Normobaric hyperoxia improves cerebral blood flow and oxygenation, and inhibits peri-infarct depolarizations in experimental focal ischaemia

Abstract

Normobaric hyperoxia is under investigation as a treatment for acute ischaemic stroke. In experimental models, normobaric hyperoxia reduces cerebral ischaemic injury and improves functional outcome. The mechanisms of neuroprotection are still debated because, (i) inhalation of 100% O2 does not significantly increase total blood O2 content; (ii) it is not known whether normobaric hyperoxia increases O2 delivery to the severely ischaemic cortex because of its short diffusion distance; and (iii) hyperoxia may reduce collateral cerebral blood flow (CBF) to ischaemic penumbra because it can cause vasoconstriction. We addressed these issues using real-time two-dimensional multispectral reflectance imaging and laser speckle flowmetry to simultaneously and non-invasively determine the impact of normobaric hyperoxia on CBF and oxygenation in ischaemic cortex. Ischaemia was induced by distal middle cerebral artery occlusion (dMCAO) in normoxic (30% inhaled O2, arterial pO2 134 +/- 9 mmHg), or hyperoxic mice (100% inhaled O2 starting 15 min after dMCAO, arterial pO2 312 +/- 10 mmHg). Post-ischaemic normobaric hyperoxia caused an immediate and progressive increase in oxyhaemoglobin (oxyHb) concentration, nearly doubling it in ischaemic core within 60 min. In addition, hyperoxia improved CBF so that the area of cortex with < or =20% residual CBF was decreased by 45% 60 min after dMCAO. Furthermore, hyperoxia reduced the frequency of peri-infarct depolarizations (PIDs) by more than 60%, and diminished their deleterious effects on CBF and metabolic load. Consistent with these findings, infarct size was reduced by 45% in the hyperoxia group 2 days after 75 min transient dMCAO. Our data show that normobaric hyperoxia increases tissue O2 delivery, and that novel mechanisms such as CBF augmentation, and suppression of PIDs may afford neuroprotection during hyperoxia.

Fig. 1

Hyperoxia increases O₂ delivery in ischaemic core. The time course of changes in core oxyHb (A) and total Hb (B) concentrations in the normoxia (squares, n = 11) and hyperoxia (circles, n = 8) groups expressed as % of baseline. Time 0 is the onset of dMCAO. Hyperoxia group inspired 100% O₂ starting from 15 min after dMCAO (horizontal bar, Experiment I). Vertical bars indicate ± SEM.

Fig. 2

Hyperoxia preserves CBF after distal middle cerebral artery occlusion. Representative speckle contrast images taken 75 min after dMCAO from normoxia (A) and hyperoxia groups (B). Superimposed in shades of blue are pixels with residual CBF ≤30%. The area of cortex with residual CBF ≤ 30% was smaller in hyperoxia group compared to normoxia. (C) The time course of changes in area of cortex with severe CBF deficit (residual CBF ≤20%) in normoxia (squares, n = 11) and hyperoxia mice (circles, n = 8). Time 0 indicates dMCAO. Horizontal bar represents 100% O₂ starting from 15 min after dMCAO (Experiment I). The area of CBF deficit was quantified using a thresholding paradigm (see ‘Methods’ section). Vertical error bars indicate ± SEM. (D) Mouse skull showing the position of imaging field (gray rectangle) from which speckle contrast images were acquired over the right hemisphere. Arrows indicate the location of microvascular clip occluding distal MCA.

Fig. 3

The effects of delayed administration or early discontinuation of normobaric hyperoxia on CBF and oxygenation. The time courses of changes in core oxyHb concentration and the area of severely ischaemic cortex after dMCAO are shown from Experiments II (B and E, 100% O₂ starting at 45 min after dMCAO; n = 5) and III (C and F, 100% O₂ between 15 and 45 min of dMCAO, n = 5), along with normoxic time controls (A and D, n = 5). Vertical bars indicate ± SEM.

