The change of microglia numbers within the mice retina, optic nerve and chiasm following intravitreal AAV2-GFP injection

Purpose To explore the optimized concentration of AAV2-GFP for sparse transfection of retinal ganglion cells (RGCs) and optic nerve (ON), and to examine the changes of microglial morphology and distribution in the retina, optic nerve and chiasm after injection. Methods We defined the optimal concentration of AAV2-GFP for sparse labeling of RGCs and axons in WT mice. We further explored the changes of microglial morphology and distribution in the retina, optic nerve and chiasm after intravitreal injection in CX3CR1+/GFP mice. Results 14 days after intravitreal injection of AAV2-GFP, live imaging of the retina showed that fundus fluorescence was very strong and dense at 2.16 × 1011 VG/retina, 2.16 × 1010 VG/retina, 2.16 × 109 VG/retina. RGCs were sparsely marked at a concentration 1:1000 (2.16 × 108 VG/retina) and fundus fluorescence was weak. The transfected RGCs and axons were unevenly distributed in the retina and significantly more RGCs were transfected near the injection site of AAV2-GFP compared to the other sites of the flat-mounted retina. Microglia density increased significantly in the retina and part of optic nerve, but not in the optic chiasm. The morphology of microglia was largely unchanged. Conclusions AAV2-GFP was highly efficient and the optimal concentration of sparsely labeled RGCs was 1:1000 (2.16 × 108 VG/retina). After intravitreal injection of AAV2-GFP, the number of microglia increased partly. The morphology of microglia was comparable.

Beyond Activation: Characterizing Microglial Functional Phenotypes

Classically, the following three morphological states of microglia have been defined: ramified, amoeboid and phagocytic. While ramified cells were long regarded as “resting”, amoeboid and phagocytic microglia were viewed as “activated”. In aged human brains, a fourth, morphologically novel state has been described, i.e. dystrophic microglia, which are thought to be senescent cells. Since microglia are not replenished by blood-borne mononuclear cells under physiological circumstances, they seem to have an “expiration date” limiting their capacity to phagocytose and support neurons. Identifying factors that drive microglial aging may thus be helpful to delay the onset of neurodegenerative diseases, such as Alzheimer’s disease (AD). Recent progress in single-cell deep sequencing methods allowed for more refined differentiation and revealed regional-, age- and sex-dependent differences of the microglial population, and a growing number of studies demonstrate various expression profiles defining microglial subpopulations. Given the heterogeneity of pathologic states in the central nervous system, the need for accurately describing microglial morphology and expression patterns becomes increasingly important. Here, we review commonly used microglial markers and their fluctuations in expression in health and disease, with a focus on IBA1 low/negative microglia, which can be found in individuals with liver disease.

Microglial morphology in Alzheimer’s disease and after Aβ immunotherapy

Microglia are the brain immune cells and their function is highly dependent on cell motility. It was hypothesised that morphological variability leads to differences in motility, ultimately impacting on the microglial function. Here, we assessed microglial morphology in 32 controls, 44 Alzheimer’s disease (AD) cases and 16 AD cases from patients immunised against Aβ42 (iAD) using 2D and 3D approaches. Our 2D assessment showed an increased number of microglia in iAD vs. AD (P = 0.032) and controls (P = 0.018). Ramified microglia were fewer in AD vs. controls (P = 0.041) but increased in iAD compared to AD (P < 0.001) and controls (P = 0.006). 3D reconstructions highlighted larger cell bodies in AD vs. controls (P = 0.049) and increased total process length in iAD vs. AD (P = 0.032), with negative correlations detected for pan-Aβ load with total process length (P < 0.001) in AD and number of primary processes (P = 0.043) in iAD. In summary, reactive/amoeboid microglia are the most represented population in the aged human brain. AD does not affect the number of microglia, but the ramified population is decreased adopting a more reactive morphology. Aβ removal by immunotherapy leads to increased ramified microglia, implying that the cells retain plasticity in an aged disease brain meriting further investigation.

An open-source pipeline for analysing changes in microglial morphology

Changes in microglial morphology are powerful indicators of the inflammatory state of the brain. Here, we provide an open-source microglia morphology analysis pipeline that first cleans and registers images of microglia, before extracting 62 parameters describing microglial morphology. It then compares control and ‘inflammation’ training data and uses dimensionality reduction to generate a single metric of morphological change (an ‘inflammation index’). This index can then be calculated for test data to assess inflammation, as we demonstrate by investigating the effect of short-term high-fat diet consumption in heterozygous Cx3CR1-GFP mice, finding no significant effects of diet. Our pipeline represents the first open-source microglia morphology pipeline combining semi-automated image processing and dimensionality reduction. It uses free software (ImageJ and R) and can be applied to a wide variety of experimental paradigms. We anticipate it will enable others to more easily take advantage of the powerful insights microglial morphology analysis provides.

