Novel Injury Scoring Tool for Assessing Brain Injury following Neonatal Hypoxia-Ischemia in Mice

Abstract

The variability of severity in hypoxia-ischemia (HI)-induced brain injury among research subjects is a major challenge in developmental brain injury research. Our laboratory developed a novel injury scoring tool based on our gross pathological observations during hippocampal extraction. The hippocampi received scores of 0-6 with 0 being no injury and 6 being severe injury post-HI. The hippocampi exposed to sham surgery were grouped as having no injury. We have validated the injury scoring tool with T2-weighted MRI analysis of percent hippocampal/hemispheric tissue loss and cell survival/death markers after exposing the neonatal mice to Vannucci's rodent model of neonatal HI. In addition, we have isolated hippocampal nuclei and quantified the percent good quality nuclei to provide an example of utilization of our novel injury scoring tool. Our novel injury scores correlated significantly with percent hippocampal and hemispheric tissue loss, cell survival/death markers, and percent good quality nuclei. Caspase-3 and Poly (ADP-ribose) polymerase-1 (PARP1) have been implicated in different cell death pathways in response to neonatal HI. Another gene, sirtuin1 (SIRT1), has been demonstrated to have neuroprotective and anti-apoptotic properties. To assess the correlation between the severity of injury and genes involved in cell survival/death, we analyzed caspase-3, PARP1, and SIRT1 mRNA expressions in hippocampi 3 days post-HI and sham surgery, using quantitative reverse transcription polymerase chain reaction. The ipsilateral (IL) hippocampal caspase-3 and SIRT1 mRNA expressions post-HI were significantly higher than sham IL hippocampi and positively correlated with the novel injury scores in both males and females. We detected a statistically significant sex difference in IL hippocampal caspase-3 mRNA expression with comparable injury scores between males and females with higher expression in females.

Keywords: Apoptosis; Brain injury; Caspase-3; Hippocampus; Hypoxia-ischemia; Injury score; MRI; Nuclei integrity; PARP; SIRT; Sirtuin.

Fig. 1.. Novel injury scoring tool.

The freshly harvested whole brain, ipsilateral (IL) and contralateral (CL) hippocampi samples were examined under a microscope and injury scores were assigned from 0 to 6. Hippocampi are scored as 0, if the IL hippocampus has identical tissue consistency and morphology with the CL hippocampus. Hippocampi are scored as 1, if the head of the IL hippocampus (approximately 1/3 of the length of the hippocampus) is slightly opaque compared to the CL hippocampus. Hippocampi are scored as 2, if the opacity in the IL hippocampus extends to the body (approximately 1/2 of the length of the hippocampus) and slight liquefaction in the head of IL hippocampus is present. Hippocampi are scored as 3, if the opacity in the IL hippocampus constitutes 2/3 of the length of the hippocampus and slight liquefaction is observed in the entire IL hippocampus. Hippocampi are scored as 4, if there is opacity and moderate liquefaction in the entire IL hippocampus, and if there is a slight loss of shape due to liquefaction. Hippocampi are scored as 5, if opacity and moderate liquefaction is present in the entire IL hippocampus along with moderate shape loss. Hippocampi are scored as 6, if there is total liquefaction in the IL hippocampus that causes total shape loss. The injury scores were then grouped together based on severity of injury. Injury scores 0 and 1 were assigned as mild, 2 and 3 were assigned as moderate, 4, 5, and 6 were assigned as severe. Sham samples were assigned as having no injury. Whole brain photographs are shown with white arrows denoting cortical infarcts.

Fig. 2.. MRI segmentation methodology with ImageJ and ITK-SNAP.

The T2-weighted MRIs were segmented using the ImageJ and ITK-SNAP programs. (a) Representative MRI coronal slices obtained from mouse brain, (b) hippocampal and (b’) hemispheric segmentation using ImageJ and (c) hippocampal and (c’) hemispheric segmentation using ITK-SNAP are shown. Red and blue represents the contralateral and ipsilateral sides, respectively (scale bar: 1 mm).

Fig. 3.. Correlation of the hemispheric and hippocampal percent tissue loss with the injury score.

Percent tissue loss analysis of the brain MRIs were performed to validate the novel injury scoring tool. Using ImageJ, (a) hemispheric (R²=0.84, p<0.0001) and (b) hippocampal (R²=0.65, p=0.0001) area loss was quantified and correlated with injury score. The data was stratified into anterior and posterior (a’) hemispheric (R²_anterior=0.80, p_anterior<0.0001, R²_posterior=0.59, p_posterior=0.0003) and (b’) hippocampal (R²_anterior=0.62, p_anterior=0.0002, R²_posterior=0.42, p_posterior=0.0051) regions and correlated with injury score. Using ITK-SNAP the (c) hemispheric (R²=0.88, p<0.0001) and (d) hippocampal (R²=0.82, p<0.0001) percent volume loss was quantified and correlated with injury score. The data was stratified into anterior and posterior (c’) hemispheric (R²_anterior=0.87, p_anterior<0.0001, R²_posterior=0.58, p_posterior=0.0004) and (d’) hippocampal (R²_anterior=0.68, p_anterior<0.0001, R²_posterior=0.61, p_posterior=0.0002) regions and correlated with injury score (n=17).

