Role for RIP1 in mediating necroptosis in experimental intracerebral hemorrhage model both in vivo and in vitro

Shen, Haitao; Liu, Chenglin; Zhang, Dongping; Yao, Xiyang; Zhang, Kai; Li, Haiying; Chen, Gang

doi:10.1038/cddis.2017.58

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Original Article
Open access
Published: 02 March 2017

Role for RIP1 in mediating necroptosis in experimental intracerebral hemorrhage model both in vivo and in vitro

Haitao Shen¹^na1,
Chenglin Liu¹^na1,
Dongping Zhang¹^na1,
Xiyang Yao¹,
Kai Zhang¹,
Haiying Li¹ &
…
Gang Chen¹

Cell Death & Disease volume 8, page e2641 (2017)Cite this article

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Abstract

Cell death is a hallmark of second brain injury after intracerebral hemorrhage (ICH); however, the mechanism still has not been fully illustrated. In this study, we explored whether necroptosis, a type of regulated necrosis, has an essential role in brain injury after ICH. We found that inhibiting receptor-interacting protein 1 (RIP1) – a core element of the necroptotic pathway – by a specific chemical inhibitor or genetic knockdown attenuated brain injury in a rat model of ICH. Furthermore, necroptosis of cultured neurons could be induced by conditioned medium from microglia stimulated with oxygen hemoglobin, and this effect could be inhibited by TNF-α inhibitor, indicating that TNF-α secreted from activated microglia is an important factor in inducing necroptosis of neurons. Undoubtedly, overexpression of RIP1 increased conditioned medium-induced necroptosis in vitro, but this effect was partially diminished in mutation of serine kinase phosphorylation site of RIP1, showing that phosphorylation of RIP1 is the essential molecular mechanism of necroptosis, which was activated in the in vitro model of ICH. Collectively, our investigation identified that necroptosis is an important mechanism of cell death in brain injury after ICH, and inhibition of necroptosis may be a potential therapeutic intervention of ICH.

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Main

Intracerebral hemorrhage (ICH) is the second largest type of stroke, accounting for ~15% of all patients with stroke.^{1, 2} ICH is associated with fast progression, high mortality and high morbidity. It has been reported that, with mortality rates close to 40% in 1 month, patients have legacy paralysis, aphasia and other severe disabilities after ICH. The incidence of ICH also increased significantly with the increase of population age.³ Primary brain injury after ICH is due to hematoma mass effect and mechanical damage to adjacent brain tissues,⁴ and the secondary brain injury is a key reason to cause nerve function damage in patients with ICH.^{5, 6} In secondary brain injury after ICH, pathological changes, including cell death, cerebral edema and blood–brain barrier (BBB) damage, occur in brain tissues surrounding hematoma.⁷ Mechanism of secondary brain injury after ICH includes excitatory amino-acid toxicity, inflammatory response, expression of proteolytic enzyme and the toxic effects of hematoma release product.^{5, 7} Cell death is an important factor in secondary brain injury after ICH.

In recent years, necrosis as another form of cell death causes more attention. The definition of necrosis is based on its pathological morphological characteristics; it is a passive cell death mostly caused by overwhelming stress such as dramatic changes in temperature or pH. Necrotic cells rapidly lose cell membrane integrity, and cell membrane swelling and mitochondrial dysfunction occur. The membrane rupture caused a large number of cytoplasmic components to leak from the cell and then induced inflammation in the surrounding tissues.⁸ Previous studies suggested that necrosis is an occasional and irregular event and is difficult to study.⁹ However, Yuan and co-workers¹⁰ reported, for the first time, necroptosis as a form of necrosis that can be regulated.¹⁰ Necroptosis has similar morphological characteristics (including early membrane integrity, cell and intracellular organelle swelling) with necrosis, but it is caspase-independent programmed cell death.¹¹

Recent studies have shown that death receptors on the cell membrane mediated different pathways of cell death depending on the state of cell or local physiological or pathophysiological microenvironment; for example, in the event of TNF-α-induced cell death, TNF-α and its receptors firstly formed a trimer, and then recruited proteins containing the death domain (DD) formed a complex, which activated the downstream apoptotic pathway. However, if caspase activity was inhibited, the cells would go to the necroptotic pathway. RIP1 was recruited to TNFR1 through its DD, which bound directly to the DD of TNFR1, and it was activated by multiple forms of ubiquitination. The activation of RIP1 led to the recruitment of RIP3, MLKL and caspase-8, forming a complex called 'necrosome' involved in necroptotic pathway. Thus, RIP1 played the role of adaptor in mediating necroptosis. Activation of RIP3 activated three key enzymes of cell metabolic pathways – glycogen phosphorylase, glutamine synthetase, and glutamate dehydrogenase – which caused overproduction of reactive oxygen species (ROS). ROS caused DNA damage, damage to mitochondrial membrane permeability and lysosome damage, eventually leading to cell death.^{11, 12}

