Limits...
Functional differences between microglia and monocytes after ischemic stroke.

Ritzel RM, Patel AR, Grenier JM, Crapser J, Verma R, Jellison ER, McCullough LD - J Neuroinflammation (2015)

Bottom Line: The lack of discriminating markers between these two myeloid populations has led many studies to generate conclusions based on the grouping of these two populations.We found that at 72 h after a 90-min middle cerebral artery occlusion (MCAO), microglia populations decrease whereas monocytes significantly increase in the stroke brain compared to sham.In summary, the resident microglia population is vulnerable to the effects of severe ischemia, show compromised cell cycle progression, and adopt a largely pro-inflammatory phenotype after stroke.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT, 06030, USA. rritzel@uchc.edu.

ABSTRACT

Background: The brain's initial innate response to stroke is primarily mediated by microglia, the resident macrophage of the CNS. However, as early as 4 h after stroke, the blood-brain barrier is compromised and monocyte infiltration occurs. The lack of discriminating markers between these two myeloid populations has led many studies to generate conclusions based on the grouping of these two populations. A growing body of evidence now supports the distinct roles played by microglia and monocytes in many disease models.

Methods: Using a flow cytometry approach, combined with ex-vivo functional assays, we were able to distinguish microglia from monocytes using the relative expression of CD45 and assess the function of each cell type following stroke over the course of 7 days.

Results: We found that at 72 h after a 90-min middle cerebral artery occlusion (MCAO), microglia populations decrease whereas monocytes significantly increase in the stroke brain compared to sham. After stroke, BRDU incorporation into monocytes in the bone marrow increased. After recruitment to the ischemic brain, these monocytes accounted for nearly all BRDU-positive macrophages. Inflammatory activity peaked at 72 h. Microglia produced relatively higher reactive oxygen species and TNF, whereas monocytes were the predominant IL-1β producer. Although microglia showed enhanced phagocytic activity after stroke, monocytes had significantly higher phagocytic capacity at 72 h. Interestingly, we found a positive correlation between TNF expression levels and phagocytic activity of microglia after stroke.

Conclusions: In summary, the resident microglia population is vulnerable to the effects of severe ischemia, show compromised cell cycle progression, and adopt a largely pro-inflammatory phenotype after stroke. Infiltrating monocytes are primarily involved with early debris clearance of dying cells. These findings suggest that the early wave of infiltrating monocytes may be beneficial to stroke repair and future therapies aimed at mitigating microglia cell death may prove more effective than attempting to elicit targeted anti-inflammatory responses from damaged cells.

No MeSH data available.


Related in: MedlinePlus

Number of microglia and monocytes in the ischemic hemisphere in the early period after stroke. A representative dot plot depicts the gating strategy used to identify both brain-resident microglia and Ly6C+ monocytes at 72 h after 90-min MCAO (a, b). Absolute cell counts of microglia (c) and monocytes (d) were quantified at 0, 24, and 72 h after stroke. For all experiments, N = 9/group. Error bars show mean SEM. Abbreviation: SEM standard error of the mean. *p < 0.05; ***p < 0.001
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4465481&req=5

Fig1: Number of microglia and monocytes in the ischemic hemisphere in the early period after stroke. A representative dot plot depicts the gating strategy used to identify both brain-resident microglia and Ly6C+ monocytes at 72 h after 90-min MCAO (a, b). Absolute cell counts of microglia (c) and monocytes (d) were quantified at 0, 24, and 72 h after stroke. For all experiments, N = 9/group. Error bars show mean SEM. Abbreviation: SEM standard error of the mean. *p < 0.05; ***p < 0.001

Mentions: We confirmed the ability to reliably distinguish CD45int microglia from CD45hi monocyte populations in the ischemic brain by generating GFP bone marrow chimeras, in which all bone marrow-derived cells were GFP-positive (Additional file 1: Figure S1A). We demonstrated that the two populations did not significantly overlap after stroke, validating this approach. Absolute leukocyte counts were obtained by flow cytometry at 24 and 72 h after MCAO in non-irradiated, intact wild type mice using the gating strategy shown in Fig. 1a, b. A significant reduction in the number of microglia (CD45intCD11b+Ly6C−) after stroke was found after 24 h (p < 0.05; Fig. 1c). Conversely, we found a dramatic increase in monocyte (CD45hiCD11b+Ly6C+Ly6G−) counts in the stroke hemisphere compared to sham brain (Fig. 1d). At 72 h, microglia expressed increased levels of Ki67, a marker of actively cycling cells (p = 0.017; Fig. 2a, b). DNA synthesis, an indicator of cell proliferation, was then measured by BRDU incorporation. Following repeated BRDU injections starting at 12 h, microglia showed little BRDU incorporation by 72 h, whereas ~90 % of monocytes in the ischemic brain were BRDU-positive (Fig. 2c, d). Stroke is known to stimulate bone marrow production of myeloid cells that are subsequently recruited to the brain [17]. We found a significant increase in the percentage of BRDU+ monocytes in the bone marrow following stroke (p = 0.016; Fig. 2e, f). These data suggest that after 90-min tMCAO, there is a significant loss of resident microglia, impaired cell cycle progression, and an increased number of newly produced, bone marrow-derived monocytes.Fig. 1


Functional differences between microglia and monocytes after ischemic stroke.

