Hypoxia-Ischemia and Hypothermia Independently and Interactively Affect Neuronal Pathology in Neonatal Piglets with Short-Term Recovery

Abstract

Therapeutic hypothermia is the standard of clinical care for moderate neonatal hypoxic-ischemic encephalopathy. We investigated the independent and interactive effects of hypoxia-ischemia (HI) and temperature on neuronal survival and injury in basal ganglia and cerebral cortex in neonatal piglets. Male piglets were randomized to receive HI injury or sham procedure followed by 29 h of normothermia, sustained hypothermia induced at 2 h, or hypothermia with rewarming during fentanyl-nitrous oxide anesthesia. Viable and injured neurons and apoptotic profiles were counted in the anterior putamen, posterior putamen, and motor cortex at 29 h after HI injury or sham procedure. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) identified genomic DNA fragmentation to confirm cell death. Though hypothermia after HI preserved viable neurons in the anterior and posterior putamen, hypothermia prevented neuronal injury in only the anterior putamen. Hypothermia initiated 2 h after injury did not protect against apoptotic cell death in either the putamen or motor cortex, and rewarming from hypothermia was associated with increased apoptosis in the motor cortex. In non-HI shams, sustained hypothermia during anesthesia was associated with neuronal injury and corresponding viable neuron loss in the anterior putamen and motor cortex. TUNEL confirmed increased neurodegeneration in the putamen of hypothermic shams. Anesthetized, normothermic shams did not show abnormal neuronal cytopathology in the putamen or motor cortex, thereby demonstrating minimal contribution of the anesthetic regimen to neuronal injury during normothermia. We conclude that the efficacy of hypothermic protection after HI is region specific and that hypothermia during anesthesia in the absence of HI may be associated with neuronal injury in the developing brain. Studies examining the potential interactions between hypothermia and anesthesia, as well as with longer durations of hypothermia, are needed.

Keywords: Brain injury; Hypothermia therapy; Hypoxic-ischemic encephalopathy; Neonatal; Neurodegeneration; Neuroprotection; Perinatal asphyxia.

Fig. 1.

Representative brain images from pigs that underwent sham surgery or HI injury. a, b Macrophotographs show the anterior and posterior levels of the putamen where viable, injured, and apoptotic profiles were counted. Photos are grayscale images of hematoxylin and eosin (H&E)-stained sections that were taken at 10× magnification. Brain section orientation is indicated at the lower right of each image. c H&E-stained putamen in a sham-operated normothermic pig. Arrows denote morphologically viable neurons. Perineuronal, pericapillary, and parenchymal neuropil integrity appears excellent at this resolution. d H&E-stained putamen in an HI normothermic pig. The arrows identify morphologically ischemic necrotic neurons, which were classified as “injured” for the analysis. Neuropil integrity is severely damaged as evidenced by the overt perineuronal, pericapillary, and parenchymal vacuoles. e H&E-stained putamen in a sham-operated hypothermic pig. Arrows denote apoptotic profiles. The neuropil appears finely vacuolated with some pallor. The inset at lower right demonstrates an apoptotic profile at higher magnification (from upper right hatched box) for better visualization of spherical chromatin clumps in the nucleus. f Representative TUNEL+ profile in a sham-operated hypothermic pig. The morphology of the DNA fragmentation and compartmentation seen as round aggregates is consistent with apoptosis. The cell size suggests that this apoptotic profile is a neuron. c–d were taken at 400× magnification. f was taken at 600× magnification with oil immersion. Scale bar is 10 μm.

Fig. 2.

Representative hematoxylin and eosin (H&E)-stained sections of motor cortex in pigs that underwent sham surgery and normothermic recovery (a), sustained hypothermia (b), or hypothermia with rewarming (c). Shown are low magnification panoramic and higher resolution (insets) views to appreciate the general anatomic locations and the cellular details. a In a sham normothermic piglet, neurons in layers 2 and 3 appear morphologically viable. b, c After hypothermia and hypothermia with rewarming, subsets of neurons are characterized by vacuolated, eosinophilic cytoplasm and nuclei that are dark and condensed but maintain nucleoli (arrows). The neuropil also shows spongiform changes. Photos taken at 200× magnification and insets taken at 600× magnification. Scale bar is 50 μm.

Fig. 3.

Interrater reliability for counting viable neurons (a, b) and apoptotic profiles (c, d) on hematoxylin and eosin (H&E)-stained sections in putamen of 15 random piglets. Two investigators counted cells in different microscope fields of the same pig. Viable neuron counts (a; r = 0.73, p = 0.002) and apoptotic profile counts (c; r = 0.83, p < 0.001) correlated between the investigators. Bland-Altman plots for viable neurons (b) and apoptotic profiles (d). Bias is shown by the solid line. The 95% limits of agreement are shown by dotted lines. The apoptotic counts had near-zero bias, whereas bias was higher for viable neuron counts.

