Experimental pediatric arterial ischemic stroke model reveals sex-specific estrogen signaling

Abstract

Background and Purpose

Pediatric stroke, birth-18 years, is a significant cause of long-term disability in the United States, however there is currently little experimental data on the pathophysiology of childhood stroke due to lack of animal models. We developed a novel mouse model of experimental childhood-onset arterial ischemic stroke (AIS) in order to characterize the sex-specific response of the adolescent brain to cerebral ischemia and assess the neuroprotective effect of estrogen at this developmental stage.

Methods

Postnatal day 20–25 (P20-25) mice were subjected to 90 minutes experimental stroke via the intraluminal filament middle cerebral artery occlusion (MCAO) model and ischemic damage assessed 22 hr after reperfusion. Real-time quantitative RT-PCR (qPCR) was performed 22 hr after MCAO to determine the effects of ischemia and estrogen treatment on the pro-apoptotic gene Bax.

Results

Ischemic injury did not differ between male and female juvenile (P20-25) mice following MCAO. However, estrogen reduced ischemic injury in female mice, while having no effect in juvenile males. No differences in estrogen receptor expression was observed between P20 males and females. In contrast, estrogen minimized the ischemia-induced increase in the pro-apoptotic gene Bax in female mice, while having no effect on Bax induction in the male brain.

Conclusions

Focal ischemia has fundamentally different effects in the juvenile brain compared to the adult, as evidenced by the lack of gender difference in ischemic injury in the murine P20-25 MCAO model and the sexually dimorphic response to estrogen neuroprotection.

Introduction

Pediatric stroke, birth through age 18 years, is a significant cause of long-term disability, with an incidence of approximately 11/100,000 children annually in the United States. A majority of pediatric strokes are arterial ischemic strokes (AIS), and include perinatal AIS (classically presenting with seizure in the first days of life) and childhood-onset AIS (occurring beyond the neonatal period). Permanent neurological deficits are observed in 50–90% of survivors ¹. It is becoming increasingly appreciated clinically that early diagnosis and appropriate rehabilitation therapies can improve outcome in children after AIS, as we are seeing in the adult population. Children are not just small adults, as evidenced by dramatic differences in AIS risk factors ². However, there are currently few viable experimental models aimed at determining etiology and modeling pathophysiology of childhood-onset AIS. There is extensive experimental literature modeling ischemia in perinatal animals ^3–5 and reports of pediatric stroke experiments in piglets ⁶, with no reports of the use of rodents to model pediatric ischemia. The current study describes a variation of the middle cerebral artery occlusion (MCAO) model modified for use in juvenile mice (modeling pre-pubertal children), providing a powerful new experimental tool to begin to study the effects of ischemia in the immature brain.

Incidence of childhood-onset AIS is sexually dimorphic, with boys at slightly greater risk ⁷; however, relative severity of outcome in boys and girls remains uncertain. In adult humans and animal models, females experience less damage following comparable ischemic insults compared to age-matched males ^{8, 9}. The relative advantage observed in adult females has been attributed to endogenous sex steroids, in particular estrogen ^8–10. Indeed, there is extensive evidence that estrogen is neuroprotective in adult male and female animals following experimental AIS (MCAO). Estrogen is a pleiotropic steroid hormone that affects several signaling cascades to exert its neuroprotective effects. An important neuroprotective mechanism of estrogen is its ability to decrease ischemia-induced apoptotic cell death ^10–12, by increasing expression of the anti-apoptotic protein Bcl-2 and decreasing the pro-apoptotic protein Bax, among others. Therefore, we utilized our novel model of experimental childhood-onset stroke to test two related hypotheses: 1) that juvenile male and female ischemic outcomes differ; and 2) that exogenous estrogen protects young brain by decreased expression of pro-apoptotic genes genes in a sex-specific manner.

