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Mechanisms of neurodegeneration after severe hypoxic-ischemic injury in the neonatal rat brain
brain research 1629 (2015) 94–103
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Research report
Mechanisms of neurodegeneration after severe hypoxic-ischemic injury in the neonatal rat brain Rand Askalana,n, Nadia Gabarina, Edward A. Armstrongb, Yuan Fang Liua, Deema Couchmana, Jerome Y. Yagerb a Neuroscience and Mental Health Program, Hospital for Sick Children Research Institute, University of Toronto, Toronto, ON, Canada b Section of Pediatric Neurosciences, Stollery Children's Hospital, University of Alberta, Edmonton, AB, Canada
ar t ic l e in f o
abs tra ct
Article history:
Purpose: Apoptosis is implicated in mild-moderate ischemic injury. Cell death pathways in
Accepted 9 October 2015
the severely ischemic brain are not characterized. We sought to determine the role of
Available online 17 October 2015
apoptosis in the severely ischemic immature brain.
Keywords: Severe ischemia Neonatal brain Apoptosis Neuroprotection
Methods: Seven-day old rats were randomly assigned to mild-moderate or severe cerebral hypoxia-ischemia (HI) group. After ligating the right common carotid artery, animals were subjected to hypoxia for 90 min in the mild-moderate HI or 180 min in the severe HI. The core and peri-infarct area were measured in H&E stained brain sections using NIS Elements software. Brain sections were processed for caspase-3, AIF and RIP3 immuno-staining. Number of positive cells were counted and compared between the two groups. Results: The core constituted a significantly higher proportion of the ischemic lesion in the severely compared to the moderately injured brain (Po0.04) up to 7 days post-injury. Apoptotic cell death was significantly higher (Po0.05) in the core than the peri-infarct of the severe HI brain. In the peri-infarct area of severe HI, AIF-induced cell death increased over time and caspase-3 and AIF equally mediated neuronal death. Necroptosis was significantly higher (P ¼0.02) in the peri-infarct of the severe HI lesion compared to the moderate HI lesion. In males, but not in females, apoptosis was higher in moderate compared to severe HI. Conclusions: Caspase-independent cell death plays an important role in severe ischemic injury. Injury severity, timing of intervention post-injury and sex of the animal are important determinants in designing neuroprotective intervention for the severely ischemic immature brain. & 2015 Elsevier B.V. All rights reserved.
Abbreviations: AIF, Apoptosis-inducing factor; HI, Hypoxia-ischemia; GFAP, Glial fibrillary acidic protein; NF, Neurofilament n Correspondence to: Hospital for Sick Children, Division of Neurology 555 University Avenue Toronto, ON M5G 1X8 (Canada). E-mail address: [email protected] (R. Askalan). http://dx.doi.org/10.1016/j.brainres.2015.10.020 0006-8993/& 2015 Elsevier B.V. All rights reserved.
brain research 1629 (2015) 94–103
1.
Introduction
Brain cell death occurs as a continuum that ranges from necrosis to apoptosis (Carloni et al., 2007; Northington et al., 2011). Generally, two major pathways of apoptotic cell death have been identified: the intrinsic and extrinsic pathways. In the intrinsic pathway, a death stimulus causes the release of cytochrome c from the mitochondria into the cytosol, which will then bind to Apaf-1 and caspase-9 leading to the activation of caspase-3 and cell death. The extrinsic pathway involves the surface receptor Fas that activates caspase-8, which in turn activates caspase-3. The initiation of the intrinsic and extrinsic pathways is different but they both converge on caspase-3, which is known as the final “executioner” of apoptosis (Eldadah and Faden, 2000; Ferrer and Planas, 2003; Li and Yuan 2008). Although caspases have been recognized as primary mediators of apoptotic neuronal death (Northington et al., 2011; Hyman and Yuan 2012), accumulating evidence suggests that pathways independent of caspases also play a role in neuronal injury (Cregan et al., 2002; Stefanis, 2005). Apoptosis-inducing factor (AIF) is a mitochondrial inter-membrane flavorprotein that is released and trans-located to the cytoplasm and then to the nucleus in response to specific death stimuli (Daugas et al., 2000a, 2000b; Otera et al., 2005). Here, it causes chromatin condensation and DNA fragmentation (Daugas et al., 2000a, b; Cregan et al., 2002; Plesnila et al., 2004). Clinical studies indicate that sexual dimorphism is an important interdependent risk factor for ischemic brain injury (Fullerton et al., 2003; Golomb et al., 2009; Westmacott et al., 2009). These studies reported that males had increased incidence of and worse outcomes in response to HI injury compared to females. The mechanisms of these sex-related differences of HI injury are poorly understood. Neuronal culture models of HI injury showed that cell death was predominantly via caspase-dependent pathway in the XX neurons versus AIF-dependent pathway in the XY neurons (Du et al., 2004; Sharma et al., 2011), indicating that the mechanism of cell death is innately different between sexes. Sex-specific differences in cell death were also confirmed using in vivo mouse model of neonatal HI injury (Zhu et al., 2006; Mirza et al., 2015). Therefore understanding these sexual differences in the mechanism of cell death is instrumental in designing effective neuroprotective therapies for HI injury. Several studies from our laboratories and others exploring the mechanisms of cell death using neonatal animal models of cerebral ischemia have shown that apoptotic cell death is more prevalent in the immature than in the mature brain (Towfighi et al., 1995; Liu et al., 2004; Zhu et al., 2005). We have previously shown that AIF, in addition to caspase-3, is implicated in delayed cell death in the ischemic neonatal rat brain (Askalan et al., 2011). Most of these studies, however, are done in animal models of moderately injured ischemic brains. These animal models have been used in experimental neuroprotective strategies, some of which have shown robust efficacy against ischemic injury in the developing brain. However, with the exception of therapeutic hypothermia, none of these neuroprotective strategies have been successfully translated to clinical practice. Furthermore, while post-
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ischemic hypothermia has proven to be effective in newborns with mild-moderate hypoxic-ischemic encephalopathy, its protection does not extend to the severely injured neonatal brain, as shown in both animal (Nedelcu et al., 2000; Sabir et al., 2012) and human studies (Shankaran et al., 2005; Azzopardi et al., 2008). Taken together, these studies highlight the necessity for a better understanding of the mechanisms of cell death in the severely injured brain. We therefore sought in this study to characterize the pathways that lead to cell death in the core and peri-infarct area and to understand the influence of biological sex on these pathways in the neonatal rat brain subjected to severe ischemic injury. It is only by understanding these pathways that we will be able to design neuroprotective therapies or modify existing ones (i.e. hypothermia) to be effective in protecting the severely injured developing brain.
2.
Results
2.1. The core constituted most of the ischemic lesion in the severely injured brain The total infarct area, the core area and peri-infarct area were measured in the moderately and severely injured brains (n¼ 4–6 pups/time point in each group) as described in Section 4 and in Fig. 1. The core constituted a higher proportion of the ischemic lesion in the severely injured (mean percent core 9671%) compared to the moderately injured (69713%) brain at 4 days post-injury (p r0.04). After 4 days, that difference disappeared and the area of the core became the same in both types of injury by 1 week postinjury and remained the same at 2 weeks post-injury.
2.2. In severe ischemic injury, cell death was prolonged and mediated by caspase-dependent and caspase-independent pathways In the severely injured brain, cell death by caspase-3 and AIF pathways was significantly higher in the core compared to the peri-infarct area. Interestingly, these two pathways behaved differently over time depending on the region of the lesion. In the core, caspase-3 activity was significantly higher in day 14 compared to day 1 post-injury. In the periinfarct area, AIF-induced cell death increased over time to reach levels of the core levels by 2 weeks postinjury (Fig. 2). These results indicate that there is a prolonged window for therapeutic intervention in the severely ischemic brain, and in order to be effective in this type of injury, neuroprotective strategies must target both caspase-3 and AIF-mediated cell death as highlighted by our subsequent set of experiments comparing the role of these two pathways in severe and moderate ischemic brain injury.
