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CCL2 upregulation triggers hypoxic preconditioning-induced protection from stroke.

Stowe AM, Wacker BK, Cravens PD, Perfater JL, Li MK, Hu R, Freie AB, Stüve O, Gidday JM - J Neuroinflammation (2012)

Bottom Line: The levels of circulating monocytes (p < 0.0001), T lymphocytes (p < 0.0001), and granulocytes were decreased 12 h after HPC, and those of B lymphocytes were increased (p < 0.0001), but the magnitude of these respective changes did not differ between wild-type and CCL2- mice.While HPC reduced infarct volumes by 27% (p < 0.01) in wild-type mice, CCL2- mice subjected to tMCAo were not protected by HPC.The early expression of CCL2 in neurons, the delayed expression of CCL2 in cerebral endothelial cells, and CCL2-mediated actions on circulating CCR2+ monocytes, appear to be required to establish ischemic tolerance to focal stroke in response to HPC, and thus represent a novel role for this chemokine in endogenous neurovascular protection.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurological Surgery, Washington University School of Medicine, 660 S, Euclid Ave,, Box 8057, St, Louis, MO 63110, USA.

ABSTRACT

Background: A brief exposure to systemic hypoxia (i.e., hypoxic preconditioning; HPC) prior to transient middle cerebral artery occlusion (tMCAo) reduces infarct volume, blood-brain barrier disruption, and leukocyte migration. CCL2 (MCP-1), typically regarded as a leukocyte-derived pro-inflammatory chemokine, can also be directly upregulated by hypoxia-induced transcription. We hypothesized that such a hypoxia-induced upregulation of CCL2 is required for HPC-induced ischemic tolerance.

Methods: Adult male SW/ND4, CCL2-, and wild-type mice were used in these studies. Cortical CCL2/CCR2 message, protein, and cell-type specific immunoreactivity were determined following HPC (4 h, 8% O2) or room air control (21% O2) from 6 h through 2 weeks following HPC. Circulating leukocyte subsets were determined by multi-parameter flow cytometry in naïve mice and 12 h after HPC. CCL2- and wild-type mice were exposed to HPC 2 days prior to tMCAo, with immunoneutralization of CCL2 during HPC achieved by a monoclonal CCL2 antibody.

Results: Cortical CCL2 mRNA and protein expression peaked at 12 h after HPC (both p < 0.01), predominantly in cortical neurons, and returned to baseline by 2 days. A delayed cerebral endothelial CCL2 message expression (p < 0.05) occurred 2 days after HPC. The levels of circulating monocytes (p < 0.0001), T lymphocytes (p < 0.0001), and granulocytes were decreased 12 h after HPC, and those of B lymphocytes were increased (p < 0.0001), but the magnitude of these respective changes did not differ between wild-type and CCL2- mice. HPC did decrease the number of circulating CCR2+ monocytes (p < 0.0001) in a CCL2-dependent manner, but immunohistochemical analyses at this 12 h timepoint indicated that this leukocyte subpopulation did not move into the CNS. While HPC reduced infarct volumes by 27% (p < 0.01) in wild-type mice, CCL2- mice subjected to tMCAo were not protected by HPC. Moreover, administration of a CCL2 immunoneutralizing antibody prior to HPC completely blocked (p < 0.0001 vs. HPC-treated mice) the development of ischemic tolerance.

Conclusions: The early expression of CCL2 in neurons, the delayed expression of CCL2 in cerebral endothelial cells, and CCL2-mediated actions on circulating CCR2+ monocytes, appear to be required to establish ischemic tolerance to focal stroke in response to HPC, and thus represent a novel role for this chemokine in endogenous neurovascular protection.