Sudden cessation of fluoxetine before alcohol drinking reinstatement alters microglial morphology and TLR4/inflammatory neuroadaptation in the rat brain

Preclinical studies on the effects of abrupt cessation of selective serotonin reuptake inhibitors (SSRIs), a medication often prescribed in alcohol use disorder (AUD) patients with depression, results in alcohol consumption escalation after resuming drinking. However, a potential neuroinflammatory component on this escalation remains unexplored despite the immunomodulatory role of serotonin. Here, we utilized a rat model of 14-daily administration of the SSRI fluoxetine (10 mg/kg/day) along alcohol self-administration deprivation to study the effects of fluoxetine cessation on neuroinflammation after resuming alcohol drinking. Microglial morphology and inflammatory gene expression were analyzed in prelimbic cortex, striatum, basolateral amygdala and dorsal hippocampus. Results indicated that alcohol drinking reinstatement increased microglial IBA1 immunoreactivity and altered morphometric features of activated microglia (fractal dimension, lacunarity, density, roughness, and cell area, perimeter and circularity). Despite alcohol reinstatement, fluoxetine cessation modified microglial morphology in a brain region-specific manner, resulting in hyper-ramified (spatial complexity of branching), reactive (lower heterogeneity and circularity)-like microglia. We also found that microglial cell area correlated with changes in mRNA expression of chemokines (Cx3cl1/fractalkine, Cxcl12/SDF1α, Ccl2/MCP1), cytokines (IL1β, IL6, IL10) and the innate immune toll-like receptor 4 (TLR4) in dorsal hippocampus. Specifically, TLR4 correlated with microglial spatial complexity assessed by fractal dimension in striatum, suggesting a role in process branching. These findings suggest that alcohol drinking reinstatement after fluoxetine treatment cessation disturbs microglial morphology and reactive phenotype associated with a TLR4/inflammatory response to alcohol in a brain region-specific manner, facts that might contribute to alcohol-induced damage through the promotion of escalation of alcohol drinking behavior.

Sudden Cessation of Fluoxetine Before Alcohol Drinking Reinstatement Alters Microglial Morphology and TLR4/inflammatory Neuroadaptation in the Rat Brain

Preclinical studies on the effects of abrupt cessation of selective serotonin reuptake inhibitors (SSRIs), a medication often prescribed in alcohol use disorder (AUD) patients with depression, results in alcohol consumption escalation after resuming drinking. However, a potential neuroinflammatory component on this escalation remains unexplored despite the immunomodulatory role of serotonin. Here, we utilized a rat model of 14-daily administration of the SSRI fluoxetine (10 mg/kg/day) along alcohol self-administration deprivation to study the effects of fluoxetine cessation on neuroinflammation after resuming alcohol drinking. Microglial morphology and inflammatory gene expression were analyzed in prelimbic cortex, striatum, basolateral amygdala and dorsal hippocampus. Results indicated that alcohol drinking reinstatement increased microglial IBA1 immunoreactivity and altered morphometric features of activated microglia (fractal dimension, lacunarity, density, roughness, and cell area, perimeter and circularity). Despite alcohol reinstatement, fluoxetine cessation modified microglial morphology in a brain region-specific manner, resulting in hyper-ramified (spatial complexity of branching), reactive (lower heterogeneity and circularity)-like microglia. We also found that microglial cell area correlated with changes in mRNA expression of chemokines (Cx3cl1/fractalkine, Cxcl12/SDF1α, Ccl2/MCP1), cytokines (IL1β, IL6, IL10) and the innate immune toll-like receptor 4 (TLR4) in dorsal hippocampus. Specifically, TLR4 correlated with microglial spatial complexity assessed by fractal dimension in striatum, suggesting a role in process branching. These findings suggest that alcohol drinking reinstatement after fluoxetine treatment cessation disturbs microglial morphology and reactive phenotype associated with a TLR4/inflammatory response to alcohol in a brain region-specific manner, facts that might contribute to alcohol-induced damage through the promotion of escalation of alcohol drinking behavior.

Wasl is crucial to maintain microglial core activities during glioblastoma initiation stages

SummaryMicroglia actively promote the growth of high-grade gliomas. Within the glioma microenvironment an activated (amoeboid) microglial morphology has been observed, however the underlying causes and the related impact on microglia functions and their tumour promoting activities is unclear. Using the advantages of the larval zebrafish model, we demonstrate that pre-neoplastic glioma cells have an immediate impact on microglial morphology and functions. Overexpression of human HRasV12 in proliferating domains of the larval brain induces an amoeboid morphology of microglia, increases microglial numbers and decreases their motility and phagocytic activity. RNA sequencing analysis revealed lower expression levels of the actin nucleation promoting factor wasla in microglia. Importantly, a microglia specific rescue of wasla expression restores microglial morphology and functions. This results in increased phagocytosis of pre-neoplastic cells and slows down tumour progression. In conclusion, we identified a mechanism that de-activates core microglial functions within the emerging glioma microenvironment.

Abi3 regulates microglial ramification and dynamic tissue surveillance in vivo.

A rare coding variant of Abelson-interactor gene family member 3 (Abi3) is associated with increased risk of late-onset Alzheimer's Disease (AD). Although Abi3 is recognised as a core microglial gene, its role in microglia is largely unknown. Here we demonstrate that Abi3 is crucial for normal microglial morphology, distribution, and homeostatic tissue surveillance activity in vivo.

An Open-Source Pipeline for Analyzing Changes In Microglial Morphology

ABSTRACTChanges in microglial morphology are powerful indicators of the inflammatory state of the brain. Here we provide an open-source microglia morphology analysis pipeline that first cleans and registers images of microglia, before extracting 62 parameters describing microglial morphology. It then compares control and “inflammation” training data and uses dimensionality reduction to generate a single metric of morphological change (an ‘inflammation index’). This index can then be calculated for test data to assess inflammation, as we demonstrate by investigating the effect of short-term high fat diet consumption in heterozygous Cx3CR1-GFP mice, finding no significant effects of diet. Our pipeline represents the first open-source microglia morphology pipeline combining semi-automated image processing and dimensionality reduction. It uses free software (ImageJ and R) and can be applied to a wide variety of experimental paradigms. We anticipate it will enable others to more easily take advantage of the powerful insights microglial morphology analysis provides.