2.3. Caspase-3 and AIF activities were differentially timedependent in the severely and moderately ischemic brain In the core of moderate ischemic injury, the number of nuclear AIF positive cells peaked early followed by a sharp
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Fig. 1 – Representative hematoxylin and eosin (H&E) sections showing the ischemic lesion in a moderately injured (a) and severely injured (b) immature brain. The total area of the core of the infarct is represented in green; the total peri-infarct area is represented in blue. For each specimen, one coronal section of 5 lm thickness was cut at the level of the hippocampus 24 h post-injury and stained with H&E. Using a Spot Flex camera attached to a Leica GZ7 stereoscope ( 10 objective lens), multiple images were taken of each section to encompass the entire infarct and its surrounding tissue (30–40 images/section). The areas of the core and peri-infarct were then measured on each image using NIS Elements software. The measured areas from the multiple images taken of the same section were then summed to determine the total areas of the core and peri-infarct for that section.
number of caspase-3 increased overtime but the number of nuclear AIF positive cells fluctuated without significant change among time points. The change in caspase-3 and AIF activity over time in the peri-infarct area of moderate ischemic injury was the same as that of the core. In the peri-infarct area of severe ischemic injury, however, AIF-induced cell death significantly increased by 2 weeks post-injury whereas caspaspe-3 activity did not significantly change overtime (Fig. 3B). These results, taken together, indicate that the type of injury and the timing post-injury may be key determinants in designing effective neuroprotective therapies against ischemic damage. Fig. 2 – In severe ischemic brain injury, caspase-3 and AIF dependent pathways mediate cell death in a time-sensitive pattern. P7 rat pups were subjected to severe HI injury and sacrificed at given time points. Brain sections were stained for active caspase-3 and AIF as described in Section 4.3. The number of positive cells for caspase-3 and nuclear AIF were counted in core and peri-infarct area of the lesion in 5–6 high-power fields ( 40) using the Image J computer software, and expressed as the mean percentage of the total number of cells per high power field. Cell death is significantly higher in the core compared to the peri-infarct area of the severely injured brain at all time points for caspase-3 pathway and on days 3 and 4 for AIF pathway. AIF-induced death in the peri-infarct area, however, increases over time to reach that of core levels by 2 weeks post-injury (mean percentage of nuclear AIF positive cells is 370.4% at 1 day verses 873% at 14 days post-injury). þ po0.05 compared to peri-infarct area; *p ¼0.03 compared to 14 days. and significant decrease by 48 h and remained low; the number of caspase-3 positive cells, on the other hand, exhibited a sharp and significant increase to beyond 2 weeks postinjury (Fig. 3A). In the core of severe ischemic injury, the
2.4. AIF was a key mediator of cell death in the peri-infarct area of the severely ischemic brain At 7 days post-injury, the mean percentage of casapase-3 positive cells was significantly (p r0.001) higher than the mean percentage of nuclear AIF positive cells in the core and peri-infarct area of the moderately injured brain (Fig. 4C and D) as well as in the core of the severely injured brain (Fig. 4A), indicating that caspase-3- was more active than AIFmediated cell death in these regions. There was no significant difference, however, between AIF and caspase-3 activity in the peri-infarct area of the severely injured brain (Fig. 4B).
2.5. Caspase-3 and AIF were equally active in mediating neuronal death in the peri-infarct area of the severely injured brain To identify the specific brain cells targeted by caspase-3 and AIF pathways, we performed double staining with the astrocytic marker anti-GFAP and the neuronal marker anti-NF. Astrocytic and neuronal deaths were predominantly mediated by caspase-3 dependent pathway in the core of the lesion (Fig. 5A). In the peri-infarct area, however, astrocytic death was predominantly due to caspase-3, whereas
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Fig. 3 – Activation of cell death pathways is sensitive to injury type and timing post-injury. P7 rat pups were subjected to moderate or severe HI injury and sacrificed at given time points. Brain sections were stained for active caspase-3 and AIF (Section 4.3). The number of positive cells for caspase-3 and nuclear AIF were counted in core and peri-infarct area of the lesion in 5–6 high-power fields ( 40) using the Image J computer software, and expressed as the mean percentage of the total number of cells per high power field. (A) In the core of moderate injury, AIF translocation peaked early followed by a significant decrease by 48 h and then stayed low (mean percentage of nuclear AIF positive cells at 1 day postinjury 2972% verses 973% at 14 days post-injury). Caspase-3, on the other hand, exhibited a steady increase to beyond 2 weeks post-injury (mean percentage of caspase-3 positive cells at 2 days post-injury 1774% versus 4375% at 14 days postinjury). *po0.01 compared to day 14. In the severely injured core, caspase-3 increased but AIF did not change significantly overtime. (B) Similar to the moderately injured core, in the moderately injured peri-infarct area AIF peaked early followed by a significant decrease whereas caspase-3 exhibited a significant increase to beyond 2 weeks post-injury. Caspase-3 in the severely injured peri-infarct area fluctuated but did not significantly increase overtime. AIF-induced cell death, however, significantly increased by 2 weeks post-injury in the severely injured peri-infarct (370.4% at 1 day postinjury verses 873% at 14 days post-injury). *po0.05 compared to day 14.
Fig. 4 – The proportions of cells dying by AIF and caspase-3 dependent pathways are similar in the peri-infarct area of the severely injured brain by 7 days postinjury. P7 rat pups were subjected to moderate or severe HI injury and sacrificed at 7 days post-injury. Brain sections were stained for active caspase-3 and AIF and the number of positive cells for caspase 3 and nuclear AIF were counted in core and peri-infarct area of the lesion in 5–6 high-power fields ( 40) using the Image J computer software (Section 4.3). The mean percentage of caspase-3 positive cells (83710%) is significantly (p r0.001) more than the mean percentage of AIF positive cells (1773%) in the core of the severely injured brain (A) as well as in the core (C) and periinfarct area (D) of the moderately injured brain indicating that caspase-3 is more active than AIF-mediated cell death in these regions. There was no significant difference, however, between AIF and caspase-3 activity in the peri-infarct of the severely injured brain (B).
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Fig. 5 – Caspase-3 and AIF-dependent pathways mediate astrocytic and neuronal cell death. Double staining with anti-AIF/antiGFAP, an astrocytic marker, anti-caspase-3/anti-GFAP, anti-AIF/anti-NF, neuronal marker and anti-caspase-3/anti-NF in the core (A) and peri-infarct area (B) of the severely injured lesion was performed in P7 rat brain as described in Section 4.4. Immunodetection was performed using immuno-fluorescence staining method (n¼6–8 pups/marker). Arrows indicate the co-localization of nuclear AIF and GFAP or NF as well as caspase-3 and GFAP or NF in the cortex at the dorsal hippocampal level.
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neuronal death was equally mediated by both caspase-3 and AIF pathways (Fig. 5B). These results provide another line of evidence on the important role of caspase-independent cell death in the severely ischemic developing brain.
2.6.
Necroptosis was not limited to the core of the lesion
Studies have shown that apoptosis and necroptosis (programmed necrosis) coexist in this model of hypoxicischemic injury (Nakajima et al., 2000; Chavez-Valdez et al., 2012a). Necroptosis was shown to be an early mechanism of cell death occurring within the first 24 h postischemic injury (Vieira et al., 2014; Chavez-Valdez et al., 2012b) and inversely linked to caspase-dependent cell death (Vieira et al., 2014; Northington et al., 2011). Therefore we examined the expression of RIP3 2 days post-injury where a decrease in caspase-3 was observed in the moderately and severely injured brains. In the moderately ischemic brain, necroptosis, as depicted by RIP3 staining, predominantly occurred in the core of the lesion (Fig. 6). Interestingly, the peri-infarct area of the severely ischemic brain had significantly more necroptosis than the peri-infarct area of the moderately injured brain and necroptotic cell death was as active in the peri-infarct area as it was in the core of the lesion (Fig. 6).
2.7. Sex of the animal is another determinant of the mechanism of ischemia-induced cell death Sex is an important determinant of many disease processes including response to cerebral ischemic injury. Studies have suggested that male and female neurons utilize different pathways in response to ischemic injury independent of hormonal factors (Du et al., 2004; Krishnan et al., 2009;
Fig. 6 – There is increased necroptotic cell death in the periinfarct area of the severely injured compared to the moderately injured brain. P7 rat pups were subjected to moderate or severe HI injury and sacrificed at 2 days postinjury. Brain sections were stained for RIP3 and the number of positive cells was counted in core and peri-infarct area of the lesion in 5–6 high-power fields ( 40) using the Image J computer software (Section 4.3). The mean percentage of RIP3 positive cells was significantly higher (*p ¼0.02) in the peri-infarct area of severe injury (1272%) compared to that in the peri-infarct area of moderate injury (472%).
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Mirza et al., 2015). We therefore sought to investigate the influence of injury type on sex-related differences in the mechanism of cell death in the ischemic neonatal brain by 7 days postinjury. The 3-way ANOVA analysis showed that sex of the animal (F¼ 4.6; p¼ 0.033; Fig. 7A) and types of cell death (F ¼5.0; p¼ 0.027; Fig. 7B) interactions were significant. Males with moderate ischemic injury had significantly more caspase-3 and AIF-induced cell death compared to females (difference ¼1.04%; po0.001; Fig. 7A). Severe injury abolished this difference between the two sexes (difference¼ 0.19%; p40.05). In both the moderately injured (difference ¼2.07; po0.001) and severely injured brain (difference ¼1.22; po0.001), caspase-3 dependent cell death was more pronounced than AIF-dependent cell death in male and female pups (Fig. 7B).
3.