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Genetic and pharmacologic evidence for CCL2 involvement in HPC-induced stroke tolerance. (A and B) HPC-induced ischemic tolerance was lacking in CCL2- animals with HPC completed 2 days prior to (A) a 45-min tMCAo (n = 6) or (B) a 35-min tMCAo (n = 9) tMCAo, versus control CCL2- mice with tMCAo but no prior HPC (n = 6 and n = 10, respectively). (C) HPC promoted ischemic tolerance to a 35-min tMCAo in CX3CR1GFP/+ wild-type mice. (D) In WT mice, HPC 2 days prior to tMCAo (n = 12) reduced infarct volume resulting from 45-min of tMCAo versus controls with tMCAo but no prior HPC (n = 15). Immunoneutralization of CCL2 prior to HPC reversed this protection relative to untreated control mice with HPC (n = 9), and relative to control mice given the IgG-isotype control antibody prior to HPC (n = 11). Individual values (black-filled circles), mean (black horizontal bars), 75% confidence interval (gray box), and values (open circles) that fell outside of the 95% confidence interval (whiskers) are given. *p < 0.05, **p < 0.0001 vs. HPC.
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Figure 4: Genetic and pharmacologic evidence for CCL2 involvement in HPC-induced stroke tolerance. (A and B) HPC-induced ischemic tolerance was lacking in CCL2- animals with HPC completed 2 days prior to (A) a 45-min tMCAo (n = 6) or (B) a 35-min tMCAo (n = 9) tMCAo, versus control CCL2- mice with tMCAo but no prior HPC (n = 6 and n = 10, respectively). (C) HPC promoted ischemic tolerance to a 35-min tMCAo in CX3CR1GFP/+ wild-type mice. (D) In WT mice, HPC 2 days prior to tMCAo (n = 12) reduced infarct volume resulting from 45-min of tMCAo versus controls with tMCAo but no prior HPC (n = 15). Immunoneutralization of CCL2 prior to HPC reversed this protection relative to untreated control mice with HPC (n = 9), and relative to control mice given the IgG-isotype control antibody prior to HPC (n = 11). Individual values (black-filled circles), mean (black horizontal bars), 75% confidence interval (gray box), and values (open circles) that fell outside of the 95% confidence interval (whiskers) are given. *p < 0.05, **p < 0.0001 vs. HPC.

Mentions: Because the neuronal upregulation of HIF-1 is neuroprotective [40], and to test our hypothesis that HPC requires the HIF-mediated upregulation of neuronal CCL2 to induce the protective phenotype, we examined the ability of HPC to induce ischemic tolerance in CCL2- mice following transient focal stroke [4]. Figure 4A shows 24 h post-stroke infarct volumes for non-preconditioned CCL2- mice, and HPC-treated CCL2- mice in response to a 45-min tMCAo. In the absence of CCL2, HPC exhibited no protective effect with respect to infarct volume (114 ± 16 mm3; n = 5) compared to non-preconditioned controls (114 ± 8 mm3; n = 6). These infarct volumes, however, are larger than those reported in prior studies of CCL2-/- mice following a 30-min tMCAo [51], and following permanent MCAo [17]. While infarct volumes in the latter study were similar to our previous experience with permanent MCAo [5], the smaller infarct volumes following tMCAo that others reported may be due to the use of different infarct quantification methods, as well as the shorter 30-min duration of ischemia. Schilling and colleagues [51] stained intermittent coronal sections with Toluidine Blue, and visually determined the infarct at the time of sectioning - which may have resulted in the exclusion of the most rostral and caudal portions of the infarct that are included using our TTC quantification protocol. We also examined another cohort of mice subjected to a 35-min tMCAo to ensure that the extent of damage caused by a 45-min occlusion did not mask or overwhelm any potential HPC-induced protection (Figure 4B). Again, HPC-treated CCL2- mice had infarct volumes (87 ± 10 mm3; n = 10) nearly identical to non-preconditioned CCL2-s (91 ± 16 mm3; n = 9). Taken together, the results from these two cohorts confirm our hypothesis that CCL2 is required for HPC-induced tolerance to transient focal stroke.


