmacological ablation model (Fig.
3); thereby suggesting that this pathology is not due to ablation of microglia per se, but, rather, represents a specific pathology observed in the genetic ablation model. We further discovered that this pathology is intrinsic to the ROSA26iDTR (iDTR) allele following Dtx treatment independent of cre expression. This pathology is not associated with the observed cytokine upregulation or the reactive astrocytes as the iDTR group subjected to Dtx that, likewise, exhibits a loss of CSF/ventricle phenotype, does not show increased cytokine or reactive astrocytes. Consistent with this observation, through 3D MRI analysis, histological analysis, and brain water content analysis, our data suggests that the loss of ventricles is not a result of parenchymal swelling. Additionally, inhibition of the KC/GRO signaling pathway, which demonstrated success in reducing edema severity and infarct development in a severe MCAo stroke model (Fig.
6A–D), was ineffective in preventing the induction of ventricle loss in the genetic microglia ablation model. Alternatively, our data suggests a CP-related pathology that is revealed by enrichment of activated IBA1 + positive cells in the CP in both iDTR mice and Cx3cr1-iDTR mice subjected to Dtx treatment. Currently, we are undertaking further detailed studies to characterize the precise cellular and molecular mechanisms contributing to this CP related pathology. Until the precise cause of pathology is established, caution is warranted when utilizing the genetic ablation model to study subtle neurological functions using Dtx mediated genetic ablation models and, particularly, the CNS cell-type specific ablation model. Specifically, caution needs to be given when utilizing this model to interpret subtle neurological functional changes that are thought to be mediated by microglia/or other neural cell types as these could, instead, be due, in part or wholly, to the CSF/ventricular loss pathology [
16,
24]; this is of particular importance in light of recent studies indicating the potentially important roles of CSF circulation in homeostasis and pathological conditions of the brain [
62‐
66]. Since ROSA26iDTR allele is responsible for the pathology after Dtx administration independent of cre expression, this phenotype is likely present in all mouse genetic cell ablation studies utilizing this mouse strain attempted to target specific-cell types with different cre drivers. Despite the wide application of this genetic ablation model, the whole brain MRI was not utilized in prior studies, likely explaining the lack of any earlier report on this pathology/phenotype. Nevertheless, the prospective confounding effect of this genetic ablation model is starting to gain attention, as one recent paper [
24] by Rubino et al. reported neuronal cell death (at 10 days post-ablation) caused by genetic ablation of microglia. Interestingly, our data in the Cx3cr1-iDTR microglia ablated brain or iDTR + Dtx brain does not show neuronal cell loss across different cortical layers (Fig.
7), which is consistent with another previous study [
16]. The reason for this discrepancy, in relation to our results plus those of Parkhurst et al. (reporting no neurodegeneration in the Cx3cr1-iDTR model) versus the Rubino et al. study remains unclear but could be due to a different dosage of Dtx (we used a lower dosage of 20 ng/g of Dtx versus the 1ug Dtx/mouse used in the Rubino et al. study) or method of neurodegeneration quantification (we used unbiased stereological counts of NeuN + cell density and the Parkhurst et al. study also evaluated NeuN + cell counts versus the immunoreactivity area fraction quantification used in Rubino et al. study). To the best of our knowledge, the work by Rubino et al. [
24] is the only published study reporting neuronal cell death and behavioral deficits related to this model. Further validation by additional independent groups is hence needed to examine neuronal cell death immediately after microglia ablation in this genetic model. Nevertheless and notable, the effects on ventricle volume decrease have not been reported previously, thereby making our observation novel and providing a new avenue for research and independent validation. We speculate that the CP related pathology and dysfunction of CSF production could potentially contribute to the observed pathology in the ROSA26iDTR and Cx3cr1-iDTR mice.