Microglial depletion does not impact alpha-synuclein aggregation or nigrostriatal degeneration in the rat preformed fibril model

Background: Parkinson’s disease (PD) is a neurodegenerative disorder that is characterized by the presence of proteinaceous alpha-synuclein (α-syn) inclusions (Lewy bodies), markers of neuroinflammation and the progressive loss of nigrostriatal dopamine (DA) neurons. These pathological features can be recapitulated in vivo using the α-syn preformed fibril (PFF) model of synucleinopathy. We have previously described the time course of microglial major-histocompatibility complex-II (MHC-II) expression and alterations in microglia morphology in the PFF model in rats. Specifically, the peaks of α-syn inclusion formation, MHC-II expression, and reactive morphology in the substantia nigra pars compacta (SNpc) all occur two months post PFF injection, months before neurodegeneration occurs. These results suggest that activated microglia may contribute to neurodegeneration and could represent a potential target for novel therapeutics. The goal of this study was to determine whether microglial depletion could impact the magnitude of α-syn aggregation, nigrostriatal degeneration, or related microglial activation during the α-syn PFF model. Methods: Male Fischer 344 rats were injected intrastriatally with either α-syn PFFs or saline. Rats were continuously administered Pexidartinib (PLX3397B, 600mg/kg), a colony stimulating factor-1 receptor (CSF1R) inhibitor, to deplete microglia for a period of either two or six months. Results: PLX3397B administration resulted in significant depletion (45–53%) of ionized calcium-binding adapter molecule 1 immunoreactive (Iba-1ir) microglia within the SNpc. Microglial depletion did not impact accumulation of phosphorylated α-syn (pSyn) within SNpc neurons and did not alter pSyn associated microglial reactivity or expression of MHC-II. Further, microglial depletion did not impact SNpc neuron degeneration. Paradoxically, long term microglial depletion resulted in increased soma size of remaining microglia in both control and PFF rats, as well as expression of MHC-II in extranigral regions. Conclusions: Collectively, our results suggest that microglial depletion is not a viable disease-modifying strategy for PD and that partial microglial depletion can induce a heightened proinflammatory state in remaining microglia.

These results suggest that activated microglia may contribute to neurodegeneration and could represent a potential target for novel therapeutics. The goal of this study was to determine whether microglial depletion could impact the magnitude of α-syn aggregation, nigrostriatal degeneration, or related microglial activation during the α-syn PFF model. Methods: Male Fischer 344 rats were injected intrastriatally with either α-syn PFFs or saline. Rats were continuously administered Pexidartinib (PLX3397B, 600mg/kg), a colony stimulating factor-1 receptor (CSF1R) inhibitor, to deplete microglia for a period of either two or six months. Results: PLX3397B administration resulted in signi cant depletion (45-53%) of ionized calcium-binding adapter molecule 1 immunoreactive (Iba-1ir) microglia within the SNpc. Microglial depletion did not impact accumulation of phosphorylated α-syn (pSyn) within SNpc neurons and did not alter pSyn associated microglial reactivity or expression of MHC-II. Further, microglial depletion did not impact SNpc neuron degeneration. Paradoxically, long term microglial depletion resulted in increased soma size of remaining microglia in both control and PFF rats, as well as expression of MHC-II in extranigral regions. Conclusions: Collectively, our results suggest that microglial depletion is not a viable disease-modifying strategy for PD and that partial microglial depletion can induce a heightened proin ammatory state in remaining microglia.