CCL2 upregulation triggers hypoxic preconditioning-induced protection from stroke.

Stowe AM, Wacker BK, Cravens PD, Perfater JL, Li MK, Hu R, Freie AB, Stüve O, Gidday JM - J Neuroinflammation (2012)

Genetic and pharmacologic evidence for CCL2 involvement in HPC-induced stroke tolerance. (A and B) HPC-induced ischemic tolerance was lacking in CCL2- animals with HPC completed 2 days prior to (A) a 45-min tMCAo (n = 6) or (B) a 35-min tMCAo (n = 9) tMCAo, versus control CCL2- mice with tMCAo but no prior HPC (n = 6 and n = 10, respectively). (C) HPC promoted ischemic tolerance to a 35-min tMCAo in CX3CR1GFP/+ wild-type mice. (D) In WT mice, HPC 2 days prior to tMCAo (n = 12) reduced infarct volume resulting from 45-min of tMCAo versus controls with tMCAo but no prior HPC (n = 15). Immunoneutralization of CCL2 prior to HPC reversed this protection relative to untreated control mice with HPC (n = 9), and relative to control mice given the IgG-isotype control antibody prior to HPC (n = 11). Individual values (black-filled circles), mean (black horizontal bars), 75% confidence interval (gray box), and values (open circles) that fell outside of the 95% confidence interval (whiskers) are given. *p < 0.05, **p < 0.0001 vs. HPC.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 4: Genetic and pharmacologic evidence for CCL2 involvement in HPC-induced stroke tolerance. (A and B) HPC-induced ischemic tolerance was lacking in CCL2- animals with HPC completed 2 days prior to (A) a 45-min tMCAo (n = 6) or (B) a 35-min tMCAo (n = 9) tMCAo, versus control CCL2- mice with tMCAo but no prior HPC (n = 6 and n = 10, respectively). (C) HPC promoted ischemic tolerance to a 35-min tMCAo in CX3CR1GFP/+ wild-type mice. (D) In WT mice, HPC 2 days prior to tMCAo (n = 12) reduced infarct volume resulting from 45-min of tMCAo versus controls with tMCAo but no prior HPC (n = 15). Immunoneutralization of CCL2 prior to HPC reversed this protection relative to untreated control mice with HPC (n = 9), and relative to control mice given the IgG-isotype control antibody prior to HPC (n = 11). Individual values (black-filled circles), mean (black horizontal bars), 75% confidence interval (gray box), and values (open circles) that fell outside of the 95% confidence interval (whiskers) are given. *p < 0.05, **p < 0.0001 vs. HPC.
Mentions: Because the neuronal upregulation of HIF-1 is neuroprotective [40], and to test our hypothesis that HPC requires the HIF-mediated upregulation of neuronal CCL2 to induce the protective phenotype, we examined the ability of HPC to induce ischemic tolerance in CCL2- mice following transient focal stroke [4]. Figure 4A shows 24 h post-stroke infarct volumes for non-preconditioned CCL2- mice, and HPC-treated CCL2- mice in response to a 45-min tMCAo. In the absence of CCL2, HPC exhibited no protective effect with respect to infarct volume (114 ± 16 mm3; n = 5) compared to non-preconditioned controls (114 ± 8 mm3; n = 6). These infarct volumes, however, are larger than those reported in prior studies of CCL2-/- mice following a 30-min tMCAo [51], and following permanent MCAo [17]. While infarct volumes in the latter study were similar to our previous experience with permanent MCAo [5], the smaller infarct volumes following tMCAo that others reported may be due to the use of different infarct quantification methods, as well as the shorter 30-min duration of ischemia. Schilling and colleagues [51] stained intermittent coronal sections with Toluidine Blue, and visually determined the infarct at the time of sectioning - which may have resulted in the exclusion of the most rostral and caudal portions of the infarct that are included using our TTC quantification protocol. We also examined another cohort of mice subjected to a 35-min tMCAo to ensure that the extent of damage caused by a 45-min occlusion did not mask or overwhelm any potential HPC-induced protection (Figure 4B). Again, HPC-treated CCL2- mice had infarct volumes (87 ± 10 mm3; n = 10) nearly identical to non-preconditioned CCL2-s (91 ± 16 mm3; n = 9). Taken together, the results from these two cohorts confirm our hypothesis that CCL2 is required for HPC-induced tolerance to transient focal stroke.

