The Effect of Sensory Deprivation on the Density of M1 Microglia in the Mouse Barrel Cortex
The Effect of SensoryDeprivation on the Density of M1 Microglia in the Mouse Barrel Cortex Yuhong Wang The Bronx High School of Science
Cells of The Central Nervous SystemNeurons Glia • Processes, transmits • Maintains homeostasis information • Includes: • Astrocytes • Schwann Cells • Microglia
Microglia• Resident macrophage of the Central Nervous System• Constantly surveying its environment• Processes engage in phagocytosis of pathogens and debris• Releases various cytokines to maintain homeostasis in the CNS
The Barrel Cortex• In the present study, we are looking at the presence of microglial cells in the barrel cortex• Barrel Cortex o A region of the somatosensory cortex that processes information from a rodent’s whiskers o Organized topographically; Each whisker is represented by a discrete aggregation of neurons called a barrel Barrel Cortex
The Effect of Sensory Deprivation• Trimming a rodent’s whiskers for the first postnatal month has been shown to result in neuronal remodeling But, it seems microglia are also impacted by sensory deprivation How So?
Previous Studies• Following trimming, there is a significant increase in cell body area and a significant decrease in process length of microglia The fact that sensory deprivation leads to a change in microglial morphology suggests that microglia are changing from a resting state phenotype to an activated phenotype
Activated Microglia• Expresses 2 distinct phenotypes o M1 phenotype, or classically activated microglia, are neurotoxic, as they release reactive oxygen species and proinflammatory cytokines o M2 phenotype, or alternatively activated microglia, are neuroprotective, as they block proinflammatory responses and instead produce high levels of anti-inflammatory cytokines and neurotropic factors.
Problem: How are M1 Microglia Impacted by Sensory Derivation? Due to the previously describedstructural arrangements in the barrelcortex following trimming, we aim todetermine if sensory deprivation willhave an significant impact on thedensity of M1 microglia.
Trimming and Perfusion• Sensory deprivation was achieved in mice by bilaterally trimming whiskers every other day for 31 days, starting from birth. Some mice were allocated to the p60 and p90 whisker regrow group in order to prove that microglial remodeling occurs in the first 30 days of development.• There were untrimmed mice in each group that served as control• Respective mice were perfused on the p31, p61, and p91 days using saline and 4% paraformaldehyde. Brains were post-fixed for 24 hours in 4% paraformaldehyde.
Sectioning and Staining• Brains were sectioned into 50 μm slices using a vibratome• Brain sections were immunohistochemically stained for microglia using primary antibody Iba-1• Sections were double stained with MHC-II antibody in order to detect microglia expressing the M1 phenotype.
Imaging and Analysis• Slices were mounted using 0.01M phosphate buffer saline medium and cover slipped• Slides were imaged using confocal microscopy and image stacks will be analyzed using computer assisted program Neurolucida 8.0 to determine the quantity and distribution of M1 microglia.
Iba-1 Rhodamine Staining Stains for all microglial cells
MHC-II FITC Staining Stains for all microglia expressing the M1 phenotype
FITC, Rhodamine overlap The overlap shows the number of M1 microglia (yellow) and the number that is not expressing that phenotype (remains red)
Results Number of Cells expressing the M1 phenotype in the sensory deprived and control 30Number of Cells per 100m3 25 20 15 10 5 0 IBA + MHCII+ IBA + MHCII+ Sensory Deprived Control Cell Type
The percentage of microglia stained MHC-II (M1 phenotype) 1.2 1.0 Percentage MHCII+ 0.8 0.6 0.4 0.2 0.0 Sensory Deprived ControlSensory Deprivation does not seem to have aneffect on M1 expression of microglial cells
Conclusions• Most microglia in the mouse barrel cortex expresses the M1 phenotype as opposed to M2• Sensory deprivation did not have a significant impact on the density of cells expressing M1 phenotype, therefore our hypothesis was proven wrong
Future Directions• M2 microglia plays just as an important role as M1 microglia does in neuroplasticity. Future experiments should characterize the effect of sensory deprivation on the distribution of M2 microglial cells.
Acknowledgements• This research was supported by the City University of New York at Queens College• Thanks to Dr.Joshua Brumberg, Racheli Wercberger and Zissy Turner for their contribution and guidance.