Motor deficits are triggered by reperfusion-reoxygenation injury as diagnosed by MRI and by a mechanism involving oxidants

Abstract

The early antecedents of cerebral palsy (CP) are unknown but are suspected to be due to hypoxia-ischemia (H-I). In our rabbit model of CP, the MRI biomarker, apparent diffusion coefficient (ADC) on diffusion-weighted imaging, predicted which fetuses will develop postnatal hypertonia. Surviving H-I fetuses experience reperfusion-reoxygenation but a subpopulation manifested a continued decline of ADC during early reperfusion-reoxygenation, which possibly represented greater brain injury (RepReOx). We hypothesized that oxidative stress in reperfusion-reoxygenation is a critical trigger for postnatal hypertonia. We investigated whether RepReOx predicted postnatal neurobehavior, indicated oxidative stress, and whether targeting antioxidants at RepReOx ameliorated motor deficits, which included testing of a new superoxide dismutase mimic (MnTnHex-2-PyP). Rabbit dams, 79% gestation (E25), were subjected to 40 min uterine ischemia. Fetal brain ADC was followed during H-I, immediate reperfusion-reoxygenation, and 4-72 h after H-I. Endpoints were postnatal neurological outcome at E32, ADC at end of H-I, ADC nadir during H-I and reperfusion-reoxygenation, and area under ADC curve during the first 20 min of reperfusion-reoxygenation. Antioxidants targeting RepReOx were administered before and/or after uterine ischemia. The new MRI-ADC biomarker for RepReOx improved prediction of postnatal hypertonia. Greater superoxide production, mitochondrial injury, and oligodendroglial loss occurred in fetal brains exhibiting RepReOx than in those without. The antioxidants, MnTnHex-2-PyP and Ascorbate and Trolox combination, significantly decreased postnatal motor deficits and extent of RepReOx. The etiological link between early injury and later motor deficits can thus be investigated by MRI, and allows us to distinguish between critical oxidative stress that causes motor deficits and noncritical oxidative stress that does not.

Figure 1.

Mitochondrial function was assessed at 20 min after H-I. A, Fetal brains were extirpated, dissociated into single cell suspension, incubated with potentiometric dye JC1, and measured on a flow cytometer. B, Addition of ionophore valinomycin to a separate aliquot collapsed mitochondria membrane potential. C, The difference in cell density between brain cell suspensions with addition of valinomycin in (B) and without valinomycin (A) was depicted on a density difference plot (C). Region with the decrease of cell density (blue on the density difference plot) after addition of valinomycin was attributed to cells with high mitochondrial membrane potential. Proportion of cells in this region on original (top) scatter plot was calculated as an index of cells with functional mitochondria.

Figure 2.

A preliminary study was done in E22 rabbit model to investigate feasibility of using Mn porphyrins. MnTnHex-2-Pyp was observed to have more protection than MnTE-2-Pyp. Experimental plan is depicted on left (A), and neurobehavioral outcome is shown on right (B).

Figure 3.

Time course of ADC in fetal brains during 40 min H-I and reperfusion. A, Fetuses were classified based on the pattern of ADC changes and especially during reperfusion. Group 1 showed no injury. Of the 7 fetuses in Group 1, none were dead or hypertonic. Group 4 had a deep fall in ADC that did not recover. Of the 9 fetuses 5 died and 4 were hypertonic. Sham fetuses were all normal. B, Showing the comparison with injury in H-I alone (Group 2 without RepReOx) and RepReOx (Group 3). Of the 22 fetuses in Group 2, 1 was dead and 13 were hypertonic, compared with that in RepReOx where of the 58 fetuses, 21 died and 29 were hypertonic. The amount of injury was estimated in RepReOx by taking the area under the curve below an arbitrary line of 0.83 × 10⁻³ mm²/s (from the threshold published before) and shown as a three-dimensional block.

Figure 4.

Comparison of all animals showing RepReOx with a group with similar H-I injury as shown by the ADC at end of 40 min H-I. The presence of RepReOx increases the chances of having hypertonia depending on the starting point at beginning of uterine reperfusion. The rate of hypertonia was 2.2 times higher in fetuses with RepReOx that in those without RepReOx (see arrow). Hypertonia only starts appearing in the No RepReOx kits if they reach an ADC <0.83 × 10⁻³ mm²/s at end of 40 min H-I. Note that the abscissa is different from the nadir of ADC, which can occur after the end of H-I.

Figure 5.