Bottom Line: The levels of circulating monocytes (p < 0.0001), T lymphocytes (p < 0.0001), and granulocytes were decreased 12 h after HPC, and those of B lymphocytes were increased (p < 0.0001), but the magnitude of these respective changes did not differ between wild-type and CCL2- mice.While HPC reduced infarct volumes by 27% (p < 0.01) in wild-type mice, CCL2- mice subjected to tMCAo were not protected by HPC.The early expression of CCL2 in neurons, the delayed expression of CCL2 in cerebral endothelial cells, and CCL2-mediated actions on circulating CCR2+ monocytes, appear to be required to establish ischemic tolerance to focal stroke in response to HPC, and thus represent a novel role for this chemokine in endogenous neurovascular protection.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurological Surgery, Washington University School of Medicine, 660 S, Euclid Ave,, Box 8057, St, Louis, MO 63110, USA.

ABSTRACT

Background: A brief exposure to systemic hypoxia (i.e., hypoxic preconditioning; HPC) prior to transient middle cerebral artery occlusion (tMCAo) reduces infarct volume, blood-brain barrier disruption, and leukocyte migration. CCL2 (MCP-1), typically regarded as a leukocyte-derived pro-inflammatory chemokine, can also be directly upregulated by hypoxia-induced transcription. We hypothesized that such a hypoxia-induced upregulation of CCL2 is required for HPC-induced ischemic tolerance.

Methods: Adult male SW/ND4, CCL2-, and wild-type mice were used in these studies. Cortical CCL2/CCR2 message, protein, and cell-type specific immunoreactivity were determined following HPC (4 h, 8% O2) or room air control (21% O2) from 6 h through 2 weeks following HPC. Circulating leukocyte subsets were determined by multi-parameter flow cytometry in naïve mice and 12 h after HPC. CCL2- and wild-type mice were exposed to HPC 2 days prior to tMCAo, with immunoneutralization of CCL2 during HPC achieved by a monoclonal CCL2 antibody.

Results: Cortical CCL2 mRNA and protein expression peaked at 12 h after HPC (both p < 0.01), predominantly in cortical neurons, and returned to baseline by 2 days. A delayed cerebral endothelial CCL2 message expression (p < 0.05) occurred 2 days after HPC. The levels of circulating monocytes (p < 0.0001), T lymphocytes (p < 0.0001), and granulocytes were decreased 12 h after HPC, and those of B lymphocytes were increased (p < 0.0001), but the magnitude of these respective changes did not differ between wild-type and CCL2- mice. HPC did decrease the number of circulating CCR2+ monocytes (p < 0.0001) in a CCL2-dependent manner, but immunohistochemical analyses at this 12 h timepoint indicated that this leukocyte subpopulation did not move into the CNS. While HPC reduced infarct volumes by 27% (p < 0.01) in wild-type mice, CCL2- mice subjected to tMCAo were not protected by HPC. Moreover, administration of a CCL2 immunoneutralizing antibody prior to HPC completely blocked (p < 0.0001 vs. HPC-treated mice) the development of ischemic tolerance.

Conclusions: The early expression of CCL2 in neurons, the delayed expression of CCL2 in cerebral endothelial cells, and CCL2-mediated actions on circulating CCR2+ monocytes, appear to be required to establish ischemic tolerance to focal stroke in response to HPC, and thus represent a novel role for this chemokine in endogenous neurovascular protection.

Show MeSH
Related in: MedlinePlus