Previous studies have confirmed that microglia was quickly activated in brain tissues after ICH, and it secreted a large number of inflammatory factors, including the TNF-α, which can induce necroptosis.¹³ Under the stimulation of TNF-α, a part of the brain cells undergo apoptosis and a part may undergo necroptosis in brain tissues after ICH. Necrotic cell death is common in a wide variety of pathological conditions, including stroke.¹⁴ Compared with numerous investigations on the mechanisms of apoptotic cell death, fewer studies have explored necrotic cell death in ICH. Here we studied the role of necroptosis, a type of regulated necrosis, in ICH.

Results

Necroptosis was activated in brain tissues after ICH

To explore the involvement of necroptosis in brain tissues after ICH, we detected propidium iodide-positive (PI+) cells in frozen brain sections at 24 h after ICH. Notably, compared with that in the Sham group, the PI+ cells were remarkably increased in brain tissues surrounding hematomas after ICH (P<0.0001, n=6; Figures 1a and b). We also determined the expression of RIP1, which is a major regulator for necroptosis in brain tissues; the results of western blot suggested that the expression levels of RIP1 were progressively upregulated and peaked at 24 h after ICH (P=0.0003 versus Sham group, n=3; Figures 1c and d). The results of immunofluorescent staining also showed that the expression level of RIP1 in neurons was remarkably increased at 24 h after ICH (Figure 1e).

We further evaluated the formation of necrosome, which is an important hallmark of activation of necroptosis in brain tissues after ICH; anti-RIP1 antibody was used by immunoprecipitation (IP), and RIP3, MLKL and caspase-8 were detected by immunoblotting. Increased interactions of RIP1 and RIP3, RIP1 and MLKL, and RIP1 and caspase-8 were observed in brain tissues after ICH (all P<0.01 versus Sham group, n=3, Figures 1f and g). It suggested that the formation of necrosome was significantly increased in brain tissues after ICH than that in the Sham group. The results of immunofluorescence also showed that expressions of these four proteins were significantly upregulated after ICH than that in the Sham group (all P<0.01 versus Sham group, n=6, Figures 1h and i). These results indicated that necroptosis was activated in brain tissues after ICH.

Inhibitor of necroptosis and apoptosis differentially attenuates brain injury after ICH

To determine whether necroptosis contributes to brain injury after ICH, the specific inhibitor of RIP1, necrostatin-1 (Nec-1), was used, and z-VAD, a caspase inhibitor, which can inhibit apoptosis, was also used as a positive control in a rat model of ICH. Nec-1 reduced the PI+ cells but had no significant effect in TUNEL+ cells, whereas z-VAD only downregulated the TUNEL+ cells, and did not affect the PI+ cells in brain tissues after ICH. The combination of z-VAD and Nec-1 both obviously reduced the PI+ cells and TUNEL+ cells (all P<0.01, n=6; Figures 2a and b). The results of IP also demonstrated that interactions of RIP1 and RIP3, RIP1 and MLKL, and RIP1 and caspase-8 were significantly inhibited by Nec-1 but not by z-VAD (all P<0.05, n=3; Figures 2c and d). These results indicated that necroptosis and apoptosis occurred in different cells at same times and were independent of each other in brain tissues after ICH.

BBB permeability was assessed using albumin extravasation, and the western blot was used to test the albumin level in brain tissues; the results also suggested that treatment with Nec-1 in ICH rats can improve albumin extravasation and BBB injury (P=0.0461, n=3; Figures 2e and f). Brain water content was calculated by the wet and dry weight method; the results showed that, compared with ICH group, the brain water content was significantly reduced in the ICH+Nec-1 group (P=0.0109 in Ipsi-CX and P=0.0077 in Ipsi-BG, n=6; Figure 2g). The neurological score of the ICH+Nec-1 group was significantly lower than that in the ICH group (P=0.0094, n=6; Table 1). The levels of TNF-α in the cerebrospinal fluid (CSF) were measured by ELISA; the results confirmed that the level of TNF-α was obviously reduced by Nec-1 treatment, indicating that Nec-1 inhibits inflammation in ICH rats (P<0.0001, n=6; Figure 2h). These results suggested that necroptosis contributes to brain injury after ICH, including neuronal dysfunction, brain water content, BBB permeability and inflammation.