Discussion
In this study we have subjected P7 rat pups to 180 min of hypoxia resulting in severe HI injury and compared mechanisms of cell death to those in rat pups subjected to 90 min of hypoxia (i.e. mild-moderate HI injury). We have shown that the core constituted most of the ischemic lesion in the severely injured brain up to 7 days post-injury. Therefore understanding the mechanisms of injury in the core of the severe ischemic lesion is essential to design proper therapies for this type of injury. Moreover, these results emphasized the importance of timely intervention for the moderately injured brain before the core of the lesion expands to resemble that of severe injury. We found that in the moderately injured animals caspase-dependent cell death was prominent up to 2 weeks post-injury whereas AIFdependent cell death was prominent in the severely injured animals for the same time period. We have demonstrated for the first time multiple lines of evidence that in severe HI injury of the developing brain 1) apoptosis is prolonged and plays a significant role in causing ischemic damage and 2) caspase-independent neuronal death is a greater contributor to ischemic damage than in moderate HI injury. The first line of evidence came from investigating the activity of caspase-dependent and -independent pathways in the two areas of the lesion overtime. In severe ischemic injury, both caspase-3 and AIF activity were high in the core compared to the peri-infarct area and AIF translocation increased in the peri-infarct area overtime to reach that of the core by 2 weeks postinjury. This was in contrast to what was observed in moderate injury where AIF-dependent pathway activity was higher early post-injury and significantly decreased by 2 days post-injury in the core and peri-infarct area of the lesion. Caspase-3 activity in the core and periinfarct area of the moderately injured brain exhibited an abrupt decrease at 2 days post-injury followed by a significant increase that was sustained up to 2 weeks post-injury. This increase in caspase activity was also observed in the core but not the peri-infarct area of the severely injured brain. Different temporal patterns of caspase-3 activity were previously reported and could not be fully explained. They may be related to the region of the brain, extent of injury (Nakajima
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Fig. 7 – The mechanism of ischemia-induced cell death is influenced by the sex of the animal. P7 male and female rat pups were subjected to moderate or severe HI injury and sacrificed at 7 days post-injury, sectioned and stained for caspase-3 and AIF as described in Section 4.3. The number of positive cells was counted in 5–6 high-power fields ( 40) using the Image J computer software. 3-way ANOVA analysis was used to determine the effect of sex, severity of injury and types of cell death on the ischemic damage. (A) Caspase-3 and AIF-mediated cell death was significantly (difference¼ 1.04%; po0.001) higher in males (23.1872) compared to females (1472) with moderate HI injury. There was no significant difference in types of cell death between males and females with severe HI injury. (B) The mean percentage (7SEM) of active caspases-3 positive cells was significantly higher than the mean percentage of AIF positive nuclei in the moderately injure (27.5572; 10.0571 respectively; difference¼ 2.07; po0.001) and the severely injured (14.6671; 7.0271; difference¼1.22; po0.001) in both sexes.
et al., 2000) and release of Cyt c (Zhu et al., 2006) or limiting the time window of the study to 24 h post-injury (Manabat et al., 2003). The importance of caspase-independent cell death in severe ischemic injury was further confirmed when we compared the number of active caspase-3 and nuclear AIF positive cells at the specific time point post-injury of 7 days. In the core of moderate and severe ischemic injury, the number of active caspase-3 positive cells was significantly higher than nuclear AIF positive cells. However, this difference was abolished in the peri-infarct area of the severe injury lesion with AIF having a greater contribution to cell death. The second line of evidence came from the specific brain cells that were affected by caspase-3 and AIF pathways in the severely injured animals. Astrocytic death was predominately mediated by the caspase-3 dependent pathway. The AIF-dependent pathway was as active as the caspase-3 dependent pathway in mediating neuronal death. Therefore, a neuroprotective approach has to address both forms of neuronal death to be effective. The third line of evidence came from investigating the expression of RIP3 postischemic injury. Programmed necrosis, known as necroptosis, is another form of cell death shown to play a role in tissue damage response (Zhou et al., 2012; Sun and Wang 2014). RIP3 is a determining factor in the necroptosis signaling pathway by initiating a series of phosphorylation events (Sun et al., 2012). The significance of these events and the mechanism by which RIP3 causes
programmed necrosis are still under investigation. As one would expect, necroptosis occurred primarily in the core of the lesion in the moderately injured ischemic neonatal brain. Interestingly, however, there was no significant difference in the number of RIP3 positive cells in the core and peri-infarct area of the lesion in the severely injured neonatal brain. Investigating the mechanism of RIP3-mediated necroptosis and the search for compounds that will block necroptosis are ongoing. The development of a necroptosis pathway inhibitor may be essential for any therapeutic approach to reduce the damage of severe ischemic injury. Taken together, these findings indicate that similar to what has been previously reported for moderate injury (Nakajima et al., 2000; Askalan et al., 2011), severe ischemic injury in the neonatal brain also has a prolonged therapeutic window for neuroprotective intervention. The timing postinjury, however, is crucial in determining the therapeutic target. Moreover, these results illustrate that this intervention cannot be the same for both injury types and highlight the necessity of taking into consideration caspaseindependent pathways of cell death when designing a neuroprotective therapy for the severely injured brain. Most therapies that have been tried to date in this animal model of neonatal brain HI injury were designed to attenuate caspase-3 dependent cell death (Han et al., 2002; Feng et al., 2003; Joly et al., 2004), which may explain why their neuroprotective effects have not been extended to animals with severe injury.
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Clinical and experimental studies indicate that sex is an important risk factor for ischemic brain injury. Recent clinical studies have reported an increased incidence of arterial ischemic stroke (Lynch et al., 2002; Golomb et al., 2009) and cerebral sinus venous thrombosis (Golomb et al., 2009) in male children. This male predominance persists even after excluding cases of posttraumatic brain ischemic injury (Fullerton et al., 2003; Golomb et al., 2009). These sexrelated differences in cerebral ischemic injury may be attributed to differences in cell death pathways activated by the injury. We have shown here increased caspase-3 and AIF activity in males compared to females with moderate injury but that there were no sex differences in the caspase-3 activity and AIF translocation in rat pups with severely injured brain. These results suggest that differences in mechanism of cell death induced by moderate ischemic brain injury may contribute to the clinical differences seen between male and female patients. Zhu et al. (2006) reported no difference in the number of AIF positive nuclei or active caspase-3 positive cells in the cortex of male and female P9 mice at 1 day and 3 days postinjury whereas other studies reported that AIF-dependent cell death was predominant in males whereas caspasedependent cell death was predominant in females (Renolleau et al., 2007; Weis et al., 2014). This discrepancy in results may be attributed to differences in the experimental HI injury model used, the duration of hypoxia (extent of injury), the time post-injury at which the animals were sacrificed or to the brain region being analyzed. The mechanisms of these sex-related differences of neonatal HI injury are poorly understood. Estrogen is unlikely to account for the observed protection in females because circulating estrogen is minimal in the neonatal female (McCullough and Hurn, 2003; Konkle and McCarthy, 2011). These results may explain what we and subsequently others (Westmacott et al., 2009; Smith et al., 2014) have reported in males having worse outcomes of HI injury compared to females. We have shown that males with neonatal ischemic stroke have more cognitive deficits compared to females with the same degree and timing of injury (Westmacott et al., 2009). Moreover, these results highlight the critical need for designing experimental studies that will take into consideration the interaction of the severity of injury and sex when investigating efficacy of neuroprotective therapies. Thus far, post-ischemic hypothermia is the most promising neuroprotective measure against ischemic injury in the developing brain. Several multi-center randomized clinical trials over the last 10 years have shown that post-ischemic hypothermia is effective in improving the outcome in newborns with perinatal asphyxia (Eicher et al., 2005; Shankaran et al., 2005; Azzopardi et al., 2008). In several analyses, however, this protective effect was largely afforded only to those infants with moderate hypoxic-ischemic encephalopathy and it did not protect those infants with severe encephalopathy (Shankaran et al., 2005; Azzopardi et al., 2008). This lack of protection was thought to occur because the majority of severe injury to the brain was actually within the core and was necrotic in nature, which was not amenable to current therapeutic interventions. However, the results presented here indicate that this may not be true, as apoptotic cell
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death plays an important role in the core of the lesion in the developing brain. Future studies to elucidate the effect of hypothermia on caspase-independent cell death will enable us to identify the most appropriate anti-apoptotic therapies that may be used in combination with hypothermia in order to amplify its neuroprotection, leading to a reduction in ischemic damage in the severely injured immature brain. In conclusion, we have shown that caspase-independent cell death has a pivotal role in severe ischemic injury of the neonatal brain of both sexes in a time-sensitive manner. For a therapy to be effective in reducing damage post severe ischemic injury, it has to target not just caspase-3 dependent cell death but also AIF-dependent cell death and necroptosis.
4.
Experimental procedure
4.1.
Neonatal model of hypoxia-ischemia
As previously described (Askalan et al., 2009, 2011), we induced hypoxic-ischemic injury in the neonatal rat brain using the Rice– Vannucci model with a prolonged duration of hypoxia to elicit severe injury. In brief, 7-day-old male and female rat pups were randomly assigned to either moderate or severe cerebral hypoxia-ischemia. In both groups, the right common carotid artery was ligated using a double 4–0 silk tie and cut between the sutures. During the procedure, all animals were anesthetized with isoflurane (4% induction, 1% maintenance). Following ligation, rat pups were allowed to recover for 2 h, after which ischemia was produced in the hemisphere ipsilateral to the carotid artery occlusion by subjecting the animal to hypoxia (8% oxygen) for 90 or 180 min for the moderate and severe HI injury groups, respectively. Mortality rates were 2.4% and 21.6% for the 90 and 180 min respectively. Body temperature was regulated in all groups at 36.570.5 1C during hypoxia by maintaining the rat pups in a servo-controlled neonatal Isolette (Narco Scientific, Hatboro, Pa, USA). Animals were sacrificed at 1, 2, 3, 4, 7 and 14 days after the HI insult using pentobarbital. Brains were rapidly removed and processed for hematoxylin and eosin (H&E) staining and immunohistochemistry. All animal procedures were approved by the health sciences animal care and use committee at the University of Alberta.
4.2.