Fig. 4

Hyperoxia preserves CBF in both core and penumbra. (A) The CBF profile between non-ischaemic cortex and ischaemic core was determined along a 5 mm line drawn between lambda (0 mm) and the microvascular clip occluding distal MCA (arrow), and plotted as % of baseline CBF and distance from lambda. The bright band at the lateral edge of ischaemic territory is artifact introduced by the insertion of microvascular clip through the temporal bone; such artefacts were manually excluded from the ROI for CBF analysis. (B) Normobaric hyperoxia (n = 8) increased CBF in both core (C, residual CBF ≤20%) and penumbra (P, residual CBF 21–30%) compared to normoxia (n = 11), without a significant impact on mildly ischaemic or non-ischaemic cortex (NI, residual CBF>30%). Data are from 75 min after dMCAO. Error bars represent ± SEM.

Fig. 5

Hyperoxia suppresses peri-infarct depolarizations. (A) Distal MCAO caused repetitive PIDs in normoxic mice, as shown in this representative CBF tracing obtained from mildly ischaemic cortex. In this particular example, six PIDs were observed (black dots). (B) The occurrence of PIDs was inhibited after the onset of 100% O₂ inhalation. The PID frequency histogram in normoxic (C, n = 16) or hyperoxic mice (D, Experiment I, n = 8; E, Experiment II, n = 5; F, Experiment III, n = 5). The frequency of PIDs is expressed as the average number of PIDs occurring during each 15 min period. Horizontal grey bars indicate 100% O₂.

Fig. 6

Hyperoxia attenuates the worsening in oxyHb and CBF during PIDs. The time course of oxyHb (A) and CBF (B) in ischaemic core during PIDs from normoxic (square, n =11) and hyperoxic (circle, n = 8) mice. The DC potential shift during a PID (approximate timing is shown by the horizontal grey bar) temporally corresponds to the hypoperfusion phase, as shown in detail previously (Shin et al., 2006); therefore, electrophysiological recordings were not undertaken in this study in order preserve cortical physiology. Hyperoxia reduced the transient reduction in oxyHb and CBF during each PID (line arrows), and augmented their recovery after the PID (block arrows). Therefore, hyperoxia ameliorated the negative impact of PIDs on oxyHb and CBF compared to normoxia (two-way ANOVA for repeated measures). The impact of PIDs on CBF and oxygenation was quantified by measuring the magnitude of CBF and oxyHb changes and their latency from the onset of hypoperfusion at the following major deflection points: the onset (time 0) and the trough of abrupt deoxygenation and hypoperfusion, the peak of subsequent increase, return to baseline and 1.5 min after return to baseline. OxyHb and CBF data were expressed as % of baseline 1–2 min before the occurrence of a PID. The time course of oxyHb and CBF changes were then plotted against their latency from the onset of abrupt deoxygenation and hypoperfusion (time 0). Data are the average of all PIDs occurring after 15 min of dMCAO in normoxic and hyperoxic groups from Experiment I. Vertical and horizontal bars indicated the SEM for the magnitude of oxyHb and CBF changes, and their latency, respectively.

Fig. 7

Normobaric hyperoxia reduces infarct size. (A) Topical TTC-stained brain from a representative normoxic mouse showing the dorsolateral cortical infarct 48 h after 75 min transient dMCAO. (B) Topical TTC-stained brain from a representative hyperoxic mouse showing the reduction in infarct size. (C) Normobaric hyperoxia (white, n = 5) starting 15 min after dMCAO reduced infarct volume compared to normoxia (grey, n = 6), as calculated by integrating the infarct areas in 1mm thick coronal sections following topical TTC staining. *P<0.05, t-test. (D) The area of infarct in individual 1mm thick coronal slice levels showing that the neuroprotective effect of hyperoxia (circle) was apparent at all coronal levels compared to normoxia (square). *P<0.05, two-way ANOVA for repeated measurses.