Table 1 Clinical behavior scores in each group (n=6)

Full size table

Blockade necroptosis by knockdown of RIP1 improves brain injury after ICH

To further define the role of RIP1-mediated necroptosis in neuronal dysfunction after ICH, we used knockdown by small interfering RNA (siRNA) interference and overexpression by recombinant adenoviruses transfection of RIP1 as treatment in rat model of ICH. Knockdown of RIP1 reduced the PI+ cells, but overexpression of RIP1 increased the PI+ cells in brain tissues after ICH (both P<0.0001, n=6; Figures 3a and b). The results of IP also demonstrated that interactions of RIP1 and RIP3, RIP1 and MLKL, and RIP1 and caspase-8 were significantly inhibited by knockdown of RIP1, but they were increased by overexpression of RIP1 (all P<0.05, n=3; Figures 3c and d). These results indicated that knockdown of RIP1 can effectively block necroptotic pathway.

The results of BBB permeability also suggested that treatment with RIP1 siRNA in ICH rats can inhibit albumin extravasation and BBB permeability (P=0.0493, n=3; Figures 3e and f). The results of brain water content declared that, compared with the ICH group, the brain water content was significantly reduced in the RIP1 siRNA group (P<0.0001 in Ipsi-CX and P=0.0209 in Ipsi-BG, n=6; Figure 3g). The neurological score of the ICH+Si-RIP1 group was significantly lower than that of the ICH group (P=0.0185, n=6; Table 1). The levels of TNF-α in the CSF were detected by ELISA; the results confirmed that the level of TNF-α was reduced by RIP1 siRNA treatment, indicating that downregulation of RIP1 could inhibit inflammation in brain tissues after ICH (P=0.0002, n=6; Figure 3h). These results further proved that necroptosis played an important role in brain injury after ICH.

Conditioned medium from activated microglia can induce necroptosis of cultured neuron in vitro

To further explore the mechanism of necroptosis after ICH, we used two in vitro models of ICH: one is oxygen hemoglobin (OxyHb) to deal directly with the neurons, and the other is to prestimulate microglia with OxyHb, collect the supernatant as conditioned medium and then treat neurons with the conditioned medium. After these treatments, neurons were digested by trypsin into cell suspension, stained with Annexin V and PI and then detected by flow cytometry. The results of flow cytometry showed that, compared with control group, there was a higher apoptotic ratio (~27.8%) in OxyHb directly stimulated neuron group, whereas there was a higher necroptotic ratio in conditioned medium treatment group (~31.5%). In addition, Nec-1 treatment, but not z-VAD, significantly reduced the conditioned medium-induced neuron necroptosis (data not shown). However, the TNF-α inhibitor pretreatment significantly reduced the percentage of necroptotic neurons (~13.5%; all P<0.01, n=3; Figures 4a and b).

The results of IP also demonstrated that, compared with the control, interactions of RIP1 and RIP3, RIP1 and MLKL, and RIP1 and caspase-8 were significantly increased by treatment with conditioned medium. However, interactions of RIP1 and RIP3, RIP1 and MLKL, and RIP1 and caspase-8 were remarkably inhibited by TNF-α inhibitor pretreatment than that in the conditioned medium group (all P<0.01, n=3; Figures 4c and d). To further clarify the interaction among RIP1-RIP3-MLKL-caspase-8, immunofluorescence staining of RIP1/MLKL/caspase-8 and RIP3/MLKL/caspase-8 was also performed on primary neurons (Figure 4e). Consistent with the results of IP, there were increased colocalizations of RIP1-MLKL-caspase-8 and RIP3-MLKL-caspase in conditioned medium-treated neurons, which were significantly inhibited by TNF-α inhibitor. PI and Hoechst staining also showed that treatment with conditioned medium increased the ratio of necroptosis in neurons, but this can be inhibited by TNF-α inhibitor pretreatment (both P<0.0001, n=6; Figures 4f and g). These results suggested that TNF-α in conditioned medium may be an important factor of inducing necroptosis in neurons.