Superoxide formation and mitochondrial injury after globa1 fetal H-I in E25 rabbit fetuses. A, Hydroethidine (HE) was administered to fetuses before H-I. Superoxide production in fetal brain was estimated by HPLC-ECD of 2-hydroxy-ethidium (2-OH-E⁺), a specific product of the reaction between HE and superoxide (O₂^.−). Ethidium (E⁺) is a nonspecific oxidation product of HE. Superoxide production was significantly higher in RepReOx group (+) compared with the one without (−) or other groups. The No RepReOx group (−) was not different from the sham and end of H-I groups. Superoxide production is specific to the RepReOx period. B, Proportion of cells with intact mitochondrial membrane potential in fetal brain in the groups depicted in Figure 1. RepReOx (Group 3) had significantly less cells with intact mitochondrial membrane potential compared with No RepReOx or other groups. Group 4 that had decreased ADC <0.65 × 10⁻³ mm²/s and did not show recovery had the least number of cells with intact mitochondrial membrane potential. C, Number of viable oligodendrocyte progenitors were significantly smaller in RepReOx group, assessed at 24 h after H-I. Brains were dissociated into single cell suspension and analyzed on flow cytometer using double staining identifying oligodendrocyte progenitors with O4 marker and dead or injured cells with propidium iodide. *p < 0.05 ANOVA with post hoc comparisons.

Figure 6.

Decrease of hypertonia rate with antioxidant treatment targeted at RepReOx. Antioxidants significantly decreased rate of hypertonia in surviving kits. When MnTnHex-2-PyP was administered after H-I there was no change in hypertonia. Pretreatment of MnTnHex-2-PyP or post-treatment with Ascorbate+Trolox (A+T) significantly decreased hypertonia rate. *p < 0.05 compared with H-I saline group on Fisher's exact test.

Figure 7.

Pretreatment with MnTnHex-2-PyP and post-treatment with Ascorbate+Trolox (A+T) decreased the area of reperfusion-reoxygenation injury on ADC curve indicating less brain injury. *p < 0.05 ANOVA with post hoc comparisons.

Figure 8.

A, Time course of the MnTnHex-2-PyP accumulation in fetal tissues after maternal intravenous infusion of 30 ml 0.085 mm of the compound within 20 min at E25. Absolute concentration of MnTnHex-2-PyP was estimated by change of T1 relaxation time multiplied by the compound T1 relaxivity, determine in tissue homogenates. Concentration of MnTnHex-2-PyP reaches 0.002 mm in fetal brains ∼30–35 min since the beginning of infusion. B, Clearance of MnTnHex-2-PyP in fetal tissues determined on serial imaging of the same dam. Concentration of MnTnHex-2-PyP in placenta and fetal brain decreased to the baseline levels within 3 h but increased and remained at high levels in fetal liver.

Figure 9.

Dams were infused with 1600 mg/kg ascorbic acid and 100 mg/kg Trolox for duration of H-I after start of aortic occlusion and continued with 60 mg/kg/h ascorbic acid and 50 mg/kg/h Trolox after H-I. Fetal blood and brain samples were taken from different fetuses before occlusion (no antioxidants), and at 5, 10, and 15 min of reperfusion. Total tissue antioxidant capacity was measures by TRAP assay and expressed in Trolox equivalents. Brain antioxidant concentration was normalized by the tissue protein concentration. Concentration of antioxidants in maternal blood after Ascorbate + Trolox infusion was above upper limit of the assay calibration for fetal tissue 2 mm.

Figure 10.

Surrogate marker of ADC at 24 h after H-I was not useful for predicting eventual outcome if antioxidants were administered. Improvement of brain function at 24 h was maximal in the MnTnHex-2-PyP groups including post-treatment. The post-MnTnHex-2-PyP findings confirm the importance of RepReOx to development of hypertonia. Ascorbate+Trolox group showed no difference with saline at 24 h, also indicating that short-lived protection of brain injury in RepReOx is important for hypertonia. There was no significant difference in ADC between groups at 4 h after H-I. *p < 0.05 difference from saline control group, ANOVA with post hoc comparisons.

Figure 11.

A, Micro-vascular blood flow change in fetal cortex measured using laser Doppler probe during H-I and reperfusion. Cortical blood flow steadily dropped during H-I phase accompanied by fetal bradicardia. With restoration of the blood flow to the uterus (arrow up), blood flow immediately began to recover and experience a period of hyper-perfusion 5–15 min later before normalization to the baseline. The experiment demonstrates that blood flow begins to recover immediately after cessation of H-I and that we did not observe the phenomenon of “no-reflow” in any fetus. B, Brain perfusion imaging with intravenous contrast infusion to dams before aortic balloon inflation and 5 min after aortic balloon deflation. Index of relative perfusion change was calculated as a ratio of slopes of signal enhancement before and after H-I. There was no significant relationship (R² = 0.166, p = 0.074) between recovery of cerebral perfusion at 5 min of reperfusion-reoxygenation and average ADC values at 4–6 min. Note that some degree of cerebral perfusion was always present at 5 min after H-I.