Therapeutic Hypothermia Provides Variable Protection against Behavioral Deficits after Neonatal Hypoxia-Ischemia: A Potential Role for Brain-Derived Neurotrophic Factor

Abstract

Background: Despite treatment with therapeutic hypothermia (TH), infants who survive hypoxic ischemic (HI) encephalopathy (HIE) have persistent neurological abnormalities at school age. Protection by TH against HI brain injury is variable in both humans and animal models. Our current preclinical model of hypoxia-ischemia (HI) and TH displays this variability of outcomes in neuropathological and neuroimaging end points with some sexual dimorphism. The detailed behavioral phenotype of this model is unknown. Whether there is sexual dimorphism in certain behavioral domains is also not known. Brain-derived neurotrophic factor (BDNF) supports neuronal cell survival and repair but may also be a marker of injury. Here, we characterize the behavioral deficits after HI and TH stratified by sex, as well as late changes in BDNF and its correlation with memory impairment.

Methods: HI was induced in C57BL6 mice on postnatal day 10 (p10) (modified Vannucci model). Mice were randomized to TH (31°C) or normothermia (NT, 36°C) for 4 h after HI. Controls were anesthesia-exposed, age- and sex-matched littermates. Between p16 and p39, growth was followed, and behavioral testing was performed including reflexes (air righting, forelimb grasp and negative geotaxis) and sensorimotor, learning, and memory skills (open field, balance beam, adhesive removal, Y-maze tests, and object location task [OLT]). Correlations between mature BDNF levels in the forebrain and p42 memory outcomes were studied.

Results: Both male and female HI mice had an approximately 8-12% lower growth rate (g/day) than shams (p ≤ 0.01) by p39. TH ameliorated this growth failure in females but not in males. In female mice, HI injury prolonged the time spent at the periphery (open field) at p36 (p = 0.004), regardless of treatment. TH prevented motor impairments in the balance beam and adhesive removal tests in male and female mice, respectively (p ≤ 0.05). Male and female HI mice visited the new arm of the Y-maze 12.5% (p = 0.05) and 10% (p = 0.03) less often than shams, respectively. Male HI mice also had 35% lower exploratory preference score than sham (p ≤ 0.001) in the OLT. TH did not prevent memory impairments found with Y-maze testing or OLT in either sex (p ≤ 0.01) at p26. At p42, BDNF levels in the forebrain ipsilateral to the HI insult were 1.7- to 2-fold higher than BDNF levels in the sham forebrain, and TH did not prevent this increase. Higher BDNF levels in the forebrain ipsilateral to the insult correlated with worse performance in the Y-maze in both sexes and in OLT in male mice (p = 0.01).

Conclusions: TH provides benefit in specific domains of behavior following neonatal HI. In general, these benefits accrued to both males and females, but not in all areas. In some domains, such as memory, no benefit of TH was found. Late differences in individual BDNF levels may explain some of these findings.

Keywords: Astrocytes; Growth rate; Memory deficits; Primitive reflexes; Sensory-motor deficits.

Figure 1

Timeline representation of experimental protocol

Figure 2. Neonatal HI, treatment with TH and growth

Following HI, injured mice grew slower than sham mice. TH ameliorated growth deficits only in female injured mice. Grouped data for weight (gr) from p16 to p39 are presented as box and whisker plot (A) and scatter plot (B). Sex-stratified weight data (gr) for p26 (C), p36 (D), and p39 (E), and growth rates (gr/day) from p16 to p26 (F), and from p16 to p39 (G) are represented as box and whiskers plots, while growth curves for males (H) and females (I) from p16 to p36 are represented as scatter plots. For box and whiskers plots, boxes represent the interquartile range (IQR) limited by the 25^th and 75^th percentile (lower and upper limit, respectively), line inside the box indicates the median and whiskers extend to 1.5 times the IQR. Outliers are not represented. White box or black discontinuous line represent shams, black box or black continuous line represents NT mice; and grey box or grey continuous line represent TH mice. *, p<0.05 vs. sham, one-way ANOVA (grouped or stratified by sex) with post-hoc analysis using Tamhane. Sample size (n) detailed below each box.