Phosphorylation of RIP1 has an essential role in activation of necroptosis in neuron in vitro

Phosphorylation of RIP1 is an essential element of activation of necroptosis.¹⁵ We detected the phosphorylation of RIP1 by IP using anti-RIP1 antibody followed by immunoblotting for anti-phosphorylation serine (p-Ser) antibody. The results of IP suggested that the phosphorylation (serine site) of RIP1 was obviously increased in the conditioned medium treatment group and could be inhibited by TNF-α inhibitor pretreatment (both P<0.01, n=3; Figures 4c and d). To further explore the molecular mechanism of RIP1 in necroptosis after ICH, we used overexpression with a mutation of phosphorylation site (S166A) of RIP1. After transfection and being cultured for another 24 h, neurons were digested by trypsin into cell suspension, stained with Annexin V and PI and then detected by flow cytometry. The results of flow cytometry revealed that upregulating the expression of RIP1 could increase the percentage of necroptosis (~66.7%) in neurons, but overexpression of mutation of phosphorylation site (S166A) of RIP1 has no obvious effect in inducing necroptosis of neurons (~34.2%), and Nec-1 treatment can also inhibit the necroptosis induced by RIP1 overexpression (~21.7%, all P<0.01, n=3; Figures 5a and b).

The results of IP also demonstrated that, compared with the control, interactions of RIP1 and RIP3, RIP1 and MLKL, and RIP1 and caspase-8 were significantly increased by overexpression of RIP1. However, interactions of RIP1 and RIP3, RIP1 and MLKL, and RIP1 and caspase-8 were remarkably inhibited by mutation of phosphorylation site (S166A) of RIP1 than those in the overexpression of wild type of the RIP1 group (all P<0.01, n=3; Figures 5c and d). PI and Hoechst staining also showed that overexpression of wild-type RIP1 increases the ratio of necroptosis in neurons, but this effect can be inhibited by mutation of the phosphorylation site (S166A) of RIP1 (all P<0.0001, n=6; Figures 5e and f). These results all suggested that phosphorylation in the 166th site of RIP1 may be an important element of inducing necroptosis in neurons.

Discussion

Cell death is the important reason leading to brain injury after ICH. Almost previous studies believed that the form of cell death is apoptosis in brain tissues after ICH; few researches put attention to the role of necrosis. Initiation factors of apoptosis include downregulation of blood flow and energy metabolism around the hematoma; a variety of enzymes that are activated in the blood after ICH activate the apoptosis signal, and mechanical damage of hematoma directly causes apoptosis.^{5, 16, 17} Apoptosis theory can partly explain the mechanism of cell death in brain tissues after ICH. However, recent study found that the dead brain cells release a series of proteins from the cytoplasm after ICH; these proteins were known as danger-associated molecular patterns. The most typical representative is the high-mobility group protein 1, which can stimulate the inflammatory response that aggravates secondary brain injury after ICH.¹⁸ These results pose challenge to apoptosis theory in ICH; scholars widely recognized that cell apoptotic process and released apoptotic bodies do not cause inflammation.¹⁵ Thus, presumably, another form of cell death in addition to apoptosis might exist in brain tissues and can stimulate inflammatory response after ICH. On the other hand, microglia were rapidly activated after ICH and released substantial inflammatory factors (such as TNF-α); previous studies have confirmed that TNF-α can not only induce apoptosis but also necroptosis; so whether necroptosis exists in brain tissues after ICH becomes a question.

Our present study showed that conditioned medium, especially the key composition TNF-α, from activated microglia induced necrosis of neurons after ICH for the first time. We found that necroptosis existed in brain tissues after ICH, and it had an important role in neuronal dysfunction, brain edema and BBB permeability after ICH. RIP1 inhibitor Nec-1 can remarkably attenuate neurological dysfunction, brain edema and BBB injury, and combinational use with apoptosis inhibitor z-VAD has a better effect, suggesting that necroptotic pathway and RIP1 may become targets for the treatment of brain injury after ICH. We also observed that treatment of ICH rats with Nec-1 does not affect the apoptosis of brain cells, and the use of z-VAD treatment of ICH rats also does not affect the necroptosis in brain tissues; these results indicated that apoptosis and necroptosis were coexisting and were relatively independent after ICH. Nec-1 and z-VAD could inhibit apoptosis and necroptosis, respectively, whereas combinational treatment of Nec-1 and z-VAD was more effective in treating ICH than each inhibitor alone. In addition, there may be a crosstalk between apoptosis and necroptosis in the progression of ICH, which needs further investigation. In addition, while our research was in progress, Su et al.¹⁹ reported that Nec-1 ameliorated brain injury after ICH in a collagenase-induced ICH model in mice.¹⁹ A recent report also showed that Nec-1 reduced neurovascular injury after ICH in a collagenase-induced ICH model in mice.²⁰ As our present study showed that, TNF-α, an important inflammatory cytokine, may be a key factor in neurons' necrosis after ICH, the side effects of collagen on inflammatory response should not be ignored in necrosis-related studies. In this study, we researched neurons' necrosis in autologous blood injection ICH model for the first time.

