Inhibition of neuronal necroptosis mediated by RIP1/RIP3/MLKL provides neuroprotective effects on kaolin‐induced hydrocephalus in mice

Abstract Objectives Necroptosis is widespread in neurodegenerative diseases. Here, we examined necroptosis in the hippocampus and cortex after hydrocephalus and found that a necroptosis pathway inhibitor alleviates necroptosis and provides neuroprotective effects. Materials and methods Hydrocephalus was induced in C57BL/6 mice by kaolin. Haematoxylin and eosin (HE), Nissl, PI and Fluoro‐Jade B (FJB) staining were used for general observations. Phosphorylated receptor‐interacting protein kinase 3 (p‐RIP3) and phosphorylated mixed lineage kinase domain‐like (p‐MLKL) were measured by Western blotting and immunohistochemistry. Scanning electron microscopy (SEM) was used to observe ependymal cilia. Magnetic resonance imaging (MRI) and the Morris water maze (MWM) test were used to assess neurobehavioral changes. Immunofluorescence was used to detect microglial and astrocyte activation. Inflammatory cytokines were measured by Western blotting and RT‐PCR. Results Obvious pathological changes appeared in the hippocampus and cortex after hydrocephalus, and expression of the necroptosis markers p‐RIP3, p‐MLKL and inflammatory cytokines increased. Necrostatin‐1 (Nec‐1) and GSK872 reduced necrotic cell death, attenuated p‐RIP3 and p‐MLKL levels, slightly improved neurobehaviours and inhibited microglial and astrocyte activation and inflammation. Conclusions RIP1/RIP3/MLKL mediates necroptosis in the cortex and hippocampus in a hydrocephalus mouse model, and Nec‐1 and GSK872 have some neuroprotective effects.


| INTRODUC TI ON
Hydrocephalus is a disease in which excessive secretion, circulation and absorption of cerebrospinal fluid (CSF) are impaired by craniocerebral diseases, resulting in increased intracranial CSF levels, expansion of the ventricular system and progressive expansion of the subarachnoid space. 1 Hydrocephalus can be primary or secondary to trauma, spontaneous bleeding, infection and tumours and can also be comorbid with neurological diseases such as dementia, Alzheimer's disease and Parkinson's disease. [2][3][4] Hydrocephalus is a great challenge in neurosurgery because of its complicated aetiology, unclear mechanism, difficult treatment and many complications. Therefore, it is important to find a suitable and effective treatment for hydrocephalus.
Apoptosis and necrosis are two major types of cell death and are traditional classifications of cell death modalities. In recent years, this viewpoint has been challenged by the discovery of other types of programmed cell death, such as necroptosis, ferroptosis and pyroptosis. [5][6][7] In vivo evidence supports that necroptosis may have important functions in many human inflammatory diseases. 8 Necroptosis can be defined as follows: upon stimulation by death signals, cells upregulate receptor-interacting protein kinase 1 (RIP1) and receptor-interacting protein kinase 3 (RIP3), which activate a signalling cascade involving mixed lineage kinase domain-like protein (MLKL) phosphorylation, which contributes to a conformational change in the pseudokinase domain and ultimately changes the plasm membrane permeability, resulting in necrosis. 9-11 RIP1 activation has been shown to mediate multiple alternative cell death mechanisms, including RIPK1-dependent apoptosis (RDA) under apoptosis-proficient conditions and necroptosis under apoptosisdeficient conditions. 12,13 Necrostatin-1 (Nec-1) is a highly specific RIP1 kinase inhibitor, and numerous studies have demonstrated its neuroprotective effects. 14 Moreover, necroptosis has been shown to be dependent on RIP3 and MLKL. 15 The selective RIP3 inhibitor GSK872 can block necroptosis in human and murine cells, and in general, both Nec-1 and GSK872 inhibit necroptosis and inflammation. 16,17 Necroptosis has been reported in various models of neurological diseases, such as amyotrophic lateral sclerosis, ischaemic brain injury, intracerebral haemorrhage and traumatic brain injury (TBI). [18][19][20][21] However, whether necroptosis occurs in hydrocephalus remains unknown.
In the present study, we demonstrated that RIP1/RIP3/MLKLmediated necroptosis contributes to necrotic cell death after kaolininduced hydrocephalus in mice. We further showed that the RIP1 kinase inhibitor Nec-1 and the RIP3 kinase inhibitor GSK872 exert neuroprotective effects against necroptosis by inhibiting the RIP1/ RIP3/MLKL pathway and inflammation after hydrocephalus.