Background
Parkinson's Disease (PD), the second most common neurodegenerative disease, affects around 1 million people in the USA with 60,000 newly diagnosed people each year (1). Pathologically, PD is characterized by the presence of proteinaceous alpha-synuclein (α-syn) inclusions (Lewy bodies) and the progressive loss of the nigrostriatal dopamine (DA) neurons (2). While the exact cause of PD is still unknown, mounting evidence has suggested that neuroin ammation, mediated by microglia, may play a signi cant role in PD progression and neuropathology. Microglia, have many roles in helping maintain healthy homeostasis in the brain, including synaptic pruning, neurogenesis, and neuronal surveillance (3)(4)(5).
However, microglia are main players in the immune response to an insult and allow for the bridging of the innate and adaptive immune system (6,7). Analysis of postmortem PD brain show increased in ammatory markers, including increases in cells immunoreactive for ionized calcium binding adaptor molecule 1 (Iba1), human leukocyte antigen (HLA-DR), and phagocytic marker CD68 in the vicinity of Lewy pathology, speci cally the substantia nigra (SN) (8-12). Patients with PD have elevated proin ammatory cytokines (i.e., interleukin 1-beta, interleukin-6, interferon gamma, and tumor necrosis factor-alpha) in their cerebral spinal uid (CSF) and plasma, all produced by microglia and immune cells (13)(14)(15)(16).
These pathological hallmarks of PD; α-syn inclusions, loss of dopaminergic neurons and neuroin ammation, can be recapitulated in vivo using the α-syn preformed bril (PFF) model of synucleinopathy (17)(18)(19)(20). We have previously described the time course of the accumulation of phosphorylated α-syn (pSyn) inclusions, nigrostriatal degeneration, and microglial reactivity in the rat PFF model (17,21). Speci cally, the peak of pSyn inclusion formation, number of major-histocompatibility complex-II immunoreactive (MHC-IIir) microglia and microglial soma size in the substantia nigra pars compacta (SNpc) occurs two months post intrastriatal PFF injection, months before the neurodegeneration phase occurring at 5-6 months (21,22). Of importance, a localized subpopulation of MHC-IIir microglia is observed immediately adjacent to nigral pSyn inclusions, with the number of reactive microglia dependent on nigral inclusion load (21). These results, along with results from other laboratories (23)(24)(25)(26) suggest that pSyn inclusions are immunogenic, provoking a microglial proin ammatory response that has the potential to contribute to ultimate nigrostriatal neurodegeneration.
Thus, therapeutic strategies that target and attenuate this microglial response to pathological α-syn may have potential to slow disease progression.
Pexidartinib (PLX3397B; Plexxikon inc.), a selective tyrosine kinase inhibitor, targets the macrophage (i.e. microglia) colony stimulating factor 1 receptor (CSF1R). The CSF1R is required for the activation, proliferation, and survival of microglia and, when inhibited, leads to microglial death resulting in microglial depletion within the brain parenchyma (27). Microglial depletion has previously been analyzed in mouse models of disease to understand the role microglia may play in disease progression (28-30).
However, microglia are required to maintain healthy brain homeostasis and as such, complete microglia depletion may not be a viable therapeutic strategy. Therefore, in the present study we examined the effect of partial microglia depletion on α-syn aggregation and neurodegeneration within the rat PFF model. We demonstrate that signi cant, partial microglia depletion (36-46%) does not affect pSyn inclusion accumulation in the SNpc 2-months following α-syn PFF injection or the inclusion-associated microglial response of increased microglial soma size and expression of MHC-II. Further, microglial depletion did not impact PFF induced degeneration of tyrosine hydroxylase immunoreactive (THir) neurons in the SNpc. Surprisingly, long term microglial depletion was associated with increased microglial soma size in remaining microglia as well as expression of MHC-II in extranigral regions. Our results do not support microglial depletion as a disease modifying strategy for PD and instead suggest that long term microglial depletion may be detrimental through induction of a proin ammatory phenotype in remaining microglia.

Experimental Overview
Rats received unilateral intrastriatal injections of either mouse α-syn PFFs or an equal volume of phosphate buffered saline (PBS) and were fed the CSF1R inhibitor PLX3397B or control chow for a period of either 60 or 180 days. At the conclusion of the experiment rats were euthanized and brain tissue analyzed. Figure 1Aillustrates the experimental design.

Rats
Three-month old, male Fischer 344 rats (Charles River) were housed, 2-3 per cage, at the Grand Rapids Research Center vivarium which is fully approved through the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Rats were housed in a room with a 12-hour light/dark cycle and provided food and water ad libitum. All procedures were done in accordance with the guidelines set by the Institutional Animal Care and Use Committee (IACUC) of Michigan State University.

Stereotaxic Injections
Unilateral intrastriatal α-syn PFF injections were conducted as previously described (17). Rats were anesthetized with iso urane (5% induction and 1.5% maintenance) and received unilateral intrastriatal injections to the left hemisphere (2 x 2 µl, AP +1.6, ML +2.0, DV -4.0; AP +0.1, ML +4.2, DV -5.0, AP and ML coordinates relative to Bregma, DV coordinates relative to dura). PFFs (4 µg/µl; 16 µg total) or an equal volume of PBS were injected at a rate of 0.5 µl/min with a pulled glass capillary tube attached to a 10 µl Hamilton syringe (34). To avoid PFF displacement, the needle was left in place for 1 minute following injection, retracted 0.5 mm and left for 2 minutes before fully retracted. All animals received analgesic (1.2 mg/kg of sustained release buprenorphine) after surgery and were monitored until euthanasia.

