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. 2022 May 15:374:109567.
doi: 10.1016/j.jneumeth.2022.109567. Epub 2022 Mar 17.

A method for the selective depletion of microglia in the dorsal hippocampus in the juvenile rat brain

Mary Beth Hall  1 , Nicola M Habash  2 , Nicole A Haas  3 , Jaclyn M Schwarz  4
Affiliations

A method for the selective depletion of microglia in the dorsal hippocampus in the juvenile rat brain

Mary Beth Hall et al. J Neurosci Methods. .

Abstract

Background: To understand the role of microglia in brain function and development, methods have emerged to deplete microglia throughout the brain. Liposome-encapsulated clodronate (LEC) can be infused into the brain to deplete microglia in a brain-region and time-specific manner.

New method: This study validates methodology to deplete microglia in the rat dorsal hippocampus (dHP) during a specific period of juvenile development. Stereotaxic surgery was performed to infuse LEC at postnatal day (P) 16 or 19 into dHP. Rat brains were harvested at various ages to determine specificity of infusion and duration of depletion.

Results: P19 infusion of LEC into dHP with a 27G syringe depleted microglia in dHP subregions CA1, dentate gyrus (DG), and CA3 from P24-P30. There was also evidence of depletion in parietal cortex above the infusion site. P16 infusion of LEC with a 32 G syringe depleted microglia only in dHP subregions CA1 and DG from P21-P40.

Comparison with existing method(s): Previous methods have infused LEC intra-hippocampally in adult rats or intra-cerebroventricularly in neonatal rats. This study is the first to publish methodology to deplete microglia in a brain-region specific manner during juvenile rat development.

Conclusions: The timing of LEC infusion during the juvenile period can be adjusted to achieve maximal microglia depletion by a specific postnatal day. A 27G needle results in LEC backflow during the infusion, but also allows LEC to reach all subregions of dHP. Infusion with a 32 G needle prevents backflow during infusion, but results in a more local spread of LEC within dHP.

Keywords: Dorsal hippocampus; Hippocampal infusion; Liposome-encapsulated clodronate; Microglia depletion; Stereotaxic surgery.

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Figures

Figure 1.
Figure 1.
Timeline of methodology. Red arrows indicate intra-dorsal hippocampal infusion of liposome-encapsulated clodronate (LEC) or control liposomes (CL) filled with PBS at postnatal day (P)19 (Experiment 1) or P16 (Experiment 2). Brain tissue was harvested for immunohistochemical analysis of microglia depletion-repopulation on P21, P24, P27, P30, and P40.
Figure 2.
Figure 2.
Target for infusion. Rats received bilateral infusions of control liposomes (CL) or liposome-encapsulated clodronate (LEC) in dorsal hippocampus (dHP) on postnatal day (P)19 (A-C) or P16 (D-F). Arrows indicate site of infusion. A & D. P21 brain atlas (Khazipov, et al., P21Atlas of the Developing Rat Brain in Stereotaxic Coordinates) showing coordinates of (A) P19 infusion at −3.60mm AP, ±2.50mm ML, −2.50mm DV and of (D) P16 infusion at −3.00mm AP, ±2.50mm ML, −2.50mm DV. B & C. Representative images of dHP at P21 of a rat treated with CL on P19 at 10× objective (B) and 5× objective (C). E & F. Representative images of dHP at P21 of a rat treated with CL on P16 at 10× objective (E) and 5× objective (F). AP: anterior-posterior; ML: medial-lateral; DV: dorsal-ventral. Scale bars are 100μm.
Figure 3.
Figure 3.
P19 Timeline (Experiment 1). Percent area of Iba-1 staining within dorsal hippocampus (dHP) subregions CA1, CA3, and dentate gyrus (DG). A. Percent area of CA1. B. Percent area of DG. C. Percent area of CA3. D. Percent area of Par. * pairwise comparison; significantly different than both CNT and CL groups (p < .05 ). # significantly different than CL group (p < .05). a significantly different than CNT group (p < .05). CNT: undisturbed home-cage control group; CL: control liposome group; LEC: liposome-encapsulated clodronate group.
Figure 4.
Figure 4.
P19 Timeline (Experiment 1). Representative images (10× objective) of microglia depletion and repopulation near site of infusion in dorsal hippocampus (dHP) CA1 and of microglia depletion in parietal cortex (Par). Rats were infused with liposome-encapsulated clodronate (LEC) or control liposomes (CL) on postnatal day (P)19. Brains from LEC-treated, CL-treated, and undisturbed home-cage control (CNT) rats were harvested at 2 days (P21), 5 days (P24), 8 days (P27), 11 days (P30) or 21 days (P40) post-infusion and tissue was stained for Iba-1 to detect microglia. Scale bars are 100μm.
Figure 5.
Figure 5.
P16 Timeline (Experiment 2). Percent area of Iba-1 staining within dorsal hippocampus (dHP) subregions CA1, CA3, and dentate gyrus (DG). A. Percent area of CA1. B. Percent area of DG. C. Percent area of CA3. D. Percent area of Par. * pairwise comparison; significantly different than both sham and CL groups (p < .05 ). # significantly different than CL group (p < .05). a significantly different than sham group (p < .05). Sham: surgical control group; CL: control liposome group; LEC: liposome-encapsulated clodronate group.
Figure 6.
Figure 6.
P16 Timeline (Experiment 2). Number of microglia cells divided by area of the region of interest for dorsal hippocampus (dHP) subregions CA1 and dentate gyrus (DG). A. Number of microglia divided by area of CA1. B. Number of microglia divided by area of DG. * pairwise comparison; significantly different than both sham and CL groups (p < .05 ). # significantly different than CL group (p < .05). a significantly different than sham group (p < .05). Sham: surgical control group; CL: control liposome group; LEC: liposome-encapsulated clodronate group.
Figure 7.
Figure 7.
P16 Timeline (Experiment 2). Representative images (10× objective) of microglia depletion and repopulation near site of infusion in dorsal hippocampus (dHP) dentate gyrus and of microglia density in parietal cortex (Par). Rats. Rats were infused with liposome-encapsulated clodronate (LEC), control liposomes (CL), or nothing (sham) on postnatal day (P)16. Brains were harvested at 5 days (P21), 8 days (P24), 11 days (P27), 14 days (P30) or 24 days (P40) post-infusion and tissue was stained for Iba-1 to detect microglia. Scale bars are 100μm.
Figure 8.
Figure 8.
P16 Timeline (Experiment 2). Percent area of Iba-1 staining within medial prefrontal cortex (mPFC) and ventral hippocampus (vHP) subregions CA1, CA3, and dentate gyrus (DG). A. Percent area of mPFC. B. Percent area of vHP CA1. C. Percent area of vHP CA3. D. Percent area of vHP DG. Sham: surgical control group; CL: control liposome group; LEC: liposome-encapsulated clodronate group.
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