In many previous studies, stimulation of neurons with OxyHb was an in vitro model of ICH.²¹ However, the inflammation is an important event in second brain injury after ICH; activated microglia released an amount of inflammatory cytokines such as TNF-α and IL-1β, which promote brain injury after ICH.²² Thus, in this study, we used OxyHb-stimulated microglia and collected the supernatant as conditioned medium, and then used conditioned medium to treat the neurons, as an ICH model in vitro. This model highlights the stimulation of inflammatory factor on neurons, and results of ELISA also suggested that the level of TNF-α was significantly increased in conditioned medium compared with the medium of nontreated microglia (data not shown). We found that necroptotic rate of neurons in this model was significantly higher than that in the OxyHb-treated neurons, suggesting that inflammatory factors released by activated microglia are a key factor leading to cell necroptosis in brain tissues. A previous study reported that, in a murine model of ICH, postinjury treatment with the TNF-α antibody resulted in less neuroinflammation and reduction in functional deficit;²³ combining with our results, we can conclude that ameliorating brain injury after ICH by blocking TNF-α is partially because of reducing the necroptosis in brain tissues. Our results also suggested that multiple stimuli factors coexisted in brain tissues after ICH, and led to cell death by different pathways; the dominant stimulus in local microenvironment might be the main reason leading to brain cell death. At the same time, our results demonstrated that use of multiple in vitro models may be more appropriate in the study of brain injury following ICH.

Unlike apoptosis, the release of cell contents will cause inflammation after the cell necroptosis. Interestingly, an important initial factor of necroptosis is stimulation of inflammatory factor (such as TNF-α), and necroptosis also can further promote the inflammation, these suggested that possibly have a positive feedback relationship between necroptosis and inflammation in brain injury after ICH. The effect of this positive feedback relationship in the pathophysiological processes after ICH needs to be further confirmed. There are also some deficiencies in this study; although the role of necroptosis in the brain injury following ICH was explored, the molecular mechanism is still uncertain. On the other hand, there are many kinds of inflammatory cytokines released by activated microglia, such as IL-1β, IL-6 and IL-18; whether these inflammatory factors also can cause cell necroptosis in brain tissues after ICH is not clear.

In summary, our study confirmed that the RIP1-mediated necroptosis exists in brain tissues, and it has an important role in brain injury following ICH; on the other hand, we used the in vitro model of ICH suggesting that the release of TNF-α from activated microglia might be an important factor inducing necroptosis in ICH (Figure 6). These findings further revealed the causes of cell death and the relationship between cell death and inflammation in brain injury after ICH and provide a potential therapeutic target for secondary brain injury after ICH.

Materials and Methods

Animals

Adult male Sprague–Dawley (SD) rats, weighing ~300 g, were provided by the Animal Center of Chinese Academy of Sciences (Shanghai, China). Experimental protocols were approved by the Animal Care and Use Committee of Soochow University, and were implemented with reference to the National Institutes of Health guidelines. The animals were freely fed and housed in a quiet environment (indoor temperature of ~18–22 °C). Additionally, we strived as much as possible to minimize the number of animals that were used and reduce their suffering. Besides, primary neuronal and microglial cultures (in vitro) were prepared using 16–18-day-old pregnant SD rats.

Experimental design

In experiment 1, 54 rats (70 rats were used, 54 rats survived after the surgery) were randomly assigned to nine groups of six rats each; the normal group, the Sham group and seven experimental groups were arranged by time – 3, 6, 12, 24, 48, 72 h and 7 days after ICH. Arriving at separate time points after SAH, all rats were killed and cerebral tissue samples were collected for analysis (Figure 7a). In experiment 2, 60 rats (75 rats were used, 60 rats survived after the surgery) were randomly divided into 10 groups – the Sham group, the ICH group, the ICH+vehicle group, the ICH+Nec-1 group, the ICH+z-VAD group, the ICH+Nec-1+z-VAD group, the ICH+Si-negative control group, the ICH+SiRNA-RIP1 group, the ICH+Ad-GFP group and the ICH+Ad-RIP1 group. At 24 h after ICH, all rats were examined for behavioral impairment, and then brain samples were collected (Figure 7b). In experiment 3, primary cultured neurons were used and partitioned into six groups – the control group, the OxyHb group, the conditioned medium group, the conditioned medium+TNF-α inhibitor group, the conditioned medium+Ad-RIP1 group and the conditioned medium+Ad-RIP1-S166A group (Figure 7c). Detailed information about each group was shown in special procedures as below.