Measurement of infarct area
For each specimen, one coronal section of 5 mm thickness was cut at the level of the hippocampus and stained with H&E. Using a Spot Flex camera attached to a Leica GZ7 stereoscope ( 10 objective lens), multiple images were taken of each section to encompass the entire infarct and its surrounding tissue (30–40 images/section). The core and the peri-infarct area of the lesion were then measured on each image using NIS Elements software. As we have previously described, normal brain parenchyma shown by H&E staining directly adjacent to the ischemic ‘core’ was defined as the peri-infarct area (Askalan et al., 2009). The measured areas from the multiple images taken of the same section were then summed to determine the total areas of the core and the peri-infarct for that section. A representation of these measurements is shown in Fig. 1.
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4.3.
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Immunohistochemistry
At the previously specified time points, brains from P7 injured rat pups were removed and immediately fixed in 10% formalin. Paraffin-embedded coronal sections were cut (5 μm) at the level of the hippocampus, de-waxed with xylene, hydrated and pretreated with the heat-induced antigen retrieval technique. Serial sections were then stained with commercially available specific primary antibodies for activated caspase-3 (rabbit polyclonal anti-cleaved caspase-3 (Asp175) antibody, 1:400, Cell Signaling), AIF (rabbit anti-AIF, 1:100, Cell Signaling) and RIP3 (rabbit anti-RIP3, 1:150, Abcam) at 4 1C overnight. Caspase-3, nuclear AIF and RIP3 were detected by appropriate horseradish peroxidase secondary antibodies (HRP, 1:100, Chemicon) for 1 h at room temperature and DAB substrate kit for Peroxidase/Vector/SK-4800. The number of positive cells for caspase-3, nuclear AIF and RIP3 were counted in 5–6 high-power fields ( 40) using the Image J computer software (National Institutes of Health, Bethesda, MD, USA) and expressed as the mean percentage7SEM of the total number of cells per high power field.
4.4.
Sources of funding This study was funded in part by the Heart and Stroke Foundation of Ontario to R.A. (3210326014)
Acknowledgment The authors would like to that Dr. Priyanka Shah for her invaluable assistance in the statistical analysis of the data.
r e f e r e n c e s
Double staining
Antibody for glial fibrillary acidic protein (GFAP), and neurofilament (NF) are well-established markers for detecting astrocytes, and neurons, respectively. Double staining with anti-GFAP/anti-caspase-3, anti-GFAP/anti-AIF, anti-NF/anticaspase-3 and anti-NF/anti-AIF were done to determine the predominant mechanism of cell death in astrocytes and neurons following ischemic injury. Frozen brain sections were incubated simultaneously with AIF antibody (rabbit anti-AIF, 1:100; cell signaling) and mouse anti-GFAP (1:1000, Sigma chemicals Co), or chicken polyclonal anti-NF (1:500, millipore). Another set of adjacent sections were incubated simultaneously with caspase-3 antibody (rabbit polyclonal anti-cleaved caspase-3 (Asp175) antibody, 1:400; Cell Signaling) and GFAP or NF. Immuno-reactivity was visualized using appropriate combinations of donkey anti-rabbit Fitc (1:200, invivogen) and donkey anti-mouse Cy2 (1:200, invivogen) or donkey anti-chicken Cy3 (1:200, chemicon) secondary antibodies and nuclei were counterstained with DAPI (Sigma). Multichannel images were captured and analyzed with Nikon NIS-Element Basic Research Image system.
4.5.
URL http://www.rstudio.com/. 2015). Experimental groups were compared using paired t-test. Results were considered to be statistically significant if two-tailed po0.05. All data were presented as means7SEM, and other statistical analyses were done using GraphPad InStat (GraphPad Software Inc., San Diego, USA).
Statistical analysis
At least six rat pups were utilized for each measurable time point in the moderately and severely injured groups. Threeway ANOVA was conducted to compare the main effects of and interaction among the type of cell death (AIF-induced, caspace-3), sex (female, male) and induced severity of the ischemic injury (moderately-injured, severely-injured) on the ischemic neonatal brain; because the distribution of the dependent variable, percentage of positive cells per HPF, was skewed, a square root transformation was performed. Significant findings in ANOVA were followed up by post-hoc tests, conducted using Tukey's honest significant differences method; these analyses were carried out in RStudio (Integrated Development for R. RStudio, Inc., Boston, MA
Askalan, R., Salweski, R., Tuor, U.I., Hutchison, J., Hawkins, C., 2009. X-linked inhibitor of apoptosis protein expression after ischemic injury in the human and rat developing brain. Pediatr. Res. 65, 21–26. Askalan, R., Wang, C., Shi, H., Armstrong, E., Yager, J.Y., 2011. The effect of postischemic hypothermia on apoptotic cell death in the neonatal rat brain. Dev. Neurosci. 33, 320–329. Azzopardi, D., Brocklehurst, P., Edwards, D., Halliday, H., Levene, M., Thoresen, M., et al., 2008. The TOBY study. Whole body hypothermia for the treatment of perinatal asphyxial encephalopathy: a randomised controlled trial. BMC Pediatr. 8, 17. Carloni, S., Carnevali, A., Cimino, M., Balduini, W., 2007. Extended role of necrotic cell death after hypoxia-ischemia-induced neurodegeneration in the neonatal rat. Neurobiol. Dis. 27, 354–361. Chavez-Valdez, R., Martin, L.J., Northington, F.J., 2012a. Programmed necrosis: a prominent mechanism of cell death following neonatal brain injury. Neurol. Res. Int. 2012, 1–12. Chavez-Valdez, R., Martin, L.J., Flock, D.L., Northington, F.J., 2012b. Necrostatin-1 attenuates mitochondrial dysfunction in neurons and astrocytes following neonatal hypoxia-ischemia. Neuroscience 219, 192–203. Cregan, S.P., Fortin, A., MacLaurin, J.G., Callaghan, S.M., Cecconi, F., Yu, S.W., et al., 2002. Apoptosis-inducing factor is involved in the regulation of caspase-independent neuronal cell death. J. Cell Biol. 158, 507–517. Daugas, E., Susin, S.A., Zamzami, N., Ferri, K.F., Irinopoulou, T., Larochette, N., et al., 2000a. Mitochondrio-nuclear translocation of AIF in apoptosis and necrosis. Faseb J. 14, 729–739. Daugas, E., Nochy, D., Ravagnan, L., Loeffler, M., Susin, S.A., Zamzami, N., et al., 2000b. Apoptosis-inducing factor (AIF): a ubiquitous mitochondrial oxidoreductase involved in apoptosis. FEBS Lett. 476, 118–123. Du, L., Bayir, H., Lai, Y., Zhang, X., Kochanek, P.M., Watkins, S.C., et al., 2004. Innate gender-based proclivity in response to cytotoxicity and programmed cell death pathway. J. Biol. Chem. 279, 38563–38570. Eicher, D.J., Wagner, C.L., Katikaneni, L.P., Hulsey, T.C., Bass, W.T., Kaufman, D.A., et al., 2005. Moderate hypothermia in neonatal encephalopathy: efficacy outcomes. Pediatr. Neurol. 32, 11–17. Eldadah, B.A., Faden, A.I., 2000. Caspase pathways, neuronal apoptosis, and CNS injury. J. Neurotrauma 17, 811–829.