| Experiment animals
One hundred and forty C57BL/6 mice (weighing 18-23 g) were provided by the Laboratory Animal Center of Sichuan University for use in this study. The mice were housed under controlled, pathogen-free conditions with free access to pellet food and water. All procedures were strictly performed according to the animal use protocols, which were approved by the Animal Care and Use Committee of Sichuan University.

| Kaolin-induced hydrocephalus models and drug administration
The hydrocephalus model was induced via kaolin injection into the cisterna magna. 22 The mice were anaesthetized with pentobarbital (40 mg/kg intraperitoneally). The skin was incised along the midline of the neck, the muscles were separated, and the cisterna magna was exposed under aseptic conditions. Then, a 30-gauge needle (Hamilton, Switzerland), which was custom bent to 30°-45°, was inserted into the cisterna magna. A 6μl volume of a sterile kaolin suspension (100 mg/mL, Sigma-Aldrich) in 0.9% sterile saline was injected for approximately 30 seconds. The experimental groups were divided into the sham group, hydrocephalus group, hydrocephalus+Nec-1 group (Nec-1 group) and hydrocephalus+GSK872 group (GSK872 group). The specific necroptosis inhibitors Nec-1 (Selleck) and GSK872 (Merck Millipore, Billerica, MA, USA) were dissolved in

Gene
Primer

| Cell death assays
The mice were deeply anaesthetized and perfused with 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS), and the brains were removed. For HE staining, the brain tissues were fixed

| Western blotting, immunohistochemistry, immunofluorescence analysis
Total proteins were harvested from the cortex and hippocampus.
The proteins were transferred to polyvinylidene fluoride membranes (Millipore) that were blocked with 5% bovine serum albumin (BSA) for 1 hour at room temperature and incubated overnight with Nuclei were counterstained with DAPI (Thermo Fisher Scientific).

| Magnetic resonance imaging (MRI) and the Evans ratio
Mice were anaesthetized using a 2% isoflurane-air mixture during

| Morris water maze test
Learning and spatial memory were assessed by the Morris water maze (MWM) test 23-28 days after kaolin injection, and mice in the sham group were evaluated at the same time. The mice were placed in a pool of water (25℃) with a depth of 50 cm and a diameter of 200 cm, which was made opaque with titanium dioxide.
The pool was divided into four equally sized quadrants, and a submerged platform (1 ~ 1.5 cm; 6 cm × 6 cm below the water surface) was fixed in the target quadrant. The mice were allowed to find the submerged platform within 180 seconds. Then, a random starting position was used for 5 days of training in the four quadrants.
The latency times of 4 trials were recorded and averaged each day. On the sixth day of testing, after the submerged platform was removed, the mice completed a 120-second probe trial. Platform crossing times, distances travelled and times in the target quadrant were calculated.

| Scanning electron microscopy (SEM)
After soaking in 2.5% glutaraldehyde for 12 hour, the target sections were removed from the brain tissue. The selected brain tissue blocks were placed into 0.1 mol/L phosphate buffer overnight, followed by gradient alcohol dehydration and isoamyl acetate replacement. The specimens were then critical-point dried in CO2, mounted on SEM stubs and sputter-coated with gold-palladium. Then, the surfaces of the brain slices were examined with an AMR-1400 scanning electron microscope (Phillips) at an accelerating voltage of 20 kV.

| Real-time quantitative polymerase chain reaction (RT-PCR)
Total RNA was extracted from the cortex and hippocampus  Table 1.

| Statistical analysis
Statistical analysis was performed using GraphPad Prism software (GraphPad Software). All data are presented as the mean ± SEM.
Statistical differences among groups were analysed using Student's t-test, and a P-value <0.05 was considered statistically significant.

| Weight loss, destruction of ependymal cilia and pathological damage in hippocampal and cortex after kaolin-induced hydrocephalus
Body weight was significantly lower in the hydrocephalus group than the sham group at different time points ( Figure 1A). We observed significant hydrocephalus in the mice 28 days after model establishment ( Figure 1B). HE staining revealed cell body shrinkage and nuclear edge aggregation in the cortex and hippocampus in the hydrocephalus group ( Figure 1C). The Nissl staining results confirmed that hydrocephalus reduced the number of surviving neurons compared with the sham group ( Figure 1D). In addition, we found that there were few TUNEL-positive cells in the hippocampus after hydrocephalus, whereas TUNEL-positive cells were obvious in the cortex ( Figure 1E,F). FJB staining further confirmed that hydrocephalus reduced the numbers of surviving neurons in the cortex and hippocampus compared with the sham group ( Figure 1G,H). Moreover, SEM showed that ependymal cilia in the hydrocephalus group were significantly destroyed and sparsely distributed ( Figure 1I).

