How long is too long for cerebral cooling after ischemia in fetal sheep?

Abstract

Therapeutic hypothermia can partially reduce long-term death and disability in neonates after hypoxic-ischemic encephalopathy. The aim of this study was to determine whether prolonging the duration of cooling from 3 days to 5 days could further improve outcomes of cerebral ischemia in near-term fetal sheep. Fetal sheep (0.85 gestation) received 30 minutes bilateral carotid artery occlusion followed by 3 days of normothermia (n = 8), 3 days of hypothermia (n = 8), or 5 days of hypothermia (n=8) started 3 hours after ischemia. Sham controls received sham ischemia followed by normothermia (n = 8). Cerebral ischemia was associated with profound loss of electroencephalography power and spectral edge, with greater and more rapid recovery in both hypothermia groups (P<0.05). Ischemia was associated with severe loss of neurons in the cortex, hippocampus and thalamus (P < 0.05), with a significant improvement in both hypothermia groups. However, the ischemia-3-day hypothermia group showed greater neuronal survival in the cortex and dentate gyrus compared with ischemia-5-day hypothermia (P < 0.05). Ischemia was associated with induction of iba1-positive microglia, which was attenuated in both hypothermia groups (P < 0.05). Extending the duration of delayed therapeutic hypothermia from 3 to 5 days did not improve outcomes after severe ischemia, and was associated with reduced neuronal survival in some regions.

Figures

Figure 1

Changes in mean arterial blood pressure (MAP), carotid blood flow (CBF), and extradural and esophageal temperature before, during and after 30 minutes of global cerebral ischemia in the ischemia-normothermia, ischemia-3-day, and ischemia-5-day hypothermia groups. There were no significant differences in MAP at any time between groups (P>0.05). A significant increase in CBF was seen in the ischemia–normothermia group between 6 and 48 hours after ischemia compared with either hypothermia group (P<0.05). At the onset of hypothermia (3 hours after ischemia), extradural temperature was significantly reduced in the ischemia-3-day hypothermia group and the ischemia-5-day hypothermia group compared with the ischemia–normothermia group (P<0.05). Extradural temperature returned to baseline after the end of the period of hypothermia at 72 hours or 120 hours in the ischemia-3-day hypothermia and ischemia-5-day hypothermia groups, respectively. A significant reduction in esophageal temperature was seen with the onset of hypothermia, with temperature remaining stable during the treatment period (P<0.05). Data are represented as mean±s.e.m., *P<0.05 ischemia-3-day hypothermia versus ischemia–normothermia group. #P<0.05 ischemia-5-day hypothermia versus ischemia-normothermia.

Figure 2

Changes in electroencephalography (EEG) activity, spectral edge frequency and impedance before, during and after 30 minutes of global cerebral ischemia in the ischemia–normothermia, ischemia-3-day and ischemia-5-day hypothermia groups. Time point zero denotes the start of ischemia. Electroencephalography activity was suppressed in all groups during ischemia. A rebound in EEG activity was seen during the seizure period between 8 and 48 hours. Electroencephalography activity in the ischemia–normothermia group was reduced for the remainder of the experiment, while both hypothermia groups showed a significant recovery of EEG power from 60 hours onwards (P<0.05). Spectral edge was suppressed in all groups during ischemia and remained suppressed until the end of the experiment in the ischemia–normothermia group. A significant increase in spectral edge was seen in both hypothermia groups between 8 and 12 hours and from 64 hours onwards compared with the ischemia–normothermia group (P<0.05). Ischemia was associated with an increase in impedance in all groups, which resolved after the end of ischemia. A significant rise in impedance was seen between 24 and 72 hours in the ischemia–normothermia group compared with the hypothermia groups followed by a significant reduction in impedance in the ischemia–normothermia group from 120 hours until the end of the experiment (P<0.05). Data are represented as mean±s.e.m., *P<0.05 versus ischemia–normothermia group. *P<0.05 vs sham control. #P<0.05 vs ischemia-3-day hypothermia. +P<0.05 vs ischemia-5-day hypothermia.

Figure 3

Neuronal cell count in the cortex, CA1, CA3, CA4, and dentate gyrus, thalamus, caudate nucleus, and putamen in the ischemia–normothermia, ischemia-3-day hypothermia, and ischemia-5-day hypothermia groups 7 days after 30  minutes of global cerebral ischemia. A significant decrease in neuronal number was seen in all regions except the caudate nucleus and putamen, in the ischemia–normothermia group compared with sham control (P<0.05). Neuronal number was significantly higher in the cortex, CA1, CA3, CA4, and dentate gyrus, caudate nucleus and thalamus in the ischemia-3-day hypothermia group compared with the ischemia–normothermia group (P<0.05). Neuronal number was significantly higher in the cortex, CA4, and thalamus in the ischemia-5-day hypothermia group compared with the ischemia–normothermia group (P<0.05). Neuronal number was significantly lower in the cortex, CA1, CA3 and dentate gyrus in the ischemia-5-day hypothermia group and in the CA1 in the ischemia-3-day hypothermia group compared with sham control (P<0.05). Neuronal number was significantly lower in the cortex and dentate gyrus in the ischemia-5-day hypothermia group compared with the ischemia-3-day hypothermia group (P<0.05). *P<0.05 vs sham control. #P<0.05 vs ischemia-3-day hypothermia. +P<0.05 vs ischemia-5-day hypothermia.

Figure 4

Photomicrograph of neuronal survival in the cortex (A, F, K, and P), CA1 (B, G, L, and Q), CA3 (C, H, M, and R), CA4 (D, I, N, and S ), and dentate gyrus (E, J, O, and T) of the hippocampus in the sham control (A–E), ischemia–normothermia (F–J), ischemia-3-day hypothermia (K–O), and ischemia-5-day hypothermia (P–T) groups 7 days after 30 minutes of global cerebral ischemia. Scale bar, 150 μm.

Figure 5

Photomicrograph of neuronal survival in the caudate nucleus (A, D, G, and J), putamen (B, E, H, and K), and thalamus (C, F, I, and L) in the sham control (A–C), ischemia–normothermia (D–F), ischemia-3-day hypothermia (G–I) and ischemia-5-day hypothermia (J–L) groups 7 days after 30 minutes of global cerebral ischemia. Scale bar, 150 μm.

Figure 6

Change in Iba1-positive microglial number in the cortex of the first parasagittal gyrus in the sham control (A), ischemia–normothermia (B), ischemia-3-day hypothermia (C), and ischemia-5-day hypothermia (D) groups. A significant increase in the number of microglia was seen in the ischemia–normothermia group compared with sham control (E). Microglial number was significantly reduced in the ischemia-3-day and ischemia-5-day hypothermia groups compared with the ischemia–normothermia group. Scale bar, 100 μm. *P<0.05 vs sham control. #P<0.05 vs ischemia-normothermia.