Ritzel RM, Patel AR, Grenier JM, Crapser J, Verma R, Jellison ER, McCullough LD - J Neuroinflammation (2015)

Number of microglia and monocytes in the ischemic hemisphere in the early period after stroke. A representative dot plot depicts the gating strategy used to identify both brain-resident microglia and Ly6C+ monocytes at 72 h after 90-min MCAO (a, b). Absolute cell counts of microglia (c) and monocytes (d) were quantified at 0, 24, and 72 h after stroke. For all experiments, N = 9/group. Error bars show mean SEM. Abbreviation: SEM standard error of the mean. *p < 0.05; ***p < 0.001
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4465481&req=5

Fig1: Number of microglia and monocytes in the ischemic hemisphere in the early period after stroke. A representative dot plot depicts the gating strategy used to identify both brain-resident microglia and Ly6C+ monocytes at 72 h after 90-min MCAO (a, b). Absolute cell counts of microglia (c) and monocytes (d) were quantified at 0, 24, and 72 h after stroke. For all experiments, N = 9/group. Error bars show mean SEM. Abbreviation: SEM standard error of the mean. *p < 0.05; ***p < 0.001
Mentions: We confirmed the ability to reliably distinguish CD45int microglia from CD45hi monocyte populations in the ischemic brain by generating GFP bone marrow chimeras, in which all bone marrow-derived cells were GFP-positive (Additional file 1: Figure S1A). We demonstrated that the two populations did not significantly overlap after stroke, validating this approach. Absolute leukocyte counts were obtained by flow cytometry at 24 and 72 h after MCAO in non-irradiated, intact wild type mice using the gating strategy shown in Fig. 1a, b. A significant reduction in the number of microglia (CD45intCD11b+Ly6C−) after stroke was found after 24 h (p < 0.05; Fig. 1c). Conversely, we found a dramatic increase in monocyte (CD45hiCD11b+Ly6C+Ly6G−) counts in the stroke hemisphere compared to sham brain (Fig. 1d). At 72 h, microglia expressed increased levels of Ki67, a marker of actively cycling cells (p = 0.017; Fig. 2a, b). DNA synthesis, an indicator of cell proliferation, was then measured by BRDU incorporation. Following repeated BRDU injections starting at 12 h, microglia showed little BRDU incorporation by 72 h, whereas ~90 % of monocytes in the ischemic brain were BRDU-positive (Fig. 2c, d). Stroke is known to stimulate bone marrow production of myeloid cells that are subsequently recruited to the brain [17]. We found a significant increase in the percentage of BRDU+ monocytes in the bone marrow following stroke (p = 0.016; Fig. 2e, f). These data suggest that after 90-min tMCAO, there is a significant loss of resident microglia, impaired cell cycle progression, and an increased number of newly produced, bone marrow-derived monocytes.Fig. 1

Bottom Line: The lack of discriminating markers between these two myeloid populations has led many studies to generate conclusions based on the grouping of these two populations.We found that at 72 h after a 90-min middle cerebral artery occlusion (MCAO), microglia populations decrease whereas monocytes significantly increase in the stroke brain compared to sham.In summary, the resident microglia population is vulnerable to the effects of severe ischemia, show compromised cell cycle progression, and adopt a largely pro-inflammatory phenotype after stroke.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT, 06030, USA. rritzel@uchc.edu.

ABSTRACT

Background: The brain's initial innate response to stroke is primarily mediated by microglia, the resident macrophage of the CNS. However, as early as 4 h after stroke, the blood-brain barrier is compromised and monocyte infiltration occurs. The lack of discriminating markers between these two myeloid populations has led many studies to generate conclusions based on the grouping of these two populations. A growing body of evidence now supports the distinct roles played by microglia and monocytes in many disease models.

Methods: Using a flow cytometry approach, combined with ex-vivo functional assays, we were able to distinguish microglia from monocytes using the relative expression of CD45 and assess the function of each cell type following stroke over the course of 7 days.

Results: We found that at 72 h after a 90-min middle cerebral artery occlusion (MCAO), microglia populations decrease whereas monocytes significantly increase in the stroke brain compared to sham. After stroke, BRDU incorporation into monocytes in the bone marrow increased. After recruitment to the ischemic brain, these monocytes accounted for nearly all BRDU-positive macrophages. Inflammatory activity peaked at 72 h. Microglia produced relatively higher reactive oxygen species and TNF, whereas monocytes were the predominant IL-1β producer. Although microglia showed enhanced phagocytic activity after stroke, monocytes had significantly higher phagocytic capacity at 72 h. Interestingly, we found a positive correlation between TNF expression levels and phagocytic activity of microglia after stroke.

Conclusions: In summary, the resident microglia population is vulnerable to the effects of severe ischemia, show compromised cell cycle progression, and adopt a largely pro-inflammatory phenotype after stroke. Infiltrating monocytes are primarily involved with early debris clearance of dying cells. These findings suggest that the early wave of infiltrating monocytes may be beneficial to stroke repair and future therapies aimed at mitigating microglia cell death may prove more effective than attempting to elicit targeted anti-inflammatory responses from damaged cells.

No MeSH data available.


Related in: MedlinePlus