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Feng, Y., Fratkin, J.D., LeBlanc, M.H., 2003. Inhibiting caspase-8 after injury reduces hypoxic-ischemic brain injury in the newborn rat. Eur. J. Pharmacol. 481, 169–173. Ferrer, I., Planas, A.M., 2003. Signaling of cell death and cell survival following focal cerebral ischemia: life and death struggle in the penumbra. J. Neuropathol. Exp. Neurol. 62, 329–339. Fullerton, H.J., Wu, Y.W., Zhao, S., Johnston, S.C., 2003. Risk of stroke in children: ethnic and gender disparities. Neurology 61, 89–194. Golomb, M.R., Fullerton, H.J., Nowak-Gottl, U., Deveber, G., 2009. Male predominance in childhood ischemic stroke: findings from the international pediatric stroke study. Stroke 40, 52–57. Han, B.H., Xu, D., Choi, J., Han, Y., Xanthoudakis, S., Roy, S., et al., 2002. Selective, reversible caspase-3 inhibitor is neuroprotective and reveals distinct pathways of cell death after neonatal hypoxic-ischemic brain injury. J. Biol. Chem. 277, 30128–30136. Hyman, B.T., Yuan, J., 2012. Apoptotic and non-apoptotic roles of caspases in neuronal physiology and pathophysiology. Nat. Rev. Neurosci. 13, 395–406. Joly, L.-M., Mucignat, V., Mariani, J., Plotkine, M., CharriautMarlangue, C., 2004. Caspase inhibition after neonatal ischemia in the rat brain. J. Cereb. Blood Flow Metab. 24, 124–131. Konkle, A.T., McCarthy, M.M., 2011. Developmental time course of estradiol, testosterone, and dihydrotestosterone levels in discreet regions of male and female rat brain. Endocrinology 152 (1), 223–235. Krishnan, S., Intlekofer, K.A., Aggison, L.K., Petersen, S.L., 2009. Central role of TRAF- interacting protein in a new model of brain sexual differentiation. Proc. Natl. Acad. Sci. USA 106, 16692–16697. Li, J., Yuan, J., 2008. Caspases in apoptosis and beyond. Oncogene 27, 6194–6202. Liu, C.L., Siesjo, B.K., Hu, B.R., 2004. Pathogenesis of hippocampal neuronal death after hypoxia-ischemia changes during brain development. Neuroscience 127, 113–123. Lynch, J.K., Hirtz, D.G., DeVeber, G., Nelson, K.B., 2002. Report of the national institute of neurological disorders and stroke workshop on perinatal and childhood stroke. Pediatrics 109, 116–123. Manabat, C., Han, B.H., Wendland, M., Derugin, N., Fox, C.K., Choi, J., et al., 2003. Reperfusion differentially induces caspase-3 activation in ischemic core and penumbra after stroke in immature brain. Stroke 34, 207–213. McCullough, L.D., Hurn, P.D., 2003. Estrogen and ischemic neuroprotection: an integrated view. Trends Endocrinol. Metab. 14, 228–235. Mirza, A.M., Ritzel, R., Xu, Y., McCullough, D.,L., Liu, F., 2015. Sexually dimorphic outcomes and inflammatory responses in hypoxic-ischemic encephalopathy. J. Neuroinflamm. 12, 32–42. Nakajima, W., Ishida, A., Lange, M.S., Gabrielson, K.L., Wilson, M. A., Martin, L.J., et al., 2000. Apoptosis has a prolonged role in the neurodegeneration after hypoxic ischemia in the newborn rat. J. Neurosci. 20, 7994–8004. Nedelcu, J., Klein, M.A., Aguzzi, A., Martin, E., 2000. Resuscitative hypothermia protects the neonatal rat brain from hypoxicischemic injury. Brain Pathol. 10, 61–71. Northington, F.J., Chavez-Valdez, R., Martin, L.J., 2011. Neuronal cell death in neonatal hypoxia-ischemia. Ann. Neurol. 69, 743–758. Otera, H., Ohsakaya, S., Nagaura, Z., Ishihara, N., Mihara, K., 2005. Export of mitochondrial AIF in response to proapoptotic
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stimuli depends on processing at the intermembrane space. EMBO J. 24, 1375–1386. Plesnila, N., Zhu, C., Culmsee, C., Groger, M., Moskowitz, M.A., Blomgren, K., 2004. Nuclear translocation of apoptosisinducing factor after focal cerebral ischemia. J. Cereb. Blood Flow Metab. 24, 458–466. Renolleau, S., Fau, S., Goyenvalle, C., Joly, L.-M., Chauvier, D., Jacotot, E., et al., 2007. Specific caspase inhibitor Q-VD-OPh prevents neonatal stroke in P7 rat: a role for gender. J. Neurochem. 100, 1062–1071. Sabir, H., Scull-Brown, E., Liu, X., Thoresen, M., 2012. Immediate hypothermia is not neuroprotective after severe hypoxiaischemia and is deleterious when delayed by 12 h in neonatal rats. Stroke 43, 3364–3370. Shankaran, S., Laptook, A.R., Ehrenkranz, R.A., Tyson, J.E., McDonald, S.A., Donovan, E.F., et al., 2005. Whole- body hypothermia for neonates with hypoxic-ischemic encephalopathy. N. Engl. J. Med. 353, 1574–1584. Sharma, J., Nelluru, G., Wilson, M.A., Johnston, M.V., Hossain, M. A., 2011. Sex-specific activation of cell death signalling pathways in cerebellar granule neurons exposed to oxygen glucose deprivation followed by reoxygenation. ASN Neuro 3, 85–97. Smith, A.L., Alexander, M., Rosenkrantz, T.S., Sadek, M.L., Fitch, R. H., 2014. Sex differences in behavioural outcome following neonatal hypoxia ischemia: insights from a clinical metaanalysis and a rodent model of induced hypoxic ischemic brain injury. Exp. Neurol. 254, 54–67. Stefanis, L., 2005. Caspase-dependent and -independent neuronal death: two distinct pathways to neuronal injury. Neuroscientist 11, 50–62. Sun, L., Wang, H., Wang, Z., He, S., Chen, S., Liao, D., et al., 2012. Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell 148, 213–227. Sun, L., Wang, X., 2014. A new kind of cell suicide: mechanisms and functions of programmed necrosis. Trends Biochem. Sci. 39, 587–593. Towfighi, J., Zec, N., Yager, J., Housman, C., Vannucci, R.C., 1995. Temporal evolution of neuropathologic changes in an immature rat model of cerebral hypoxia: a light microscopic study. Acta Neuropathol. 90, 375–386. Vieira, M., Fernandes, J., Carreto, L., Anuncibay-Soto, B., Santos, M., Han, J., et al., 2014. Ischemic insults induce necroptotic cell death in hippocampal neurons through the up-regulation of endogenous RIP3. Neurobiol. Dis. 68, 26–36. Weis, S.N., Toniazzo, A.P., Ander, B.P., Zhan, X., Careaga, M., Ashwood, P., et al., 2014. Autophagy in the brain of neonates following hypoxia-ischemia shows sex-and region specific effects. Neuroscience 256, 201–209. Westmacott, R., MacGregor, D., Askalan, R., deVeber, G., 2009. Late emergence of cognitive deficits after unilateral neonatal. Stroke 40, 2012–2019. Zhou, Z., Han, V., Han, J., 2012. New components of the necroptotic pathway. Protein Cell 3, 811–817. Zhu, C., Wang, X., Xu, F., Bahr, B.A., Shibata, M., Uchiyama, Y., et al., 2005. The influence of age on apoptotic and other mechanisms of cell death after cerebral hypoxia-ischemia. Cell Death Diff. 12, 162–176. Zhu, C., Xu, F., Wang, X., Shibata, M., Uchiyama, Y., Blomgren, K., et al., 2006. Different apoptotic mechanisms are activated in male and female brains after neonatal hypoxia-ischaemia. J. Neurochem. 96, 1016–1027.
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Research report
Mechanisms of neurodegeneration after severe hypoxic-ischemic injury in the neonatal rat brain Rand Askalana,n, Nadia Gabarina, Edward A. Armstrongb, Yuan Fang Liua, Deema Couchmana, Jerome Y. Yagerb a Neuroscience and Mental Health Program, Hospital for Sick Children Research Institute, University of Toronto, Toronto, ON, Canada b Section of Pediatric Neurosciences, Stollery Children's Hospital, University of Alberta, Edmonton, AB, Canada
ar t ic l e in f o
abs tra ct
Article history:
Purpose: Apoptosis is implicated in mild-moderate ischemic injury. Cell death pathways in
Accepted 9 October 2015
the severely ischemic brain are not characterized. We sought to determine the role of
Available online 17 October 2015
apoptosis in the severely ischemic immature brain.
Keywords: Severe ischemia Neonatal brain Apoptosis Neuroprotection
Methods: Seven-day old rats were randomly assigned to mild-moderate or severe cerebral hypoxia-ischemia (HI) group. After ligating the right common carotid artery, animals were subjected to hypoxia for 90 min in the mild-moderate HI or 180 min in the severe HI. The core and peri-infarct area were measured in H&E stained brain sections using NIS Elements software. Brain sections were processed for caspase-3, AIF and RIP3 immuno-staining. Number of positive cells were counted and compared between the two groups. Results: The core constituted a significantly higher proportion of the ischemic lesion in the severely compared to the moderately injured brain (Po0.04) up to 7 days post-injury. Apoptotic cell death was significantly higher (Po0.05) in the core than the peri-infarct of the severe HI brain. In the peri-infarct area of severe HI, AIF-induced cell death increased over time and caspase-3 and AIF equally mediated neuronal death. Necroptosis was significantly higher (P ¼0.02) in the peri-infarct of the severe HI lesion compared to the moderate HI lesion. In males, but not in females, apoptosis was higher in moderate compared to severe HI. Conclusions: Caspase-independent cell death plays an important role in severe ischemic injury. Injury severity, timing of intervention post-injury and sex of the animal are important determinants in designing neuroprotective intervention for the severely ischemic immature brain. & 2015 Elsevier B.V. All rights reserved.
Abbreviations: AIF, Apoptosis-inducing factor; HI, Hypoxia-ischemia; GFAP, Glial fibrillary acidic protein; NF, Neurofilament n Correspondence to: Hospital for Sick Children, Division of Neurology 555 University Avenue Toronto, ON M5G 1X8 (Canada). E-mail address: [email protected] (R. Askalan). http://dx.doi.org/10.1016/j.brainres.2015.10.020 0006-8993/& 2015 Elsevier B.V. All rights reserved.
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1.