Methods

Experimental Animals

All experimental protocols were approved by the Institutional Animal Care and Use Committee and conformed to the National Institutes of Health guidelines for the care and use of animals in research. To assess the effect of prolonged, controlled levels of hormones, 17β-estradiol (E2; 12.6 µg) was administered via subcutaneous silastic implants two days prior to experiments. We chose this dose based upon previous studies in our laboratory ¹³. All MCAO experiments utilizing vehicle or E2 were performed in a blinded, randomized manner using male and female C57Bl/6 mice at postnatal days 20–25.

Middle Cerebral Artery Occlusion

Methods are as previously published in mouse adult ¹⁴, with minor variations to accommodate the small size of P20-25 mice (including 6-0 nylon suture that was heat-blunted and coated with silicone gel to obtain a smaller filament diameter of ~0.18 mm). Cerebral ischemia was induced for 90 minutes of reversible MCAO via the intraluminal suture method under isoflurane anesthesia. Adequacy of MCAO was confirmed by laser Doppler flowmetry measured (>70% drop required for inclusion) over the ipsilateral parietal cortex in all mice and by neurological deficit scoring at end of occlusion. Additionally, neurological deficits were measured 22 hr after reperfusion. Neurological deficit scored as follows: 0=no deficit, 1=failure to extend forelimb, 2=circling, 3=unilateral weakness, 4=no spontaneous activity ¹⁵, ¹⁶.

Infarct Volume Analysis

After the period of reperfusion, the mice were anesthetized with isoflurane (2.0% to 3.0%), and animals were then decapitated for brain removal. Each brain was sliced into five 2 mm-thick coronal sections. The sections were placed in a 1.2% solution of 2,3,5-triphenyltetrazolium chloride (TTC, Sigma, St Louis, MO, USA) for 30 min at 37°C and fixed in 10% formalin for 24 h. Both sides of each stained coronal slice were photographed using a digital camera, and infarction was measured with digital image analysis software (SigmaScan Pro; Jandel, San Rafael, CA, USA) and integrated across all five slices. To account for the effect of edema, the infarcted volume was estimated and expressed as a percentage of the contralateral structure.

Quantitative Reverse Transcriptase-PCR (qPCR)

For quantitative PCR measurement of estrogen receptor transcripts and apoptotic gene expression, cortical tissue was dissected from the penumbra of the ischemic hemisphere 22 hr after MCAO, and the corresponding cortical region was isolated from the non-ischemic contralateral hemisphere. The penumbra was identified by cutting an adjacent 1 mm thick slice and staining with TTC (see above) in order to identify the infarcted region in each animal, as described previously. Total RNA was isolated using the RNAqueous-4 PCR kit (Ambion, Austin, TX, USA) per the manufacturer’s instructions. Briefly, approximately 1–3 mg of tissue was lysed in lysis buffer and total RNA was isolated and eluted from a column with 50µL RNase-free elution buffer, and further treated with Turbo DNase (Ambion, Austin, TX, USA). First strand cDNA was reverse transcribed from 500ng total RNA with High Capacity cDNA archive Kit (Applied Biosystems, Foster City, CA, USA). Real-time PCR reactions using 6-carboxyfluorescein (FAM) labeled primer/probe sets were performed on ABI Prism 7000 sequence detection system in triplicate using 50ng cDNA. Probe/Primer sets used to detect Bax, Bcl2 and the estrogen receptors were synthesized by Invitrogen. The housekeeping gene 18s was also assayed for each sample using 5ng of cDNA. 18s RNA was chosen as the normalizer due to its abundance and stability, making it highly unlikely to be effected by ischemia. Cycle parameters used were 50°C for 2min, 95°C for 10min followed by 40 cycles of 95°C for 15s and 60°C for 1min. Expression levels were calculated as the ratio of the target gene to 18S.

