Therapeutic hypothermia and hypoxia-ischemia in the term-equivalent neonatal rat: characterization of a translational preclinical model

Abstract

Background: Hypoxic-ischemic encephalopathy (HIE) is a major cause of morbidity in survivors. Therapeutic hypothermia (TH) is the only available intervention, but the protection is incomplete. Preclinical studies of HIE/TH in the rodent have relied on the postnatal day (P) 7 rat whose brain approximates a 32-36 wk gestation infant, less relevant for these studies. We propose that HIE and TH in the term-equivalent P10 rat will be more translational.

Methods: P10-11 rat pups were subjected to unilateral hypoxia-ischemia (HI) and 4 h recovery in normothermic (N) or hypothermic (TH) conditions. Brain damage was assessed longitudinally at 24 h, 2 wk, and 12 wk. Motor function was assessed with the beam walk; recognition memory was measured by novel object recognition.

Results: Neuroprotection with TH was apparent at 2 and 12 wk in both moderately and severely damaged animals. TH improved motor function in moderate, but not severe, damage. Impaired object recognition occurred with severe damage with no evidence of protection of TH.

Conclusion: This adaptation of the immature rat model of HI provides a reproducible platform to further study HIE/TH in which individual animals are followed up longitudinally to provide a useful translational preclinical model.

Figure 1. TH is not protective at 24 hours after HI

P10–11 rats were subjected to HI and recovered in normothermic (white bars) or hypothermic (black bars) chambers, n=6/group, and sacrificed at 24 hours later. H&E stained cryosections were analyzed for DS (ipsilateral) by 2 reviewers for whole hemisphere (WH), cortex (CTX), thalamus-striatum (Th-St), hippocampus (Hipp).

Figure 2. Effect of TH at 2 weeks

DS was assessed in all animals from T₂ images demonstrating more animals with lower DS following TH (black bars) relative to normothermic (white bars) recovery.

Figure 3. Effect of TH on Mild, Moderate, and Severe Damage

Experiments fell into 3 groups based on mean DS in normothermic (N) animals: A, Mild; B, Moderate; C, Severe p<0.01, ANOVA. DS in the moderate and severe groups was reduced with TH (p< 0.01 for both, unpaired t-test).

Figure 4. TH improves brain volumes at 2 weeks except for hippocampus

Volumes of healthy brain tissue in contralateral (white bars) and ipsilateral (black bars) hemispheres were calculated from 2 week MRI images as described in Methods. A: Moderate, B: Severe; depicting volumes for: a, whole hemisphere; b, cortex; c, thalamus-striatum; d, hippocampus. TH reduced brain volume loss for all regions except hippocampus, *p<0.05, ANOVA. C: Paired ipsilateral/contralateral ratios, for normothermic (white bars) and TH (black bars) for: a, Moderate; b, Severe cohorts; * p< 0.05.

Figure 5. TH reduces progression of injury following HI: Sequential T₂MRI and Histology

Representative MRI images obtained from 1 N (A–C) and 1 TH (D–F) pup at 24 hours (A,D) and 2 weeks (B, E) are compared with images from final histology of each animal at 12 weeks (C, F). Each panel depicts an anterior and posterior coronal section. Scale bar = 5 mm.

Figure 6. Effects of HI and TH on long term DS

Rats from Moderate (A) and Severe (B) cohorts were sacrificed at 12 weeks post HI. Cryosections were stained with cresyl violet and analyzed for hemispheric and regional DS as described. Change in DS between 2 and 12 weeks was calculated for each animal in Moderate (C) and Severe (D). White bars, normothermic; black bars, TH. * p<0.05, unpaired t-test.

Figure 7. Motor and Cognitive Function in TH- and N-treated HI rats

Therapeutic hypothermia (TH) improves motor performance on the beam test. (A) Beam difficulty at first failure. Most rats in control (Cntrl) and moderate (-M) groups pass to level 2 (white bars); severe (-S) groups fail at (grey bars)/before (black bars) level 1. (B) Contralateral hindlimb slips (left panel) on the difficult beam are increased in rats with moderate HIE with normothermic recovery (*p < 0.05, N-M, grey bars > Cntrl, white bars); TH-M (black bars) rats are less impaired († p< 0.05, TH-M < N-M). No difference in ipsilateral (right) hindlimb slips (right panel). Object encoding and recognition is impaired by HI. (C) Cntrl (white bars) and Moderate HIE rats (TH-M, black bars; N-M, grey bars) show above-chance recognition of the novel object (‡ p< 0.05 DI_NOR > 0); severe HIE rats do not (** p< 0.05, severe HIE:N-S (hatched bar) and TH-S (dark grey bar) vs all other groups). (D) Object contact time for Moderate HIE rats is less than for Cntrls (white bar) during the sample phase. There is a trend for TH (black bar) to mitigate this effect. * p < 0.05, moderate HIE vs Cntrl; † p< 0.1,TH-M > N-M (grey bar).