Pexidartinib Dosing
Pexidartinib chow was generously provided by Plexxikon, Inc. Rats were fed Pexidartinib chow (PLX3397B, 600mg/kg; Plexxikon Inc.; Research Diets Inc.) or control chow ad libitum for either 60 or 180 days starting on the day of PFF injections. Rat weights and collective cage food intake was tracked weekly (Supplemental Figures 1A-B, 2A-B).

Total Enumeration for pSyn and MHC-II
Due to heterogeneity in the distribution of both pSyn and MHC-II immunoreactive pro les within the SN, total enumeration rather than stereological counting frames was used for quanti cation. The investigator was blinded to treatment groups. reported as the number of pixels per outlined microglia soma. The HALO® (Indica Labs) image analysis module "Area quanti cation v1.0 for bright eld" was used to calculate total pSyn signal in the striatum and MHC-II signal in the mesencephalon.

Statistical Analysis
All statistical tests were completed using GraphPad Prism software (version 9, GraphPad, La Jolla, CA). Outliers were assessed via the absolute deviation from the median method (37) utilizing the very conservative difference of 2.5X median absolute deviation as the exclusion criteria. Statistical signi cance was set to α ≤ 0.05. Comparisons were made across all groups using two-way analysis of variance (ANOVA) with a post-hoc Tukey test with the following exceptions: Two-way ANOVA with repeated measures was used for comparisons of food intake over time, Student's T-test (two-tailed) was used for comparisons in pSyn accumulation in the striatum between PFF injected PLX3397B and control rats, two-way ANOVA with post-hoc Tukey test comparisons in THir neurons in the SNpc were made within each brain hemisphere separately.

Impact of microglial depletion during peak aggregation in the SNpc
Two months of Pexidartinib (PLX3397B) partially depletes microglia in both α-syn PFF and PBS injected rats PFF injected rats displayed substantial accumulation of pSyn within the SNpc ipsilateral to PFF injection as well as signi cantly more microglia compared to PBS rats, regardless of chow treatment (p<0.04, Figure 2A-E). Speci cally, PFF injection was associated with ~19% and ~37% more microglia in the SNpc of control and PLX3397B chow rats, respectively. Treatment with Pexidartinib (PLX3397B; 600mg/kg) for 2 months led to a signi cant depletion of microglia within the SNpc in both PBS and α-syn PFF injected rats. PBS PLX3397B rats displayed 45% fewer microglia (p=0.001) and PFF PLX3397B rats displayed 36.6% fewer microglia (p<0.001) compared to the control fed rats in their respective surgical treatment groups ( Figure 2E). These data suggest that inclusion-associated increases in microglia persist despite signi cant depletion of microglia due to 2 months of PLX3397B treatment.
Microglial depletion does not impact accumulation of pSyn aggregates in nigral neurons or early loss of TH phenotype Intrastriatal injection of mouse α-syn PFFs results in peak pSyn accumulation in the ipsilateral SNpc at two months (17,21,22). In the present study we observed pSyn accumulation in the ipsilateral SNpc of PFF injected rats ( Figure 2B, 2D, 3A) but not in PBS control rats (Figure 2A, C). PLX3397B treatment had no impact on the number of pSynir neurons within the SNpc of PFF rats (p>0.05, Figure 3B). PFF rats fed control chow possessed 4826 ± 229.3 pSyn containing neurons in the ipsilateral SNpc whereas PFF PLX3397B rats possessed 4760 ± 148.8.
We next examined whether PFF injection or PLX3397B treatment for two months impacted THir neurons in the SNpc. Utilizing identical PFF surgical parameters in rats we have previously observed ~0-25% loss of THir SNpc neurons at two months after PFF injection, however parallel neuronal counts revealed that this represents loss of TH phenotype in the absence of overt degeneration (17,38). In the present study, two months following PFF injection we observed a 24-33% reduction (p<0.04) in THir neurons in the ipsilateral SNpc as compared to the ipsilateral SNpc of PBS injected rats ( Figure 3C, D) both with and without PLX3397B treatment. No differences in THir neurons were observed due to PLX3397B treatment (p>0.05). These results suggest that microglial depletion does not impact THir neurons in control rats, nor does it prevent the modest loss of TH phenotype associated with the aggregation phase of the PFF model.
Microglial depletion does not impact reactive microglia morphology or MHC-II expression associated with α-syn inclusions in the SNpc pSyn inclusions in the SNpc are associated with an increase in microglial soma size and a localized expression of MHC-II that correlates with α-syn inclusion load (21,38). In the present study, we observed numerous MHC-IIir microglia within the SNpc after intrastriatal α-syn PFF injection whereas very few MHC-IIir microglia were observed in PBS control rats ( Figure 4A). Signi cantly more MHC-IIir microglia were observed in both PFF control and PFF PLX3397B SNpc compared to PBS injected rats (p<0.0001, Figure 4B). No signi cant differences were observed in the number of MHC-II-ir microglia due to PLX3397B treatment (p>0.05, Figure 4B). Rats with nigral pSyn inclusions exhibited signi cantly larger microglial soma size in the ipsilateral SNpc compared microglia in the ipsilateral SNpc of PBS control rats, regardless of PLX3397B treatment (p<0.0001, Figure 4C-G). In general, Iba-1 immunoreactive microglia were ~ 15-20% larger in the SNpc of PFF injected rats. No signi cant differences in microglial soma size were observed within PBS or PFF treatment groups due to PLX3397B (p>0.05). Collectively, these results suggest that despite signi cant depletion of microglia, the localized in ammatory response to pSyn inclusions in the SNpc is preserved.