Measurement of 17β-estradiol (E2)

200–400 mg of brain tissue was homogenized using on liquid nitrogen and kept at 4°C during the whole procedure. Following homogenization, the samples were centrifuged at 13000xg and 4°C for 10 minutes. An Agilent HPLC system consisting of 3 HPLC pumps, a column thermostat and a 6 port switching valve coupled to an AB-Sciex API5500 was used for the analysis of estrogen. A Zorbax XDB-C8 guard column (12*4.6 mm) was used for an inline extraction and a Zorbax XDB-C8 analytical column (3.5 micron, 4.6*150mm) was used for the separation of analytes (both columns from Agilent Technologies, Palo Alto, CA). 20% of methanol and 80% of 0.1% formic acid were initially pumped at 500 µL/min during the injection for loading the analytes onto the inline extraction column. Atmospheric pressure photo ionization (APPI) was used for the ionization of steroids. Toluene at the flow rate of 200 µL/min was used as dopant. The API5500 mass spectrometer was operated in positive multiple reaction monitoring (MRM) mode. The following parent-fragment ions were monitored: estrone and estriol 271.1 > 133.1, estradiol 255.1 > 159.1. Analyst Software version 1.5.2 was used for data acquisition and data processing. All calibration curves had a correlation coefficient R >0.99.

Statistical Analysis

All data are presented as mean ± SD. Histological damage was compared using one-way and two-way ANOVA followed by Newman-Keuls Multiple Comparison Test to determine the source of variance. Student’s t-test was used to compare Bax and Bcl-2 mRNA expression. P values less than 0.05 were considered statistically significant.

Results

Male and female mice at postnatal age 20–25 days were subjected to 90 minutes of MCAO and infarct volume was analyzed after 22 hr reperfusion. Male and female mice were implanted with subcutaneous 17β-estradiol (E2) or vehicle 2 days prior to MCAO. A total of 85 mice were used for the study (69 for histological analysis and 16 used for brain E2 measurements); 5 were excluded for insufficient laser Doppler flow reduction; 5 were excluded due to premature mortality (2 male oil, 2 male E2 and 1 female oil mouse); additionally, 1 female E2 and 1 male E2 were excluded due to the absence of behavioral deficit. No differences in intra-ischemic physiological parameters, LDF or behavioral assessments were observed among the resulting experimental groups (Table 1). E2 implanted mice had significantly elevated levels of brain 17β-estradiol measured 2 days after implantation with either oil or E2 (Table 2).

Table 1. LDF, temporal muscle temperature.

LDF, temporalis muscle temperature and behavioral assessment in 90 min MCAO. Data are presented as mean±SEM. V, vehicle, E2, 17β estradiol, LDF, laser-Doppler flometry, MCAO, middle cerebral artery occlusion. No differences were found among groups.

Group	LDF (%)	Temporal muscle temperature (°C)		Behavior Score
	MCAO 85 mins	Pre MCAO	MCAO 85 mins	MCAO 85 mins	Reperfusion 22 hr
Male V (n=6)	20 ± 6	36.5 ± 0.2	36.8 ± 0.1	2.3 ± 0.2	2.0 ± 0.3
Male E2 (n=8)	22 ± 4	36.8 ± 0.3	36.8 ± 0.1	2.1 ± 0.2	2.3 ± 0.2
Female V (n=7)	25 ± 5	36.8 ± 0.1	36.5 ± 0.1	2.3 ± 0.3	2.4 ± 0.2
Female E2 (n=7)	18 ± 4	36.4 ± 0.4	36.9 ± 0.1	2.3 ± 0.2	2.3 ± 0.3

Values are mean ± s.e.m.

Table 2. Brain 17β-Estradiol.

Data are presented as mean±SEM. V, vehicle, E2, 17β estradiol. * indicates P < 0.05 compared to V and $ indicates P < 0.05 compared to Male E2.

Group
Male V (n=3)	2.1 ± 1.3
Male E2 (n=5)	336.2 ± 41.7*
Female V (n=4)	8.8 ± 3.3
Female E2 (n=4)	454.5 ± 25.3*$

Values are mean ± s.e.m.