Establishment of ICH model

ICH model in vivo was established by injection of autologous blood.²⁴ After anesthesia with intraperitoneal injection of 4% chloral hydrate at a dosage of 1 ml/100 g, 100 μl of autologous blood was collected from the heart, and then the rats were fixed in the stereotaxic frame (Zhenghua Biological Equipment Co. Ltd, Anhui, China). The scalp was exposed and made drilling a hole corresponding to right basal ganglia (0.2 mm anterior to the intersection between the coronal suture and sagittal midline and 3.5 mm to the right of the sagittal suture). A microsyringe was affixed to the stereotactic frame and a needle was slowly inserted (5.5 mm in depth), and then 100 μl of autologous blood was slowly injected (20 μl/min). Before slowly withdrawing the needle, it was kept in place for another 5 min. Rats in the Sham group were intracerebrally injected with 100 μl physiological saline solution. The bone hole was sealed with bone wax, and skin incision was disinfected and sutured. During the establishment of the model, rats’ vital signs were monitored and maintained in normal level.

Primary neuron- and microglia-enriched cultures

Neuron-enriched cultures were prepared from brains of fetal rats (from 16–18-day-old pregnant SD rats).²⁵ The meninges and blood vessels were removed from the brain and then brain tissues were digested with 0.25% trypsin (with EDTA) for 5 min at 37 °C. The tissues were washed three times with PBS to terminate trypsin digestion. Then, brain tissue suspensions were centrifuged at 1500 r.p.m. for 5 min, and the cells were suspended in a Neurobasal-A medium containing 2% B27, 2 mM l-glutamine, 50 U/ml penicillin and 50 U/ml streptomycin (all from Gibco, Carlsbad, CA, USA). Finally, cells were plated in 6- or 12-well plates in a fresh medium and later half the medium was changed with fresh medium every 2 days.

For primary microglial cultures, the whole brains of 1- day-old rats were used.²⁶ The digestion was similar to neurons, but the pellets after centrifugation were suspended in DMEM/F12 containing 10% fetal bovine serum, 1 mM sodium pyruvate, 2 mM l-glutamine, 100 mM nonessential amino acids, 50 U/ml penicillin and 50 U/ml streptomycin (all from Gibco). Then, cells were seeded into 150 cm² culture flask in a fresh medium and half the medium was changed with fresh medium every 2 days. Two weeks after initial seeding, a confluent polylayer of glial cells could be observed. The microglia was separated from astrocytes by shaking the flask at 150 r.p.m. for 4 h, and then it was collected by centrifugation and reseeded in 12-well plates with fresh medium.

Drug administration

Nec-1 was prepared in DMSO at a concentration of 1 μg/3 μl,²⁷ and z-VAD (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA) was prepared in DMSO at a concentration of 100 μ M.¹⁰ At 1–2 h before ICH, both the inhibitors were injected into the lateral cerebral ventricle at a volume of 3 μl. Equal volumes of DMSO were used as vehicle. For in vitro experiments, the TNF-α inhibitor (Santa Cruz Biotechnology) was also dissolved in DMSO at a final concentration of 50 μM in a neuronal medium. The final concentration of Nec-1 and z-VAD was 30 and 100 μM, respectively, in a neuronal medium.

ICH models in vitro

An ICH model in vitro was established by neuronal stimulation using OxyHb.²¹ Neurons were treated with OxyHb (10 μM) for 6 h at 37 °C in 5% CO₂, and then cell medium was removed, washed three times with PBS and followed by other experiments.

The other in vitro ICH model used conditioned medium to treat neurons. First, microglia were treated with OxyHb (10 μM) and then incubated for 24 h at 37 °C with 5% CO₂. The supernatant collected was centrifuged at 10 000 r.p.m. for 5 min and then was transferred to a conditioned medium. Half of the neuronal medium was changed with conditioned medium and incubated for another 6 h at 37 °C with 5% CO₂. The cell medium was removed, washed three times with PBS and followed by other experiments.

