Effects of preoperative hypoxia on white matter injury associated with cardiopulmonary bypass in a rodent hypoxic and brain slice model

Abstract

Background: White matter (WM) injury is common after cardiopulmonary bypass or deep hypothermic circulatory arrest in neonates who have cerebral immaturity secondary to in utero hypoxia. The mechanism remains unknown. We investigated effects of preoperative hypoxia on deep hypothermic circulatory arrest-induced WM injury using a combined experimental paradigm in rodents.

Methods: Mice were exposed to hypoxia (prehypoxia). Oxygen-glucose deprivation was performed under three temperatures to simulate brain conditions of deep hypothermic circulatory arrest including ischemia-reperfusion/reoxygenation under hypothermia.

Results: WM injury in prenormoxia was identified after 35 °C-oxygen-glucose deprivation. In prehypoxia, injury was displayed in all groups. Among oligodendrocyte stages, the preoligodendrocyte was the most susceptible, while the oligodendrocyte progenitor was resistant to insult. When effects of prehypoxia were assessed, injury of mature oligodendrocytes and oligodendrocyte progenitors in prehypoxia significantly increased as compared with prenormoxia, indicating that mature oligodendrocytes and progenitors that had developed under hypoxia had greater vulnerability. Conversely, damage of oligodendrocyte progenitors in prehypoxia were not identified after 15 °C-oxygen-glucose deprivation, suggesting that susceptible oligodendrocytes exposed to hypoxia are protected by deep hypothermia.

Conclusion: Developmental alterations due to hypoxia result in an increased WM susceptibility to injury. Promoting WM regeneration by oligodendrocyte progenitors after earlier surgery using deep hypothermia is the most promising approach for successful WM development in congenital heart disease patients.

Figure 1

The combined experimental paradigm using a rodent hypoxic and brain slice model to investigate the cellular effects of preoperative hypoxia on WM oligodendrocyte injury. (a) Study design and equivalent time period for WM development between the mouse and human. (b) Perfusion protocol for oxygen-glucose deprivation under hypothermia. (c) Antibody markers used to immunostain distinct developmental stages of oligodendrocyte lineage cells.

Figure 2

Preoperative hypoxia increases developing WM vulnerability to ischemia-reperfusion/reoxygenation injury. (a–h) WM caspse3⁺ cells in different OGD groups after Pre-Normoxia and Pre-Hypoxia. (i) Caspase3⁺ cell number after OGD experiments following Pre-Hypoxia (n=6–12 each). (j) Changes of caspase3⁺ cells from Control after OGD experiments between Pre-Normoxia (white columns) and Pre-Hypoxia (gray columns; n=6–9 each). *P < .01, **P < .001, vs. Control by one-way ANOVA with Bonferroni comparisons. † P ≤ .01, ‡P < .001 by student t-test. Scale bar = 50μm.

Figure 3

The pre-oligodendrocyte is the most susceptible stage within the oligodendrocytes lineage. (a) CNP⁺PDGFRa⁺Caspase3⁺ OPCs (rectangular box). (b) CNP⁺CC1⁻Caspase3⁺ immature oligodendrocytes (upper rectangular box) and CNP⁺CC1⁺Caspase3⁺ mature oligodendrocytes (lower rectangular box). (c) Caspase3⁺ cell number after OGD with different oligodendrocyte lineage antibody (n=22–23, each). (d–k) CNP⁺CC1⁻Caspase3⁺ oligodendrocytes after different OGD experiments following Pre-Normoxia and Pre-Hypoxia. (l) OGD-induced change of CNP⁺CC1⁻Caspase3⁺ cell number from Control between Pre-Normoxia (white columns) and Pre-Hypoxia (gray columns, n=5–9 each). *P < .001 by one-way ANOVA with Bonferroni comparisons. Scale bar = 50μm.

Figure 4

Pre-operative hypoxia increases the vulnerability of OPCs and mature oligodendrocytes to ischemia-reperfusion/reoxygenation; however, susceptible oligodendrocytes exposed to preoperative hypoxia are protected by deep hypothermia. (a–d) CNP⁺CC1⁺Caspase3⁺ oligodendrocytes in different OGD groups after Pre-Hypoxia. (e–h) CNP⁺PDGFRa⁺Caspase3⁺ OPCs in different OGD groups after Pre-Hypoxia. (i) CNP⁺CC1⁺Caspase3⁺ cell number after OGD experiments following Pre-Hypoxia (n=6–12, each). (j) OGD-induced change of CNP⁺CC1⁺Caspase3⁺ cell number from Control between Pre-Normoxia (white columns) and Pre-Hypoxia (gray columns, n=5–9 each). (k) CNP⁺PDGFRa⁺Caspase3⁺ cell number after OGD experiments in Pre-Hypoxia group (n=5–9 each). (l) OGD-induced change of CNP⁺PDGFRa⁺Caspase3⁺ cell number from Control between Pre-Normoxia (white columns) and Pre-Hypoxia (gray columns, n=5–9 each). *P < .05, **P < .001 vs. Control and 15°C OGD by one-way ANOVA with Bonferroni comparisons. §P < .001 by one-way ANOVA with Bonferroni comparisons. †P < .05, ‡P <. 001 by student t-test. Scale bar = 50μm.

Figure 5

Other cell populations contribute to WM injury after DHCA in the brain that has developed under hypoxic conditions. (a–d) CNP⁺Caspase3⁺ oligodendrocytes in different OGD groups after Pre-Hypoxia. (e) CNP⁺Caspase3⁺ cell number after OGD experiments following Pre-Hypoxia (n=6–12, each). *P < .05, **P < .01 vs. Control and 15°C OGD by one-way ANOVA with Bonferroni comparisons. Scale bar = 50μm.