In order to characterize possible sex differences in ischemic outcome in childhood experimental stroke, male and female mice of the same age (P20-25) were exposed to 90 minutes MCAO. Male and female mice exhibited nearly identical ischemic damage following MCAO, with edema-corrected hemispheric infarct volumes of 41±12% (n=9) and 41±14% (n=8), respectively (Figure 1). Ischemic damage was not different between sexes in the cortex (52±12% in male and 66±8.2% in female) and striatum (65±17% in male and 73±12% in female).

Figure 1.

No sex difference in infarct volumes following MCAO. Representative TTC-stained brain slices in male (A) and female (B) mice. C) Quantification of infarct volume in male (n=9) and female (n=8) mice. Data were presented as % infarct relative to contralateral structure, mean±SD.

In a separate cohort of mice, we examined the neuroprotective effectiveness of E2 in young male and female mice. E2 treated female mice had significantly smaller infarct volumes than vehicle treated female mice, exhibiting corrected hemispheric infarct volumes of 48±8.9% (n=7) in vehicle and 25±9.7% (n=7; P < 0.05) in E2 treated females (Figure 2A). Estrogen was equally as protective in cortex (67±15% in vehicle and 39±25% in E2) and striatum (88±16% in vehicle and 38±23% in E2). In contrast, E2 had no effect on male infarct volume in any of the brain regions analyzed (Figure 2A). Importantly, infarct volume of E2 treated female pediatric brain remained reduced when analyzed 3 days after reperfusion, exhibiting 51±8.1% (n=6) in vehicle and 27±16% (n=6, P < 0.05) in E2 treated female (Figure 2B).

Figure 2.

E2 decreases infarct volume in female brain and has no effect in male brain. A) Quantification of infarct volume analyzed 22 h after reperfusion in male and female mice treated with either vehicle (n=6 male; n=7 female) or E2 (n=8 male; n=7 female). B) Quantification of infarct volume analyzed 3 days after reperfusion in female mice treated with either vehicle (n=6) or E2 (n=6). Data presented as % infarct relative to contralateral structure, mean±SD. *P < 0.05 compared to vehicle. #P < 0.05 male compared to female.

To determine the relative expression of estrogen receptors alpha and beta (ERα and ERβ) in the juvenile mouse brain, quantitative real-time RT-PCR (qPCR) was performed from cortex of untreated males and females. Figure 3A demonstrates that a comparison of expression of each receptor in male and female cortex revealed a trend towards greater expression of each receptor in the male brain compared to the female brain (male ERα expression was 184±99% (n=5; p=0.13) that of female ERα and male ERβ was 165±44% (n=5; p=0.07) that of female ERβ). However, relative expression of ERα to ERβ was similar between males and females (ERα/ERβ ratio was approximately 20 in males and 18 in females).

Figure 3.

E2 decreases ischemia-induced increase in Bax. A) Quantification of estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) mRNA in the cortex of postnatal day 20–25 male (n=5) and female (n=5) mice. B) Quantification of ischemia-induced changes in Bax expression in the penumbra (cortex) of male and female mice treated with vehicle (n=6 male; n=5 female) or E2 (n=6 male; n=5 female). Data expressed as ratio of ipsilateral (ischemic penumbra) over corresponding contralateral cortex. C) Quantification of ischemia-induced changes in Bcl-2 expression in the penumbra (cortex) of male and female mice treated with vehicle (n=6 male; n=5 female) or E2 (n=6 male; n=5 female). Data expressed as ratio of ipsilateral (ischemic penumbra) over corresponding contralateral cortex. Data presented as mean±SD. *P < 0.05 compared to vehicle.