Impact of microglial depletion during nigrostriatal degeneration phase
Six months of Pexidartinib (PLX3397B) partially depletes microglia in both α-syn PFF and PBS injected rats PFF injected rats displayed modest accumulation of pSyn within the SNpc ipsilateral to PFF injection, however microglia number was not increased due to PFF injection (p>0.05, Figure 5A-E). Similar to the effect of 2 months of PLX3397B treatment, 6 months of PLX3397B led to a signi cant depletion of Iba-1 immunoreactive microglia in both PBS and α-syn PFF injected rats ( Figure 5E, p<0.001). PBS PLX3397B rats displayed 56% fewer microglia and PFF PLX3397B rats displayed 36% fewer microglia compared to control fed rats in their respective surgical treatment groups. Further, PLX3397B PFF rats possessed signi cantly more microglia than PLX3397B PBS rats (51% increase, p=0.001). Our results con rm successful depletion of microglia using PLX3397B over the 6-month interval and suggest that during PFF-induced nigrostriatal degeneration PLX3397B is less effective in microglial depletion.

Microglial depletion does not impact pSyn inclusion triggered degeneration of nigral dopamine neurons
Our previous work has demonstrated that few pSyn inclusions remain in the SNpc 6 months following PFF injection due to the loss of the SNpc neurons that were initially seeded (17,21). In general, the number of pSyn immunoreactive SNpc neurons observed at 6 months represents 10-20% what is observed during the peak 2-month aggregation phase (17). In the present study we similarly observed an approximate 80% reduction in pSyn immunoreactive neurons in the SNpc at 6 months when compared to 2 months post α-syn PFF injection (p<0.0001). A modest yet signi cant increase in pSynir SNpc neurons was observed in PFF PLX3397B rats compared to PFF rats fed control chow (p=0.0470; Figure 6A,B). We also evaluated the impact of PLX3397B on pSyn accumulation in the striatum, a structure in which pSyn accumulation is abundant at the 6-month time point (17). No signi cant differences were observed in pSyn accumulation in the striatum of PFF PLX3397B rats compared to PFF control chow rats (p>0.05, Supplemental Figure 3). These data suggest that 6 months of PLX3397 treatment results in little to no impact on pSyn accumulation following PFF injection.
Previous rat PFF model studies using identical surgical parameters reveal signi cant loss of ipsilateral SNpc THir neurons 5-6 months post intrastriatal α-syn PFF injection that parallels frank neuronal loss (17). In the present study, 6 months following surgery, we observed a 52-55% reduction in THir neurons in the ipsilateral SNpc of PFF rats as compared to the ipsilateral hemisphere of PBS injected rats (p<0.0001), both with and without PLX3397B treatment (p<0.0001, Figure 6C Analysis of the microglial soma size at six months revealed that PFF injected rats possessed signi cantly larger microglia in the SNpc compared to PBS control rats regardless of PLX3397B treatment (p=0.0194, Figure 7A-E). Further, six months of microglial depletion led to a signi cant increase in microglial soma size in both PBS and PFF injected animals (p<0.001). We next analyzed the number of MHC-IIir microglia in the SNpc ipsilateral to injection. MHC-IIir microglia peak in abundance in the SNpc 2 months after intrastriatal PFF injection, in immediate proximity to pSyn inclusions (21). Although the number of MHC-IIir microglia decrease in abundance over time, MHC-IIir microglia remain elevated compared to controls during the degenerative phase at 6 months (21). In alignment with these earlier observations, in the present experiment we observed a signi cant decrease in the number of MHC-IIir microglia in the SNpc of PFF injected rats at six months compared to two months (p<0.0001), representing a reduction of approximately 70%. Despite the reduced population of MHC-IIir microglia, we observed a signi cant increase in MHC-IIir microglia in PFF rats compared to controls, in both PLX3397B treated (p=0.0001) and untreated (p<0.0001) groups ( Figure 7F, G). Speci cally, PFF control chow rats possessed 302% more MHC-IIir microglia than PBS control chow rats, whereas PFF PLX3397B rats possessed 214% more MHC-IIir microglia than PBS PLX3397B rats. Within surgical treatment groups, no signi cant differences were observed in the number of MHC-IIir microglia in the SNpc due to PLX3397B treatment (p>0.05, Figure 7G). Further, in rats that received PLX3397B chow (both PFF and PBS) we also noticed MHC-II expression in the mesencephalon outside the nigral region ( Figure 7H). Quanti cation of MHC-II expression in the extranigral mesencephalon revealed a signi cant increase associated with long term PLX3397CB treatment (p=0.0006; Figure 7I). Collectively, these results suggest that despite signi cant microglial depletion, the localized in ammatory response to nigral degeneration normally observed following PFF injection is preserved. Further, long term microglial depletion may produce an enhanced proin ammatory phenotype in remaining microglia.