| Necroptosis, necrotic cell death and inflammatory cytokines are increased in the cortex and hippocampus after kaolin-induced hydrocephalus
Western blotting showed that the expression levels of p-RIP3 and p-MLKL in the hippocampus and cortex were increased at 28 days compared with the sham group (Figure 2A,B,E,F). Similarly, the immunohistochemical results showed that the expression of p-RIP3 and p-MLKL in the hippocampus and the cortex were higher in the hydrocephalus group than in the sham group ( Figure 2K-N). In addition, necrotic cell death was examined by PI labelling. The results showed that compared with those in the sham group, hydrocephalus triggered significant increases in the numbers of PI-positive cells in the cortex and hippocampus ( Figure 2I,J). Furthermore, we assessed several proinflammatory cytokines, such as IL-1β, IL-6 and TNFα, by Western blotting, and the results showed that the expression levels of IL-1β, IL-6 and TNFα in the cortex and hippocampus were increased in the hydrocephalus group compared with the sham group at 28 days (Figure 2C,D,G,H).

| Phosphorylated RIP3 and MLKL are mainly expressed by neuronal cells in the cortex and hippocampus after kaolin-induced hydrocephalus
To investigate the cell types with activated p-RIP3 and p-MLKL after hydrocephalus, we used double immunofluorescence staining to observe the cellular localization of p-RIP3 and p-MLKL in three kinds of cells, including NeuN + neurons, Iba-1 + microglia and GFAP + astrocytes, in the cortex and hippocampal DG, CA1 and CA3 regions.
We found that p-RIP3 and p-MLKL were mainly colocalized with NeuN+neurons in the hippocampus and cortex after hydrocephalus induction ( Figure 3A,B). We also found that p-RIP3 and p-MLKL rarely colocalized with Iba-1 + microglia or GFAP + astrocytes at the same time point ( Figure 3C-F).

| Nec-1 and GSK872 reduce necrotic cell death and attenuate the levels of phosphorylated RIP3 and MLKL in the cortex and hippocampus after hydrocephalus
PI was impermeable to the intact cell membrane and was used to

| Nec-1 and GSK872 improves neurobehavioral performance slightly and does not significantly reduce the size of the lateral ventricle
The ventricular volumes were significantly enlarged in the hydrocephalus group compared with the sham group. However, compared with that in the hydrocephalus group, Nec-1 and GSK872 did not significantly reduce the ventricle volume ( Figure 6A,B). Moreover, we found that Nec-1 and GSK872 could not reverse weight loss in mice after model induction ( Figure 6C). Furthermore, we assessed the neurocognitive functions by the MWM test ( Figure 6D). In the probe quadrant trial, the mice in the hydrocephalus group spent less time in the target quadrant than those in the sham group, while the reference memory deficits were increased by Nec-1 and GSK872 treatment ( Figure 6E). Moreover, compared with the sham group, the hydrocephalus group performed fewer platform crossings, while the mice in the Nec-1 and GSK872 groups did not perform significantly more platform crossings ( Figure 6F). The escape latencies of mice in the hydrocephalus group were increased compared with those in the sham group. The escape latencies of mice in the Nec-1 and GSK872 groups were decreased compared with the hydrocephalus group ( Figure 6G). The swim distance of mice in the hydrocephalus group was also increased compared with the sham group, and the swim distances of mice in the Nec-1 and GSK872 groups were decreased compared with the hydrocephalus group ( Figure 6H). Working model of necroptosis after hydrocephalus is shown in Figure 7.