Introduction
Brain cell death occurs as a continuum that ranges from necrosis to apoptosis (Carloni et al., 2007; Northington et al., 2011). Generally, two major pathways of apoptotic cell death have been identified: the intrinsic and extrinsic pathways. In the intrinsic pathway, a death stimulus causes the release of cytochrome c from the mitochondria into the cytosol, which will then bind to Apaf-1 and caspase-9 leading to the activation of caspase-3 and cell death. The extrinsic pathway involves the surface receptor Fas that activates caspase-8, which in turn activates caspase-3. The initiation of the intrinsic and extrinsic pathways is different but they both converge on caspase-3, which is known as the final “executioner” of apoptosis (Eldadah and Faden, 2000; Ferrer and Planas, 2003; Li and Yuan 2008). Although caspases have been recognized as primary mediators of apoptotic neuronal death (Northington et al., 2011; Hyman and Yuan 2012), accumulating evidence suggests that pathways independent of caspases also play a role in neuronal injury (Cregan et al., 2002; Stefanis, 2005). Apoptosis-inducing factor (AIF) is a mitochondrial inter-membrane flavorprotein that is released and trans-located to the cytoplasm and then to the nucleus in response to specific death stimuli (Daugas et al., 2000a, 2000b; Otera et al., 2005). Here, it causes chromatin condensation and DNA fragmentation (Daugas et al., 2000a, b; Cregan et al., 2002; Plesnila et al., 2004). Clinical studies indicate that sexual dimorphism is an important interdependent risk factor for ischemic brain injury (Fullerton et al., 2003; Golomb et al., 2009; Westmacott et al., 2009). These studies reported that males had increased incidence of and worse outcomes in response to HI injury compared to females. The mechanisms of these sex-related differences of HI injury are poorly understood. Neuronal culture models of HI injury showed that cell death was predominantly via caspase-dependent pathway in the XX neurons versus AIF-dependent pathway in the XY neurons (Du et al., 2004; Sharma et al., 2011), indicating that the mechanism of cell death is innately different between sexes. Sex-specific differences in cell death were also confirmed using in vivo mouse model of neonatal HI injury (Zhu et al., 2006; Mirza et al., 2015). Therefore understanding these sexual differences in the mechanism of cell death is instrumental in designing effective neuroprotective therapies for HI injury. Several studies from our laboratories and others exploring the mechanisms of cell death using neonatal animal models of cerebral ischemia have shown that apoptotic cell death is more prevalent in the immature than in the mature brain (Towfighi et al., 1995; Liu et al., 2004; Zhu et al., 2005). We have previously shown that AIF, in addition to caspase-3, is implicated in delayed cell death in the ischemic neonatal rat brain (Askalan et al., 2011). Most of these studies, however, are done in animal models of moderately injured ischemic brains. These animal models have been used in experimental neuroprotective strategies, some of which have shown robust efficacy against ischemic injury in the developing brain. However, with the exception of therapeutic hypothermia, none of these neuroprotective strategies have been successfully translated to clinical practice. Furthermore, while post-
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ischemic hypothermia has proven to be effective in newborns with mild-moderate hypoxic-ischemic encephalopathy, its protection does not extend to the severely injured neonatal brain, as shown in both animal (Nedelcu et al., 2000; Sabir et al., 2012) and human studies (Shankaran et al., 2005; Azzopardi et al., 2008). Taken together, these studies highlight the necessity for a better understanding of the mechanisms of cell death in the severely injured brain. We therefore sought in this study to characterize the pathways that lead to cell death in the core and peri-infarct area and to understand the influence of biological sex on these pathways in the neonatal rat brain subjected to severe ischemic injury. It is only by understanding these pathways that we will be able to design neuroprotective therapies or modify existing ones (i.e. hypothermia) to be effective in protecting the severely injured developing brain.
2.
Results
2.1. The core constituted most of the ischemic lesion in the severely injured brain The total infarct area, the core area and peri-infarct area were measured in the moderately and severely injured brains (n¼ 4–6 pups/time point in each group) as described in Section 4 and in Fig. 1. The core constituted a higher proportion of the ischemic lesion in the severely injured (mean percent core 9671%) compared to the moderately injured (69713%) brain at 4 days post-injury (p r0.04). After 4 days, that difference disappeared and the area of the core became the same in both types of injury by 1 week postinjury and remained the same at 2 weeks post-injury.
2.2. In severe ischemic injury, cell death was prolonged and mediated by caspase-dependent and caspase-independent pathways In the severely injured brain, cell death by caspase-3 and AIF pathways was significantly higher in the core compared to the peri-infarct area. Interestingly, these two pathways behaved differently over time depending on the region of the lesion. In the core, caspase-3 activity was significantly higher in day 14 compared to day 1 post-injury. In the periinfarct area, AIF-induced cell death increased over time to reach levels of the core levels by 2 weeks postinjury (Fig. 2). These results indicate that there is a prolonged window for therapeutic intervention in the severely ischemic brain, and in order to be effective in this type of injury, neuroprotective strategies must target both caspase-3 and AIF-mediated cell death as highlighted by our subsequent set of experiments comparing the role of these two pathways in severe and moderate ischemic brain injury.
2.3. Caspase-3 and AIF activities were differentially timedependent in the severely and moderately ischemic brain In the core of moderate ischemic injury, the number of nuclear AIF positive cells peaked early followed by a sharp
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Fig. 1 – Representative hematoxylin and eosin (H&E) sections showing the ischemic lesion in a moderately injured (a) and severely injured (b) immature brain. The total area of the core of the infarct is represented in green; the total peri-infarct area is represented in blue. For each specimen, one coronal section of 5 lm thickness was cut at the level of the hippocampus 24 h post-injury and stained with H&E. Using a Spot Flex camera attached to a Leica GZ7 stereoscope ( 10 objective lens), multiple images were taken of each section to encompass the entire infarct and its surrounding tissue (30–40 images/section). The areas of the core and peri-infarct were then measured on each image using NIS Elements software. The measured areas from the multiple images taken of the same section were then summed to determine the total areas of the core and peri-infarct for that section.
number of caspase-3 increased overtime but the number of nuclear AIF positive cells fluctuated without significant change among time points. The change in caspase-3 and AIF activity over time in the peri-infarct area of moderate ischemic injury was the same as that of the core. In the peri-infarct area of severe ischemic injury, however, AIF-induced cell death significantly increased by 2 weeks post-injury whereas caspaspe-3 activity did not significantly change overtime (Fig. 3B). These results, taken together, indicate that the type of injury and the timing post-injury may be key determinants in designing effective neuroprotective therapies against ischemic damage. Fig. 2 – In severe ischemic brain injury, caspase-3 and AIF dependent pathways mediate cell death in a time-sensitive pattern. P7 rat pups were subjected to severe HI injury and sacrificed at given time points. Brain sections were stained for active caspase-3 and AIF as described in Section 4.3. The number of positive cells for caspase-3 and nuclear AIF were counted in core and peri-infarct area of the lesion in 5–6 high-power fields ( 40) using the Image J computer software, and expressed as the mean percentage of the total number of cells per high power field. Cell death is significantly higher in the core compared to the peri-infarct area of the severely injured brain at all time points for caspase-3 pathway and on days 3 and 4 for AIF pathway. AIF-induced death in the peri-infarct area, however, increases over time to reach that of core levels by 2 weeks post-injury (mean percentage of nuclear AIF positive cells is 370.4% at 1 day verses 873% at 14 days post-injury). þ po0.05 compared to peri-infarct area; *p ¼0.03 compared to 14 days. and significant decrease by 48 h and remained low; the number of caspase-3 positive cells, on the other hand, exhibited a sharp and significant increase to beyond 2 weeks postinjury (Fig. 3A). In the core of severe ischemic injury, the
2.4. AIF was a key mediator of cell death in the peri-infarct area of the severely ischemic brain At 7 days post-injury, the mean percentage of casapase-3 positive cells was significantly (p r0.001) higher than the mean percentage of nuclear AIF positive cells in the core and peri-infarct area of the moderately injured brain (Fig. 4C and D) as well as in the core of the severely injured brain (Fig. 4A), indicating that caspase-3- was more active than AIFmediated cell death in these regions. There was no significant difference, however, between AIF and caspase-3 activity in the peri-infarct area of the severely injured brain (Fig. 4B).
2.5. Caspase-3 and AIF were equally active in mediating neuronal death in the peri-infarct area of the severely injured brain To identify the specific brain cells targeted by caspase-3 and AIF pathways, we performed double staining with the astrocytic marker anti-GFAP and the neuronal marker anti-NF. Astrocytic and neuronal deaths were predominantly mediated by caspase-3 dependent pathway in the core of the lesion (Fig. 5A). In the peri-infarct area, however, astrocytic death was predominantly due to caspase-3, whereas
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Fig. 3 – Activation of cell death pathways is sensitive to injury type and timing post-injury. P7 rat pups were subjected to moderate or severe HI injury and sacrificed at given time points. Brain sections were stained for active caspase-3 and AIF (Section 4.3). The number of positive cells for caspase-3 and nuclear AIF were counted in core and peri-infarct area of the lesion in 5–6 high-power fields ( 40) using the Image J computer software, and expressed as the mean percentage of the total number of cells per high power field. (A) In the core of moderate injury, AIF translocation peaked early followed by a significant decrease by 48 h and then stayed low (mean percentage of nuclear AIF positive cells at 1 day postinjury 2972% verses 973% at 14 days post-injury). Caspase-3, on the other hand, exhibited a steady increase to beyond 2 weeks post-injury (mean percentage of caspase-3 positive cells at 2 days post-injury 1774% versus 4375% at 14 days postinjury). *po0.01 compared to day 14. In the severely injured core, caspase-3 increased but AIF did not change significantly overtime. (B) Similar to the moderately injured core, in the moderately injured peri-infarct area AIF peaked early followed by a significant decrease whereas caspase-3 exhibited a significant increase to beyond 2 weeks post-injury. Caspase-3 in the severely injured peri-infarct area fluctuated but did not significantly increase overtime. AIF-induced cell death, however, significantly increased by 2 weeks post-injury in the severely injured peri-infarct (370.4% at 1 day postinjury verses 873% at 14 days post-injury). *po0.05 compared to day 14.