Discussion
Imaging and histological studies provide support for the presence of ongoing neuroin ammatory processes in PD (12,15,16,(39)(40)(41). The ability to attenuate in ammatory processes through microglial depletion has yielded mixed results in both AD (Tau; (30)) and PD (MPTP;(42)) animal models. In some studies, microglial depletion has led to the exacerbation of neurodegeneration (43)(44)(45) whereas in others neuroprotection is observed (42,46). Previous studies using CSF1R inhibitors in mice employed dosing strategies that resulted in near complete microglial depletion (~90%) (27,46,47). However, microglia play many roles in maintaining healthy homeostasis in the brain (3,4,48)and thus complete microglia depletion may not be a safe therapeutic strategy. Therefore, in the present study we employed a PLX3397B dosing strategy that elicited partial (~40%) microglial depletion in the SNpc. Our results demonstrate that partial microglial depletion does not prevent α-syn aggregation in the SNpc or the striatum, attenuate the in ammatory response to aggregation or degeneration, or prevent nigral degeneration following intrastriatal PFF injection.
Our previous studies have revealed that microglia react to the aggregation and degeneration phases of the rat α-syn PFF model in a consistent, measurable manner (21,22,38). During the peak aggregation phase in the SNpc at 2 months, microglia increase in number, soma size and MHC-II expression. The MHC-II response of microglia to intraneuronal pSyn aggregates is heterogeneous, limited to a subpopulation of microglia within the immediate vicinity of the SNpc inclusions. The heterogeneity of in ammatory responses within individual microglia (49) and between different brain regions (4) has been well-documented. The number of MHC-IIir microglia positively correlates to the number of pSyn immunoreactive SNpc neurons and is markedly decreased during the nigral degeneration phase (21). In the present study, microglial depletion with PLX3397B attenuated the increase in microglia associated with aggregation and degeneration in the SNpc, as would be expected, but the soma size of the remaining microglia increased in rats that received PLX3397B for 6 months. Further, the MHC-IIir microglial subpopulation was not impacted by microglial depletion. We had initially hypothesized that with ~40% depletion of microglia we would observe ~40% reduction in MHC-IIir microglia with PLX3397B. The maintenance of the pSyn inclusion associated MHC-IIir microglia subpopulation, despite signi cant microglial depletion, suggests that the remaining microglia maintain the capacity to mount a similar proin ammatory response. This nding is not unique to this study, as other microglia depletion studies have shown similar maintenance or an increase in in ammatory responses when general microglial populations are depleted (27,30,50) along with increases in adaptive immune cells within the brain (51).
Our ndings point to a need to understand the full phenotype of the inclusion associated MHC-IIir microglial subpopulation that is maintained despite signi cant general microglial depletion.
The approach of microglia repopulation as a therapeutic strategy in order to "reset" microglia has been recently proposed with the goal of exchanging dysfunctional with functional microglia. However, the results from repopulation studies vary (28,47) and suggest that repopulation comes from the remaining microglia. Our data suggests a microglia repopulation strategy would not be bene cial, and that the in ammatory response to pSyn inclusions and nigrostriatal degeneration would be maintained.
Our study is unique in that the microglial depletion was sustained for a period of 6 months, whereas most previous microglial depletion studies use much shorter depletion intervals (7-28 days (28, 29,43,47,50). We observed evidence of a more pronounced in ammatory state in our 6-month microglial depletion study compared to our 2-month microglial depletion study. Speci cally, after 6 months of microglial depletion, microglia soma size was increased, even within control PBS injected rats. Further, after 6 months of microglial depletion we observed MHC-IIir cells in multiple brain regions, and also in control rats. Normally, except for border associated macrophages (52,53), MHC-II immunoreactive cells are not often observed in uninjured brain regions in control rats. The increased MHC-II expression we observe with long term microglial depletion may be attributable to microglia or to in ltrating monocytes, border associated macrophages or perivascular macrophages (24,(54)(55)(56). Future investigation is required to ascertain the identity of the cells that respond to microglial depletion with upregulated MHC-II expression.