| D ISCUSS I ON
Hydrocephalus induced by kaolin is still the most commonly used model to mimic obstructive hydrocephalus. 24 The present study Hydrocephalus compacts the cerebral cortex and hippocampus and changes their output neurons, which is associated with spatial learning and memory impairment. 25  Therefore, this time point is the focus of this study.
The detailed mechanisms of cell death or neuronal loss after hydrocephalus remain unclear. Necroptosis has been described as the basis of ischaemic injury, neurodegenerative diseases, viral infection and other diseases. [28][29][30] The phosphorylation of RIP3 and MLKL plays an important role in the development of necroptosis. 31 We further found that p-RIP3 and p-MLKL expression in the cortex and hippocampus increased after hydrocephalus. Moreover, p-RIP3 and p-MLKL were mainly colocalized with neurons but not astrocytes or microglia in the hippocampal cortex after hydrocephalus. Another feature of necroptosis is cell temperature, which can be detected by PI staining. We found that PI-positive cells were increased in the hippocampus and cortex after hydrocephalus.
These results suggest that necroptosis may be involved in cell death after hydrocephalus. This study is the first to report necroptosis in kaolin-induced hydrocephalus. Emerging evidence suggests that necroptosis can also trigger an inflammatory response. 32 Targeted interventions in necroptosis may ameliorate inflammatory and neurodegenerative diseases. 33 Inflammation plays an important role in the occurrence and development of hydrocephalus. [34][35][36][37] A previous study demonstrated that the expression of the proinflammatory cytokine IL-1β is significantly increased in the cortex and hippocampus in chronic hydrocephalus in juvenile rats. 38 In this study, we found inhibitor that is widely used in the study of necroptosis. 45 Studies have shown that GSK872 reduces the phosphorylation of RIP3 and MLKL, thereby ameliorating brain injury, antioxidant capacity and proinflammatory cytokines. 46-49 RIP3-and MLKL-dependent necrosis exacerbates tissue damage and inflammation in some animal models.
We therefore anticipated that necroptosis would play a proinflammatory role after hydrocephalus. In a hydrocephalus mouse model, Nec-1 and GSK872 reduce necrotic cell death, effectively inhibit the phosphorylation of RIP3 and MLKL, and reduce the levels of inflammatory factors such as IL-1β, IL-6 and TNFα in the cortex and hippocampus.
Moreover, the results showed that Nec-1 can significantly reduce the number of TUNEL-positive cells in the cortex after hydrocephalus, whereas GSK872 has no similar effect. Astrogliosis and microgliosis have been reported in kaolin-induced hydrocephalus in mice, rats and ferrets of different ages. 50,51 Reactive astrogliosis in the brain is characterized by GFAP upregulation and astrocytic process hypertrophy. 52 The level of GFAP is elevated in the CSF of human patients, and cortical biopsies of patients with chronic hydrocephalus show increased astrogliosis. 53,54 Activated microglia are associated with some nervous system diseases, such as Alzheimer's disease and chronic pain. 55 Previous reports have shown that inhibiting necroptosis can reduce not only the inflammatory response but also the expression of the astrocytic marker GFAP and the microglial marker Iba-1. 22,56 Moreover, there is evidence that necroptosis causes neuronal damage by affecting microglia or astrocytes. 57,58 In this study, we found that microglia and astrocytes were activated in the hippocampus and cortex of mice after hydrocephalus, and this response was alleviated by Nec-1 and GSK872. We hypothesize that this effect may be associated with the inhibition of the necroptosis-mediated inflammatory response after hydrocephalus. In addition, we found that both Nec-1 and GSK872 improved the neurobehaviours of mice slightly after hydrocephalus but had no significant effects on body weight or lateral ventricular enlargement. Taken together, these data indicate that Nec-1 and GSK872 have certain neuroprotective effects after hydrocephalus, such as anti-inflammatory effects, but some of the effects of blocking necroptosis on behaviour appear to be relatively minor, suggesting that necroptosis is perhaps not a major contributor to the impact of hydrocephalus on behaviour.
There are some limitations in the present study. First, we focused on one time point and did not observe potential changes at different time intervals after hydrocephalus. Second, we only showed pharmacological inhibition of necroptosis, and genetic inhibition was not further verified in gene knockout mice, such as RIP3 -/and MLKL -/mice. More accurate results may be obtained by gene inhibition.

F I G U R E 7
The mechanism of necroptosis after hydrocephalus. After hydrocephalus, RIP1 recruits and promotes RIP3 phosphorylation. Then, a body consisting of RIP1, RIP3 and MLKL is formed. MLKL is phosphorylated by p-RIP3, located at position 357 of threonine and position 358 of serine, and is considered to be the executor of necroptosis. p-MLKL finally leads to the exposure of cells by changing permeabilization in plasm membrane and result in necrosis of the cell. However, the effects were improved after the use necroptosis inhibitors Nec-1 and GSK872 Additionally, one of the major limitations is the descriptive nature of the experiments, which did not explore the mechanism by which hydrocephalus causes necroptosis. Therefore, these issues need to be clarified in future studies.
In summary, in line with increasing evidence of neuroprotection after inhibition of necroptosis, we found that necroptosis occurs after hydrocephalus. Although necroptosis may not be the major mode of cell death after hydrocephalus, inhibition of necroptosis may still produce some neuroprotective effects. Therefore, reducing necroptosis has potential value for the management of hydrocephalus.

E TH I C A L A PPROVA L A N D CO N S E NT TO PA RTI CI PATE
All experiments were reviewed and approved by the Ethics Committee of Sichuan University.

ACK N OWLED G EM ENTS
The work was funded by 1.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest. The sponsors had no role in the design, execution, interpretation or writing of the study, or in the decision to publish the results. Data; Writing -Project Administration, Liangxue Zhou, Aiping Tong.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.