Transfection of siRNA and adenoviruses in vivo and in vitro

The following three kinds of recombinant adenoviruses were used: (1) adenoviruses containing rat RIP1 (Ad-RIP1; Genbank ID: 157824040) that was used to overexpress RIP1 protein; (2) adenovirus with rat RIP1 with mutation of S166A (Ser166 of RIP1 was mutated to alanine, Ad-RIP1-S166A); and (3) adenovirus with human GFP (Ad-GFP) as a control to Ad-RIP1. Ad-RIP1 and Ad-RIP1-S166A (both 6 × 10⁹ PFU/ml) and Ad-GFP (2 × 10⁹ PFU/ml) were produced by Genescript (Nanjing, China). All of them were stored at −80 °C and diluted to 1 × 10⁹ PFU/ml in an enhanced transfection solution (Genescript) before intracerebroventricular injection in vivo and diluted to 1 × 10⁸ PFU/ml before being transfected to the cultured neuron.

The following two kinds of siRNAs were used: (1) disorganizing rat RIP1 mRNA (Si-RIP1) to silence its transcription and (2) scramble siRNA (Si-negative control) (both from Genescript). According to the manufacturer’s instructions for Entranster-in vivo RNA transfection reagent (Engreen, Shanghai, China), 500 pmol RIP1 siRNA and 500 pmol scramble siRNA were dissolved in 5 μl RNase-free water. Then, 10 μl Entranster-in vivo RNA transfection reagent was added to 5 μl siRNA or 5 μl scramble siRNA. After mixing for another 15 min, Entranster-in vivo-siRNA mixture was injected intracerebroventricularly 24 h before ICH. RIP1 siRNA sequences were as follows: sense, 5′-GGAACAACGGAGUAUAUAAdTdT-3′ and Antisense: 3′-dTdTCCUUGUUGCCUCAUAUAUU-5′.

Western blot analysis

Western blot analysis was performed as indicated previously.²⁵ Briefly, the brain samples or extracted cells were mechanically lysed in RIPA lysis buffer (Beyotime, Shanghai, China). Then we used enhanced BCA Protein Assay Kit (Beyotime) to measure protein concentrations by the bicinchoninic acid method. The protein samples (50 μg per lane) were then loaded onto a 10% SDS-polyacrylamide gel, separated and electrophoretically transferred to a polyvinylidene difluoride membrane (Millipore Corporation, Billerica, MA, USA), which was then blocked with 5% bovine serum albumin (BioSharp, Anhui, China) (1 h at room temperature). Then, the membrane was incubated for 12 h at 4 °C with primary antibodies. The primary antibodies used were RIP1, RIP3, MLKL, caspase-8, p-Ser (all from Santa Cruz Biotechnology) and albumin (Abcam, Cambridge, UK; ab106582). Besides, the β-tubulin was also detected and served as a loading control. Later, the membrane was incubated with related HRP-conjugated secondary antibody (Santa Cruz Biotechnology) for 2 h at room temperature. We revealed the band signals via the Enhanced Chemiluminescence (ECL) Kit (Beyotime), and the relative quantity of proteins was analyzed via the Image J Software (NIH, Bethesda, MD, USA) and normalized to that of the loading control as discussed previously. In addition, the levels of phosphorylation were evaluated as the ratio of phosphoprotein to total protein.

Immunoprecipitation

Briefly, according to our previous report,²⁵ the brain samples were lysed in ice-cold RIPA lysis buffer (Beyotime). For IP, the lysate was incubated with specific antibodies against RIP1 or rabbit IgG (negative control) overnight at 4 °C with agitation. Protein A+G Sepharose beads were then added to each immune complex, and the lysate–bead mixture was incubated for 4 h at 4 °C under rotary agitation. SDS-PAGE and immunoblotting were then performed for further protein separation and detection.

Immunofluorescent staining

The brain samples were fixed in 4% paraformaldehyde, embedded in paraffin and then cut into 4 μm sections. The cultured neurons were fixed in 4% paraformaldehyde. Then, the sections and neurons were incubated with primary antibody against RIP1, RIP3, MLKL and caspase-8 and secondary antibodies. Normal rabbit IgG, normal mouse IgG and normal goat IgG were used as negative controls for the immunofluorescence assay (data not shown). Nuclei were stained with DAPI mounting medium. Finally, the sections and neurons were observed in a fluorescence microscope (OLYMPUS BX50/BX-FLA/DP70; Olympus Co., Tokyo, Japan). The relative fluorescence intensity was analyzed with the Image J program. The quantitative analysis was performed by an observer who was blinded to the experimental group.

