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Stereological estimation of total microglia number in mouse hippocampus
J M Long1, A N Kalehua, N J Muth
1Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA. LongJ@vax.grc.nia.nih.gov
Abstract:
Microglia are brain cells of considerable interest because of their role in CNS inflammatory responses and strong association with neuritic plaques in Alzheimer's disease (AD). In the present study, immunocytochemistry was combined with unbiased stereology to estimate the mean total number of microglia in dentate gyrus and CA1 regions of the mouse hippocampus. Systematic-uniform-random (SUR) sections were cut through the hippocampal formation of male C57BL/6J mice (n = 7, 4-5 months) and immunostained with Mac-1, an antibody to the complement subunit 3 receptor (CR3). The total number of Mac-1 immunopositive cells was determined using the optical fractionator method. The mean total number of microglia in the mouse dentate gyrus was estimated to be 20,300 (CV = 0.21) with a mean coefficient of error (CE) = 0.09. The mean total number of microglia in the mouse CA1 was estimated to be 43,200 (CV = 0.24) with a CE = 0.11. Comparison of total number estimates, derived from fraction- or volume-based methods, supported stereological theory regarding the equivalence of the two techniques. The time required to determine total microglia number in both hippocampal sub-regions was approximately 6 h per mouse from stained sections. The combination of immunocytochemistry and stereology provides a reliable means to assess microglia number that can easily be adopted for studies of transgenic and lesion-based models of aging and neurodegenerative diseases.
Insights
This study quantifies microglia, or immune cells in the brain, within specific mouse hippocampus regions using advanced imaging and stereology. This method offers a reliable way to count these crucial cells for neurodegenerative disease research.
Area of Science:
- Neuroscience
- Immunology
Background:
- Microglia are key brain immune cells involved in CNS inflammation.
- Microglia are associated with Alzheimer's disease (AD) pathology, specifically neuritic plaques.
Purpose of the Study:
- To estimate the total number of microglia in the dentate gyrus and CA1 regions of the mouse hippocampus.
- To validate a combined immunocytochemistry and stereology method for microglia quantification.
Main Methods:
- Used immunocytochemistry with Mac-1 antibody to label microglia.
- Employed unbiased stereology, specifically the optical fractionator method, on systematic-uniform-random sections.
- Analyzed hippocampus from male C57BL/6J mice (n=7, 4-5 months).
Main Results:
- Estimated mean microglia number in the dentate gyrus: 20,300 (CV=0.21).
- Estimated mean microglia number in the CA1 region: 43,200 (CV=0.24).
- Quantification took approximately 6 hours per mouse for both regions.
Conclusions:
- The combination of immunocytochemistry and stereology is a reliable method for assessing microglia numbers.
- This technique is adaptable for research on aging and neurodegenerative diseases, including transgenic and lesion models.
- Confirms stereological theory on the equivalence of fraction- and volume-based estimation methods.