Conclusions
In ammatory microglia may contribute to PD progression and microglial based in ammation has been under investigation in order to identify therapeutic targets. One limitation of the present study is that the response of other cell types (peripheral macrophages, astrocytes, adaptive immune cells) to microglial depletion was not examined. Previous studies have indicated that near complete microglial depletion can impact astrocytes and adaptive immune cells (30). Another limitation of the present study is that the magnitude of microglial depletion was not that which has been previously achieved in mouse studies (~90%) (27,46,47). It is possible that near complete levels of microglial depletion may have yielded different outcomes. Despite these limitations, the present study suggests that partial microglial depletion may not be an effective, disease-modifying approach for PD and may instead induce a heightened proin ammatory state in remaining microglia.
Abbreviations   Inclusion-associated increases in microglia persist in the SNpc despite PLX3397B depletion of microglia.   Localized in ammatory response to pSyn inclusions in the SNpc is preserved despite microglial depletion.
A: Major histocompatibility complex II immunoreactive (MHC-IIir) cells in the ipsilateral SNpc of α-syn PFF or PBS injected rats with or without PLX3397B treatment. B: Quanti cation of MHC-IIir microglia in the ipsilateral SNpc demonstrates a signi cant increase in PFF compared to PBS rats at 2-months that is unaffected by PLX3397B treatment. C-F: Ionized calcium-binding adaptor molecule 1 (Iba1, green) and phosphorylated alpha-synuclein at serine 129 (pSyn, red) immuno uorescence in the ipsilateral substantia nigra pars compacta (SNpc) two months after intrastriatal alpha-synuclein preformed bril (α-syn PFF) of phosphate buffered saline (PBS) injection, with or without Pexidartinib (PLX3397B). G:Quanti cation of Iba1 immunoreactivity (Iba1ir) microglia soma size demonstrates a signi cant increase following α-syn PFF injection as compared to PBS that is unaffected by PLX3397B treatment.

Figure 5
PLX3397B is less effective in microglial depletion during nigrostriatal degeneration.
A-D: Ionized calcium-binding adaptor molecule 1 (Iba1, green) and phosphorylated alpha-synuclein (pSyn, red) immuno uorescence in the substantia nigra pars compacta (SNpc) six months following intrastriatal alpha-synuclein preformed bril (α-syn PFF) or phosphate buffered saline (PBS injection), with or without PLX3397B. Modest accumulation of pSyn immunoreactive neurons in the ipsilateral SNpc is evident following α-syn PFF injection. E. Quantitation of Iba1 immunoreactive microglia in the SNpc in all treatment groups. Six months of PLX3397B treatment resulted in signi cant microglial depletion in both PBS and PFF rats. PFF PLX3397B rats display signi cantly more microglia compared to PBS PLX3397B rats. Values represent the mean ± SEM. No PLX3397B = black outline, PLX3397B = green outline. ****p<0.0001 ***p=0.0001. Scale bars in Panels A-D are 100µm.  Long-term microglial depletion increases microglial soma size and extranigral major histocompatibility complex II expression.