Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is a very rare inherited neurodegenerative and demyelinating disease. Due to its symptoms of cognitive impairment, bradykinesia, tremors, and others, it is usually misdiagnosed as multiple sclerosis, Parkinson’s disease, or frontotemporal dementia. It is caused by autosomal dominant pathogenic variants in CSF1R, which is expressed by microglia. While the disease mechanisms are not fully known, damage to myelin, microglia, and astrocytes has been observed in brain lesions. Reactive astrocytes upregulate a marker called GFAP, which is not typically expressed by most cortical astrocytes. To study the link between dysfunctional CSF1R and brain pathology, we used a CSF1R haploinsufficiency mouse model, where one of the CSF1R alleles is deleted. To mimic the phenotype of ALSP, demyelination was triggered using a cuprizone-supplemented diet over a period of four weeks, followed by a regular diet for four more weeks to allow remyelination. To retrieve and prepare the brain slices, transcardiac perfusion is performed to euthanize the mice, followed by cryosectioning and immunofluorescence staining for cell-specific markers. The somatosensory cortex of these samples was then imaged using an epifluorescence microscope, and the 500 x 500 micron regions were divided into 100 micron layers relative to the brain surface. Next, the cell bodies of astrocytes (labeled with GFAP) were manually counted. The somatosensory cortex was explicitly chosen due to its high concentration of myelinated axons in the adult mouse brain. An example of the processed images can be seen on the poster, with red marking astrocytes and blue (DAPI) marking all nuclei. Astrocytes were counted when the colocalization of red and blue was evident in the images. As seen with the samples of images in the results section of the poster, there is a noticeable increase in the number of GFAP+ astrocytes of CSF1R (+/-) mice four weeks after cuprizone treatment and two weeks into recovery relative to the baseline mice before cuprizone was administered – these are “reactive” astrocytes. In Graph 1, the number of GFAP+ astrocytes quantified at each time-point is divided into 100 micron layers. In CSF1R haploinsufficient mice, there are more reactive astrocytes after demyelination relative to the low levels at baseline, following a similar pattern of upregulation after cuprizone and persistence into recovery as WT mice. The overall upregulation in both CSF1R haploinsufficient and WT mice after cuprizone and recovery periods is further seen in Graph 2. In Graph 1, there are more reactive astrocytes deeper into the somatosensory cortex after four and six weeks, in the regions from 200-500 microns, in both WT and CSF1R heterozygous mice. Notably, more myelin in the deep cortex leads to more debris after cuprizone, which may explain why astrocytes react in this region. However, it does not fully explain why this reactivity persists through recovery. In addition, we found that more oligodendrocytes (the cells that make myelin) are present at two weeks recovery in the Csf1r haploinsufficiency mouse compared to WT – so it is unexpected that astrocyte response would be similar to wild-type. In future directions, we will examine how microglia change function in this model and how these cell types change after five weeks of recovery. The conclusions from this research may be used to pave the way for other research projects looking at the involvement of other glial cells in ALSP.