Fig. 4 – The proportions of cells dying by AIF and caspase-3 dependent pathways are similar in the peri-infarct area of the severely injured brain by 7 days postinjury. P7 rat pups were subjected to moderate or severe HI injury and sacrificed at 7 days post-injury. Brain sections were stained for active caspase-3 and AIF and the number of positive cells for caspase 3 and nuclear AIF were counted in core and peri-infarct area of the lesion in 5–6 high-power fields ( 40) using the Image J computer software (Section 4.3). The mean percentage of caspase-3 positive cells (83710%) is significantly (p r0.001) more than the mean percentage of AIF positive cells (1773%) in the core of the severely injured brain (A) as well as in the core (C) and periinfarct area (D) of the moderately injured brain indicating that caspase-3 is more active than AIF-mediated cell death in these regions. There was no significant difference, however, between AIF and caspase-3 activity in the peri-infarct of the severely injured brain (B).
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Fig. 5 – Caspase-3 and AIF-dependent pathways mediate astrocytic and neuronal cell death. Double staining with anti-AIF/antiGFAP, an astrocytic marker, anti-caspase-3/anti-GFAP, anti-AIF/anti-NF, neuronal marker and anti-caspase-3/anti-NF in the core (A) and peri-infarct area (B) of the severely injured lesion was performed in P7 rat brain as described in Section 4.4. Immunodetection was performed using immuno-fluorescence staining method (n¼6–8 pups/marker). Arrows indicate the co-localization of nuclear AIF and GFAP or NF as well as caspase-3 and GFAP or NF in the cortex at the dorsal hippocampal level.
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neuronal death was equally mediated by both caspase-3 and AIF pathways (Fig. 5B). These results provide another line of evidence on the important role of caspase-independent cell death in the severely ischemic developing brain.
2.6.
Necroptosis was not limited to the core of the lesion
Studies have shown that apoptosis and necroptosis (programmed necrosis) coexist in this model of hypoxicischemic injury (Nakajima et al., 2000; Chavez-Valdez et al., 2012a). Necroptosis was shown to be an early mechanism of cell death occurring within the first 24 h postischemic injury (Vieira et al., 2014; Chavez-Valdez et al., 2012b) and inversely linked to caspase-dependent cell death (Vieira et al., 2014; Northington et al., 2011). Therefore we examined the expression of RIP3 2 days post-injury where a decrease in caspase-3 was observed in the moderately and severely injured brains. In the moderately ischemic brain, necroptosis, as depicted by RIP3 staining, predominantly occurred in the core of the lesion (Fig. 6). Interestingly, the peri-infarct area of the severely ischemic brain had significantly more necroptosis than the peri-infarct area of the moderately injured brain and necroptotic cell death was as active in the peri-infarct area as it was in the core of the lesion (Fig. 6).
2.7. Sex of the animal is another determinant of the mechanism of ischemia-induced cell death Sex is an important determinant of many disease processes including response to cerebral ischemic injury. Studies have suggested that male and female neurons utilize different pathways in response to ischemic injury independent of hormonal factors (Du et al., 2004; Krishnan et al., 2009;
Fig. 6 – There is increased necroptotic cell death in the periinfarct area of the severely injured compared to the moderately injured brain. P7 rat pups were subjected to moderate or severe HI injury and sacrificed at 2 days postinjury. Brain sections were stained for RIP3 and the number of positive cells was counted in core and peri-infarct area of the lesion in 5–6 high-power fields ( 40) using the Image J computer software (Section 4.3). The mean percentage of RIP3 positive cells was significantly higher (*p ¼0.02) in the peri-infarct area of severe injury (1272%) compared to that in the peri-infarct area of moderate injury (472%).
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Mirza et al., 2015). We therefore sought to investigate the influence of injury type on sex-related differences in the mechanism of cell death in the ischemic neonatal brain by 7 days postinjury. The 3-way ANOVA analysis showed that sex of the animal (F¼ 4.6; p¼ 0.033; Fig. 7A) and types of cell death (F ¼5.0; p¼ 0.027; Fig. 7B) interactions were significant. Males with moderate ischemic injury had significantly more caspase-3 and AIF-induced cell death compared to females (difference ¼1.04%; po0.001; Fig. 7A). Severe injury abolished this difference between the two sexes (difference¼ 0.19%; p40.05). In both the moderately injured (difference ¼2.07; po0.001) and severely injured brain (difference ¼1.22; po0.001), caspase-3 dependent cell death was more pronounced than AIF-dependent cell death in male and female pups (Fig. 7B).
3.
Discussion
In this study we have subjected P7 rat pups to 180 min of hypoxia resulting in severe HI injury and compared mechanisms of cell death to those in rat pups subjected to 90 min of hypoxia (i.e. mild-moderate HI injury). We have shown that the core constituted most of the ischemic lesion in the severely injured brain up to 7 days post-injury. Therefore understanding the mechanisms of injury in the core of the severe ischemic lesion is essential to design proper therapies for this type of injury. Moreover, these results emphasized the importance of timely intervention for the moderately injured brain before the core of the lesion expands to resemble that of severe injury. We found that in the moderately injured animals caspase-dependent cell death was prominent up to 2 weeks post-injury whereas AIFdependent cell death was prominent in the severely injured animals for the same time period. We have demonstrated for the first time multiple lines of evidence that in severe HI injury of the developing brain 1) apoptosis is prolonged and plays a significant role in causing ischemic damage and 2) caspase-independent neuronal death is a greater contributor to ischemic damage than in moderate HI injury. The first line of evidence came from investigating the activity of caspase-dependent and -independent pathways in the two areas of the lesion overtime. In severe ischemic injury, both caspase-3 and AIF activity were high in the core compared to the peri-infarct area and AIF translocation increased in the peri-infarct area overtime to reach that of the core by 2 weeks postinjury. This was in contrast to what was observed in moderate injury where AIF-dependent pathway activity was higher early post-injury and significantly decreased by 2 days post-injury in the core and peri-infarct area of the lesion. Caspase-3 activity in the core and periinfarct area of the moderately injured brain exhibited an abrupt decrease at 2 days post-injury followed by a significant increase that was sustained up to 2 weeks post-injury. This increase in caspase activity was also observed in the core but not the peri-infarct area of the severely injured brain. Different temporal patterns of caspase-3 activity were previously reported and could not be fully explained. They may be related to the region of the brain, extent of injury (Nakajima
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Fig. 7 – The mechanism of ischemia-induced cell death is influenced by the sex of the animal. P7 male and female rat pups were subjected to moderate or severe HI injury and sacrificed at 7 days post-injury, sectioned and stained for caspase-3 and AIF as described in Section 4.3. The number of positive cells was counted in 5–6 high-power fields ( 40) using the Image J computer software. 3-way ANOVA analysis was used to determine the effect of sex, severity of injury and types of cell death on the ischemic damage. (A) Caspase-3 and AIF-mediated cell death was significantly (difference¼ 1.04%; po0.001) higher in males (23.1872) compared to females (1472) with moderate HI injury. There was no significant difference in types of cell death between males and females with severe HI injury. (B) The mean percentage (7SEM) of active caspases-3 positive cells was significantly higher than the mean percentage of AIF positive nuclei in the moderately injure (27.5572; 10.0571 respectively; difference¼ 2.07; po0.001) and the severely injured (14.6671; 7.0271; difference¼1.22; po0.001) in both sexes.
et al., 2000) and release of Cyt c (Zhu et al., 2006) or limiting the time window of the study to 24 h post-injury (Manabat et al., 2003). The importance of caspase-independent cell death in severe ischemic injury was further confirmed when we compared the number of active caspase-3 and nuclear AIF positive cells at the specific time point post-injury of 7 days. In the core of moderate and severe ischemic injury, the number of active caspase-3 positive cells was significantly higher than nuclear AIF positive cells. However, this difference was abolished in the peri-infarct area of the severe injury lesion with AIF having a greater contribution to cell death. The second line of evidence came from the specific brain cells that were affected by caspase-3 and AIF pathways in the severely injured animals. Astrocytic death was predominately mediated by the caspase-3 dependent pathway. The AIF-dependent pathway was as active as the caspase-3 dependent pathway in mediating neuronal death. Therefore, a neuroprotective approach has to address both forms of neuronal death to be effective. The third line of evidence came from investigating the expression of RIP3 postischemic injury. Programmed necrosis, known as necroptosis, is another form of cell death shown to play a role in tissue damage response (Zhou et al., 2012; Sun and Wang 2014). RIP3 is a determining factor in the necroptosis signaling pathway by initiating a series of phosphorylation events (Sun et al., 2012). The significance of these events and the mechanism by which RIP3 causes
programmed necrosis are still under investigation. As one would expect, necroptosis occurred primarily in the core of the lesion in the moderately injured ischemic neonatal brain. Interestingly, however, there was no significant difference in the number of RIP3 positive cells in the core and peri-infarct area of the lesion in the severely injured neonatal brain. Investigating the mechanism of RIP3-mediated necroptosis and the search for compounds that will block necroptosis are ongoing. The development of a necroptosis pathway inhibitor may be essential for any therapeutic approach to reduce the damage of severe ischemic injury. Taken together, these findings indicate that similar to what has been previously reported for moderate injury (Nakajima et al., 2000; Askalan et al., 2011), severe ischemic injury in the neonatal brain also has a prolonged therapeutic window for neuroprotective intervention. The timing postinjury, however, is crucial in determining the therapeutic target. Moreover, these results illustrate that this intervention cannot be the same for both injury types and highlight the necessity of taking into consideration caspaseindependent pathways of cell death when designing a neuroprotective therapy for the severely injured brain. Most therapies that have been tried to date in this animal model of neonatal brain HI injury were designed to attenuate caspase-3 dependent cell death (Han et al., 2002; Feng et al., 2003; Joly et al., 2004), which may explain why their neuroprotective effects have not been extended to animals with severe injury.