Figure 3. Negative geotaxis and open field behavior

Injured mice regardless of treatment or sex turned up hill in the negative geotaxis test. Only female injured mice spent more time in the periphery of the open field. Sex-stratified negative geotaxis data (in sec) at p16 (A) and p26 (B) and open field data (in %) for time in periphery (C) and center (D) are represented as box and whiskers plots, where boxes represent the interquartile range (IQR) limited by the 25^th and 75^th percentile (lower and upper limit, respectively), line inside the box indicates the median and whiskers extend to 1.5 times the IQR. Outliers are not represented. White boxes represent shams, black boxes represents NT mice; and grey boxes represent TH mice. *, p<0.05 vs. sham, one-way ANOVA (grouped or stratified by sex) with post-hoc analysis using Tamhane. Sample size (n) detailed below each box.

Figure 4. Motor outcomes and protection by TH

TH protects against motor impairment in both male mice (balance beam performance) and female mice (adhesive removal test) following neonatal HI. Sex-stratified balance beam data (sec to cross) 12 mm (A), 6 mm (B) and round (C) beams at p24 and adhesive removal data (sec to remove) from right (C) and left (D) paw at p36 are represented as box and whiskers plots, where boxes represent the interquartile range (IQR) limited by the 25^th and 75^th percentile (lower and upper limit, respectively), line inside the box indicates the median and whiskers extend to 1.5 times the IQR. Outliers are not represented. White boxes represent shams, black boxes represents NT mice; and grey boxes represent TH mice. *, p<0.05 vs. sham, one-way ANOVA (grouped or stratified by sex) with post-hoc analysis using Tamhane. Sample size (n) detailed below each box.

Figure 5. Memory outcomes and TH

Following neonatal HI, TH does not protect against memory deficits assessed by Y-maze and object location test (OLT) in either sex. Sex-stratified Y-maze data for total number of visits (phase 1, A) and percent SAP (phase 1, B) and percent visit to novel arm (phase 2, C) at p22-p26 and OLT data for EP score (D) and discrimination index (E) at p23 are represented as box and whiskers plots, where boxes represent the interquartile range (IQR) limited by the 25^th and 75^th percentile (lower and upper limit, respectively), line inside the box indicates the median and whiskers extend to 1.5 times the IQR. Outliers are not represented. White boxes represent shams, black boxes represents NT mice; and grey boxes represent TH mice. *, p<0.05 vs. sham, one-way ANOVA (grouped or stratified by sex) with post-hoc analysis using Tamhane. Sample size (n) detailed below each box.

Figure 6. Late BDNF changes after HI

Higher BDNF levels observed in the forebrain of the hemisphere ipsilateral to the injury correlates with worse memory outcomes regardless of treatment. Grouped BDNF data for ipsilateral (A), contralateral (B) and ipsilateral/ contralateral (I/C) ratio (C) and sex-stratified I/C ratio (D) are represented as box and whiskers plots, where boxes represent the interquartile range (IQR) limited by the 25^th and 75^th percentile (lower and upper limit, respectively), line inside the box indicates the median and whiskers extend to 1.5 times the IQR. Outliers are not represented. White boxes represent shams, black boxes represent NT mice, and grey boxes represent TH mice. *, p<0.05 vs. sham, one-way ANOVA (grouped or stratified by sex) with post-hoc analysis using Tamhane (n=14 per group for panels A, B and C, and n = 7 per group/ per sex for panel D). Subject-by subject correlation between BDNF levels in ipsilateral hemisphere (E, H), contralateral hemisphere (F, I) and I/C ratio (G, J) vs. % entries to novel arm (Y-maze phase 2, group data) and EP score (OLT, male mice). Analysis by Spearman Rho. r, correlation coefficient. Significance determined by p<0.05. Representative immunoblots for pTrkB, TrkB and loading control with β-actin (n=5/group) shows no significant receptor activation despite BDNF increase in hemisphere ipsilateral to injury.

Figure 7. Late activation of astrocytes

Representative microphotographs of sham (A), NT (B) and TH (C) mice and details (A’, B’, and C’) in hippocampal CA1 region. Large numbers of hypertrophic and densely GFAP-stained astrocytes primarily throughout the hippocampus of injured mice treated with NT. Active astrocytes are mostly seen around glial scaring in injured mice treated with TH. Comparative GFAP staining of the hemispheres ipsilateral and contralateral to the HI injury are shown (D). In contrast to contralateral staining, abundant reactive astrocytes were visualized in the ipsilateral hemisphere of injured mice (D’ and D”). Scale bars are shown. LM, lacunosum moleculare layer; PL, pyramidal cell layer; RL, radiatum layer.