Fig. 4.. Correlations of ImageJ and ITK-SNAP percent tissue loss measurements.

Following MRI, percent area loss was calculated using ImageJ and percent volume loss was calculated using ITK-SNAP. The percent (a) hemispheric and (b) hippocampal tissue loss obtained using each program were correlated with each other. The hemispheric and hippocampal measurements had a statistically significant positive correlation (p<0.0001) with an R² value of 0.82 and 0.82, respectively (n=17).

Fig. 5.. SIRT1, Caspase-3, PARP1 mRNA expressions in CL and IL hippocampi at 3 days post-HI.

At 3 days post-HI, hippocampi were extracted and the CL and IL hippocampi were probed for SIRT1, caspase-3, PARP1, and Hprt. Hippocampal mRNA expressions were quantified using RT-qPCR. The mean CL and IL SIRT1 (a, a’), caspase-3 (b, b’), and PARP1 (c, c’) mRNA expression ± SEM values are reported (Sham n=6–8 per sex, HI n=10–12 per sex) with 2^−ΔΔCt values on the Y-axis and the experimental groups on the X-axis. The average injury scores were 2.18±1.30 for male HI hippocampi and 2.25±1.26 for female HI hippocampi (p=0.856). The IL hippocampal SIRT1 and caspase-3 mRNA expressions were statistically significantly higher than CL hippocampi in both males (p_SIRT1=0.0002, p_caspase-3=0.0016) and females (p_SIRT1=0.0006, p_caspase-3=0.0001) post-HI. There was a statistically significant difference in IL hippocampal SIRT1 and caspase-3 mRNA expressions between sham and HI groups in male (p_SIRT1=0.0001, p_caspase-3=0.007) and female (p_SIRT1=0.0002, p_caspase-3=0.0002) mice. Asterisk (*) represents higher IL hippocampal caspase-3 mRNA expressions in females compared to males post-HI (b’) (p=0.0204). No statistically significant induction of PARP1 mRNA expression was detected post-HI (c, c’). There was no statistically significant difference detected among the CL groups.

Fig. 6.. Correlations of IL hippocampal SIRT1 and caspase-3 mRNA expressions with injury severity post-HI.

The male and female IL hippocampi were grouped as no injury, mild, moderate, and severe according to their injury severities as explained in the methods section. Regression analyses were performed to assess the correlation between IL hippocampal SIRT1 (a, a’), caspase-3 (b, b’) mRNA 2^−ΔΔCt values and the injury severity (Sham n=6–8 per sex, HI n=10–12 per sex). The 2^−ΔΔCt values are shown on the Y-axis and the injury severity groups are shown on the X-axis. The IL hippocampal SIRT1 mRNA 2^−ΔΔCt values positively correlated with injury severity in (a) males (p<0.05, R²=0.25) and (a’) females (p<0.05, R²=0.21). IL hippocampal caspase-3 mRNA 2^−ΔΔCt values positively correlated with injury severity in (b) males (p<0.05, R²=0.31) and (b’) females (p<0.05, R²=0.68).

Fig. 7.. Quantification of percent good quality hippocampal nuclei post-HI.

At 3 days post-HI, the male and female IL hippocampi were extracted and utilized for nuclei isolation. Three fields per hippocampus were imaged using 20X magnification and the images were quantified using the ImageJ software. (a) The bright field nuclei images were converted to 8-bit format and then (b) threshold images were created. (c) The particles with an area greater than 150 pixels were considered as nuclei. (d) The nuclei with an intact membrane and >75% circularity were considered as good quality nuclei. Nuclei obtained from the IL sham, (e) mildly, (f) moderately, and (g) severely injured hippocampi were used to assess the changes in nuclei morphology according to the injury severity (scale bar: 0.05 mm). The red arrowheads show good quality nuclei, and the white arrowheads show damaged nuclei. (h) The percentage of good quality nuclei were calculated and correlated with the injury severity. Counts obtained from each field per injury severity were plotted in the figure. The injury severity was shown on X-axis and the percent good quality nuclei was shown on Y-axis. The percent good quality nuclei negatively correlated with the injury severity (p<0.0001, R²= 0.71) (n=4 hippocampi per group, total n=16). The average percent good quality nuclei was % 56 ± 3 for sham, % 42 ± 3 for mildly injured, % 33 ± 2 for moderately injured, and % 17 ± 2 for severely injured hippocampi.