PI and TUNEL staining

For all experiments, PI (Sigma-Aldrich, St. Louis, MO, USA) was administered intraperitoneally (1 μg/g) while the ICH model was established.²⁸ According to the manufacturer’s protocol (Roche, Mannheim, Germany), we detected cell apoptosis via TUNEL staining. The brain was removed and frozen in liquid nitrogen and then stored at −80 °C. Then, brain sections (12 μm) were cut on a cryostat 150 to 200 μm apart along the anterior–posterior lesion and were placed on poly-l-lysine-coated glass slides (−80 °C). Next, the sections were incubated with TUNEL staining (37 °C for 1 h). After washing three times with PBST, sections were visualized by a fluorescence microscope (OLYMPUS BX50/BX-FLA/DP70; Olympus Co.). In conclusion, PI/TUNEL+ cells were counted by observers blinded to the groups of experiment. To evaluate necropoptosis/apoptosis of cells, we examined and photographed six microscopic fields per sample and the index was defined as the average number in each section. As reported previously, PI+/TUNEL− is defined as the pure necropoptosis, PI−/TUNEL+ is defined as pure apoptosis, and PI+/TUNEL+ is defined as the mixed cell death.²⁹

Brain edema and BBB injury

As described previously,²¹ the wet–dry method was adopted to evaluate the index of brain edema and BBB injury. Briefly, after brain tissues were removed and collected, the samples were weighed immediately (wet weight), dried (100 °C for 72 h) and then reweighed (dry weight). Thus, brain edema was calculated as ((wet weight−dry weight)/wet weight) × 100%. Meanwhile, the level of albumin in brain tissues was regarded as the index of BBB injury. The western blot was used to test the level of albumin.

Neurobehavioral evaluation

At 24 h after ICH, all the rats in the experiments were examined for behavioral impairment using a scoring system and were monitored for appetite, activity and neurological defects, as reported previously³⁰ (for more details see Table 2).

Table 2 Neurobehavioral evaluation

Full size table

Assay of inflammatory cytokines (TNF-α)

We used specific Rat Tumor necrosis factor α, TNF-α ELISA KIT (Bio-Swamp; Hubei, China) to quantify the levels of TNF-α in the CSF according to the manufacturer's instructions.

Annexin V and PI staining in vitro

After various treatments, neurons were trypsinized by 0.25% trypsin (without EDTA) and centrifuged at 1500 r.p.m. for 5 min, and the resulting cell pellet was resuspended in 500 μl binding buffer. Later, 5 μl Annexin V and 5 μl PI (Beyotime, Shanghai, China) were added to the cell suspension. After 20 min of incubation at 37 °C in the dark, the cells were analyzed by flow cytometry (FACS Cabibur; BD, San Diego, CA, USA) and at least 20 000 events per sample were recorded.

PI and Hoechst staining in vitro

After various treatments, add 5 μl Hoechst and 5 μl PI (Beyotime, Shanghai, China) to the cell medium. After 20 min of incubation at 4 °C in the dark, the cells were analyzed by a fluorescence microscope (OLYMPUS BX50/BX-FLA/DP70; Olympus Co., Tokyo, Japan.)

Statistical analysis

All data are presented as mean±S.E.M. GraphPad Prism 5.0 software (GraphPad, San Diego, CA, USA) was used for statistical analysis. Data sets were tested for normality of distribution with Kolmogorov–Smirnov test. Data groups (two groups) with normal distribution were compared using two-sided unpaired Student’s t-test, and the Mann–Whitney U-test was used for nonparametric data. P<0.05 indicates a statistically significant difference.

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Acknowledgements

This work was supported by Suzhou Key Medical Center (Szzx201501), grants from the National Natural Science Foundation of China (No. 81601007), Scientific Department of Jiangsu Province (No. BL2014045), Education Department of Jiangsu Province (No. 16KJB320008), Suzhou Government (No. SZS201413 and SYS201608) and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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Haitao Shen, Chenglin Liu and Dongping Zhang: These authors contributed equally to this work.

Authors and Affiliations

Department of Neurosurgery and Brain and Nerve Research Laboratory, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu Province, China
Haitao Shen, Chenglin Liu, Dongping Zhang, Xiyang Yao, Kai Zhang, Haiying Li & Gang Chen

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Haitao Shen
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Chenglin Liu
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Dongping Zhang
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Xiyang Yao
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Kai Zhang
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Haiying Li
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Gang Chen
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Shen, H., Liu, C., Zhang, D. et al. Role for RIP1 in mediating necroptosis in experimental intracerebral hemorrhage model both in vivo and in vitro. Cell Death Dis 8, e2641 (2017). https://doi.org/10.1038/cddis.2017.58

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Received: 27 October 2016
Revised: 28 December 2016
Accepted: 03 January 2017
Published: 02 March 2017
Issue Date: March 2017
DOI: https://doi.org/10.1038/cddis.2017.58

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