Because estrogen inhibits cell death following ischemia in adult animals by decreasing apoptosis, we analyzed the expression of Bax and Bcl-2, known contributors to apoptosis signaling, in our juvenile mouse MCAO model. Expression of Bax and Bcl-2 was analyzed in ischemic (penumbra) and non-ischemic cortex 22 hr after MCAO. We observed a significant increase in Bax mRNA expression in the penumbra relative to corresponding non-ischemic region in both male and female brains (Bax increased to 160±56% (n=6) of contralateral region in males and to 199±52% (n=5) in females). Consistent with the neuroprotective effect of E2 in females, E2 treatment significantly reduced the ischemia-induced increase in Bax expression, reducing Bax in the penumbra from 199±52% of non-ischemic cortex to 130±23% of corresponding E2 treated non-ischemic cortex (n=6; P < 0.05 compared to vehicle; Figure 3B). In contrast, E2 treatment did not significantly alter Bax expression in male animals (Figure 3B). Figure 3C demonstrates the minimal effects of ischemia and E2 treatment on Bcl-2 expression in the young brain.

Discussion

Our results indicate that ischemic injury is comparable between young male and female mice exposed to identical ischemic duration. This observation is in stark contrast to the adult experimental stroke literature which provides overwhelming evidence that females are relatively resistant to ischemia compared to males. The simplest explanation for the lack of sexual dimorphism in our experimental pediatric model is the low level of endogenous estrogen during childhood. Estrogen levels are comparable in boys and girls before puberty, at which time girls begin producing large levels of estrogen and boys begin producing androgens. Female mice experience a sharp increase in estrogen production, ie ‘puberty’, at approximately 5 weeks of age (P28-49) ¹⁷, ¹⁸. We used mice before postnatal day 25 and observed a lack of ‘benefit’ in females, consistent with the presumed similar levels of estrogen between male and female mice at this developmental stage. To directly test this hypothesis we administered estrogen to female mice, hypothesizing that we would produce a sex difference in ischemic outcome reminiscent of that observed in the adult. Indeed, female mice in the presence of exogenous estrogen have significantly smaller infarcts compared to their male counterparts.

We made the notable observation that estrogen failed to protect the juvenile male brain from transient focal ischemia. This is in contrast to the large body of literature that reports potent estrogen neuroprotection in adult male and female animals following experimental ischemia ^{8, 9, 19}. While the molecular mechanism of this novel observation warrants further study, our findings suggest that the failure of estrogen to have a neuroprotective effect on the juvenile male brain following AIS is not due to a lack of receptor expression, as we found robust expression of estrogen receptors in the male brain at this age. This suggested that a downstream mediator of estrogen signaling was implicated, which differed fundamentally between the juvenile male and female brain. There is strong evidence in the adult stroke literature that estrogen neuroprotection in both males and females is in part mediated by its potent anti-apoptotic actions ^10–12. However, we observe female-specific regulation of apoptosis in our novel model of childhood-onset AIS. E2 regulation of Bax in female brain and not male brain indicates a female-specific neuroprotective signaling that may underlie the reduced ischemia observed histologically in this study. Interestingly, we did not observe an effect of E2 on Bcl-2 expression in either male or female mice after MCAO. This finding may provide insight in to E2 neuroprotection in the young brain, implicating cell-extrinsic apoptosis signaling in the cell death and neuroprotection pathways engaged in childhood-onset AIS. Further research is necessary to fully elucidate this intriguing observation.

Summary/Conclusions

We have developed a new experimental model of childhood-onset arterial ischemic stroke in mice that will provide an important and clinically relevant platform for the study of ischemic signaling in the young brain. Our data demonstrates that the overall injury following transient focal ischemia is not different in young male and females; however we revealed fundamental sex differences in signaling. Female-specific estrogen neuroprotection in our juvenile mouse MCAO model is an important observation because it provides the first experimental evidence that protective strategies developed in the adult may not translate directly to children, making it extremely important to obtain experimental preclinical data in this age group.