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Clinical and experimental studies indicate that sex is an important risk factor for ischemic brain injury. Recent clinical studies have reported an increased incidence of arterial ischemic stroke (Lynch et al., 2002; Golomb et al., 2009) and cerebral sinus venous thrombosis (Golomb et al., 2009) in male children. This male predominance persists even after excluding cases of posttraumatic brain ischemic injury (Fullerton et al., 2003; Golomb et al., 2009). These sexrelated differences in cerebral ischemic injury may be attributed to differences in cell death pathways activated by the injury. We have shown here increased caspase-3 and AIF activity in males compared to females with moderate injury but that there were no sex differences in the caspase-3 activity and AIF translocation in rat pups with severely injured brain. These results suggest that differences in mechanism of cell death induced by moderate ischemic brain injury may contribute to the clinical differences seen between male and female patients. Zhu et al. (2006) reported no difference in the number of AIF positive nuclei or active caspase-3 positive cells in the cortex of male and female P9 mice at 1 day and 3 days postinjury whereas other studies reported that AIF-dependent cell death was predominant in males whereas caspasedependent cell death was predominant in females (Renolleau et al., 2007; Weis et al., 2014). This discrepancy in results may be attributed to differences in the experimental HI injury model used, the duration of hypoxia (extent of injury), the time post-injury at which the animals were sacrificed or to the brain region being analyzed. The mechanisms of these sex-related differences of neonatal HI injury are poorly understood. Estrogen is unlikely to account for the observed protection in females because circulating estrogen is minimal in the neonatal female (McCullough and Hurn, 2003; Konkle and McCarthy, 2011). These results may explain what we and subsequently others (Westmacott et al., 2009; Smith et al., 2014) have reported in males having worse outcomes of HI injury compared to females. We have shown that males with neonatal ischemic stroke have more cognitive deficits compared to females with the same degree and timing of injury (Westmacott et al., 2009). Moreover, these results highlight the critical need for designing experimental studies that will take into consideration the interaction of the severity of injury and sex when investigating efficacy of neuroprotective therapies. Thus far, post-ischemic hypothermia is the most promising neuroprotective measure against ischemic injury in the developing brain. Several multi-center randomized clinical trials over the last 10 years have shown that post-ischemic hypothermia is effective in improving the outcome in newborns with perinatal asphyxia (Eicher et al., 2005; Shankaran et al., 2005; Azzopardi et al., 2008). In several analyses, however, this protective effect was largely afforded only to those infants with moderate hypoxic-ischemic encephalopathy and it did not protect those infants with severe encephalopathy (Shankaran et al., 2005; Azzopardi et al., 2008). This lack of protection was thought to occur because the majority of severe injury to the brain was actually within the core and was necrotic in nature, which was not amenable to current therapeutic interventions. However, the results presented here indicate that this may not be true, as apoptotic cell
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death plays an important role in the core of the lesion in the developing brain. Future studies to elucidate the effect of hypothermia on caspase-independent cell death will enable us to identify the most appropriate anti-apoptotic therapies that may be used in combination with hypothermia in order to amplify its neuroprotection, leading to a reduction in ischemic damage in the severely injured immature brain. In conclusion, we have shown that caspase-independent cell death has a pivotal role in severe ischemic injury of the neonatal brain of both sexes in a time-sensitive manner. For a therapy to be effective in reducing damage post severe ischemic injury, it has to target not just caspase-3 dependent cell death but also AIF-dependent cell death and necroptosis.
4.
Experimental procedure
4.1.
Neonatal model of hypoxia-ischemia
As previously described (Askalan et al., 2009, 2011), we induced hypoxic-ischemic injury in the neonatal rat brain using the Rice– Vannucci model with a prolonged duration of hypoxia to elicit severe injury. In brief, 7-day-old male and female rat pups were randomly assigned to either moderate or severe cerebral hypoxia-ischemia. In both groups, the right common carotid artery was ligated using a double 4–0 silk tie and cut between the sutures. During the procedure, all animals were anesthetized with isoflurane (4% induction, 1% maintenance). Following ligation, rat pups were allowed to recover for 2 h, after which ischemia was produced in the hemisphere ipsilateral to the carotid artery occlusion by subjecting the animal to hypoxia (8% oxygen) for 90 or 180 min for the moderate and severe HI injury groups, respectively. Mortality rates were 2.4% and 21.6% for the 90 and 180 min respectively. Body temperature was regulated in all groups at 36.570.5 1C during hypoxia by maintaining the rat pups in a servo-controlled neonatal Isolette (Narco Scientific, Hatboro, Pa, USA). Animals were sacrificed at 1, 2, 3, 4, 7 and 14 days after the HI insult using pentobarbital. Brains were rapidly removed and processed for hematoxylin and eosin (H&E) staining and immunohistochemistry. All animal procedures were approved by the health sciences animal care and use committee at the University of Alberta.
4.2.
Measurement of infarct area
For each specimen, one coronal section of 5 mm thickness was cut at the level of the hippocampus and stained with H&E. Using a Spot Flex camera attached to a Leica GZ7 stereoscope ( 10 objective lens), multiple images were taken of each section to encompass the entire infarct and its surrounding tissue (30–40 images/section). The core and the peri-infarct area of the lesion were then measured on each image using NIS Elements software. As we have previously described, normal brain parenchyma shown by H&E staining directly adjacent to the ischemic ‘core’ was defined as the peri-infarct area (Askalan et al., 2009). The measured areas from the multiple images taken of the same section were then summed to determine the total areas of the core and the peri-infarct for that section. A representation of these measurements is shown in Fig. 1.
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4.3.
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Immunohistochemistry
At the previously specified time points, brains from P7 injured rat pups were removed and immediately fixed in 10% formalin. Paraffin-embedded coronal sections were cut (5 μm) at the level of the hippocampus, de-waxed with xylene, hydrated and pretreated with the heat-induced antigen retrieval technique. Serial sections were then stained with commercially available specific primary antibodies for activated caspase-3 (rabbit polyclonal anti-cleaved caspase-3 (Asp175) antibody, 1:400, Cell Signaling), AIF (rabbit anti-AIF, 1:100, Cell Signaling) and RIP3 (rabbit anti-RIP3, 1:150, Abcam) at 4 1C overnight. Caspase-3, nuclear AIF and RIP3 were detected by appropriate horseradish peroxidase secondary antibodies (HRP, 1:100, Chemicon) for 1 h at room temperature and DAB substrate kit for Peroxidase/Vector/SK-4800. The number of positive cells for caspase-3, nuclear AIF and RIP3 were counted in 5–6 high-power fields ( 40) using the Image J computer software (National Institutes of Health, Bethesda, MD, USA) and expressed as the mean percentage7SEM of the total number of cells per high power field.
4.4.
Sources of funding This study was funded in part by the Heart and Stroke Foundation of Ontario to R.A. (3210326014)
Acknowledgment The authors would like to that Dr. Priyanka Shah for her invaluable assistance in the statistical analysis of the data.
r e f e r e n c e s
Double staining
Antibody for glial fibrillary acidic protein (GFAP), and neurofilament (NF) are well-established markers for detecting astrocytes, and neurons, respectively. Double staining with anti-GFAP/anti-caspase-3, anti-GFAP/anti-AIF, anti-NF/anticaspase-3 and anti-NF/anti-AIF were done to determine the predominant mechanism of cell death in astrocytes and neurons following ischemic injury. Frozen brain sections were incubated simultaneously with AIF antibody (rabbit anti-AIF, 1:100; cell signaling) and mouse anti-GFAP (1:1000, Sigma chemicals Co), or chicken polyclonal anti-NF (1:500, millipore). Another set of adjacent sections were incubated simultaneously with caspase-3 antibody (rabbit polyclonal anti-cleaved caspase-3 (Asp175) antibody, 1:400; Cell Signaling) and GFAP or NF. Immuno-reactivity was visualized using appropriate combinations of donkey anti-rabbit Fitc (1:200, invivogen) and donkey anti-mouse Cy2 (1:200, invivogen) or donkey anti-chicken Cy3 (1:200, chemicon) secondary antibodies and nuclei were counterstained with DAPI (Sigma). Multichannel images were captured and analyzed with Nikon NIS-Element Basic Research Image system.
4.5.
URL http://www.rstudio.com/. 2015). Experimental groups were compared using paired t-test. Results were considered to be statistically significant if two-tailed po0.05. All data were presented as means7SEM, and other statistical analyses were done using GraphPad InStat (GraphPad Software Inc., San Diego, USA).
Statistical analysis
At least six rat pups were utilized for each measurable time point in the moderately and severely injured groups. Threeway ANOVA was conducted to compare the main effects of and interaction among the type of cell death (AIF-induced, caspace-3), sex (female, male) and induced severity of the ischemic injury (moderately-injured, severely-injured) on the ischemic neonatal brain; because the distribution of the dependent variable, percentage of positive cells per HPF, was skewed, a square root transformation was performed. Significant findings in ANOVA were followed up by post-hoc tests, conducted using Tukey's honest significant differences method; these analyses were carried out in RStudio (Integrated Development for R. RStudio, Inc., Boston, MA
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