Fig. 4.

Viable neuron and apoptotic profile counts in anterior (a, b) and posterior (c, d) putamen and in motor cortex (e, f) of naïve and sham pigs. Counts were made on hematoxylin and eosin (H&E)-stained sections. The viable neuron counts were similar in naïve unanesthetized and sham normothermic piglets (a, p = 0.693 for anterior putamen; (c), p = 0.311 for posterior putamen). Naïve piglets had more apoptosis than did sham piglets, with a difference in medians of 0.75 apoptotic profiles per microscope field between groups in anterior putamen (b, * p < 0.05). Apoptotic profile counts did not differ in posterior putamen (d, p = 1.00). e, f Viable neuron and apoptotic profile counts did not differ in the motor cortex. Each circle represents 1 piglet. Box plots with IQRs and 5–95th percentile whiskers are shown.

Fig. 5.

The effects of temperature and HI on viable neuron (a, d), apoptotic profile (b, e), and injured neuron (c, f) counts in anterior (a–c) and posterior (d–f) putamen on hematoxylin and eosin (H&E)-stained sections. a HI and temperature significantly interacted in their effect on the number of viable neurons (p < 0.001). * p < 0.05 and ** p < 0.01. b HI independently affected the number of apoptotic profiles (p = 0.004), and HI and temperature significantly interacted (p = 0.003). ** p ≤ 0.01. c HI and temperature significantly interacted in their effect on injured neurons (p < 0.001). * p < 0.05 and ** p < 0.01. d In the posterior putamen, HI and temperature significantly interacted in their effect on the number of viable neurons (p = 0.003). * p < 0.05 and ** p < 0.01. e HI and temperature interacted in their effect on apoptosis (p = 0.008) *p < 0.05. f HI and temperature did not affect the number of ischemic neurons in the posterior putamen (p > 0.05 for all comparisons). Each circle represents 1 piglet. Box plots with IQRs and 5–95th percentile whiskers are shown. Analyses were conducted by 2-way analysis of variance and post hoc pairwise comparisons with Holm-Sidak tests. NormoT, sustained normothermia; HypoT, sustained hypothermia; Rewarm, hypothermia followed by rewarming.

Fig. 6.

Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining to identify cell death in anterior putamen (a–c) and posterior putamen (d–f). a Temperature and HI interacted in their effect on the number of TUNEL+ cells (p = 0.035). * p < 0.05. b Interrater reliability analysis showed significant correlation in counting TUNEL+ cells and apoptotic profiles by hematoxylin and eosin (H&E) at the anterior level (r = 0.65, p < 0.0001). c Bland-Altman plot for TUNEL+ cells and apoptotic cells by H&E stain at the anterior level. Bias is shown by the solid line. The 95% limits of agreement are shown by dotted lines. Bias was near-zero with high agreement. d Temperature independently affected the number of TUNEL+ cells (p < 0.001), and HI and cooling had an interactive effect (p < 0.001) ** p < 0.01. e Interrater reliability analysis showed significant correlation in counting TUNEL+ cells and apoptotic profiles by H&E at the posterior level (r = 0.57, p < 0.0001). f Bland-Altman plot for TUNEL+ cells and apoptotic cells by H&E stain at the posterior level. Bias is shown by the solid line. The 95% limits of agreement are shown by dotted lines. Bias was near-zero with high agreement. Each circle represents 1 piglet. Data in (a, b) are graphed as box plots with IQRs and 5–95th percentile whiskers. Analyses in (a, b) were conducted by 2-way analysis of variance and post hoc pairwise comparisons with Holm-Sidak tests. NormoT, sustained normothermia; HypoT, sustained hypothermia; Rewarm, hypothermia followed by rewarming.

Fig. 7.

The effect of temperature and HI on viable neuron (a), apoptotic profile (b), and injured neuron (c) counts in the motor cortex. a HI had an independent (p = 0.007) and interactive effect with temperature (p < 0.001) on the number of viable neurons. * p < 0.05, ** p < 0.01, and *** p < 0.001. b HI (p < 0.001) and temperature (p = 0.009) independently and interactively (p < 0.001) affected the number of apoptotic profiles. * p < 0.05 and *** p < 0.001. c HI (p < 0.001) and temperature (p = 0.014) independently and interactively (p < 0.001) affected injured neuron counts. * p < 0.05, ** p < 0.01, *** p < 0.001. Each circle represents 1 piglet. Box plots with IQRs and 5–95th percentile whiskers are shown. Analyses were conducted by 2-way analysis of variance and posthoc pairwise comparisons with Holm-Sidak tests. NormoT, sustained normothermia; HypoT, sustained hypothermia; Rewarm, hypothermia followed by rewarming.