Necrostatin-1 decreases necroptosis and inflammatory markers after intraventricular hemorrhage in mice

Abstract

Necrostatin-1, an inhibitor of necroptosis, can effectively inhibit necrotic apoptosis in neurological diseases, which results in the inhibition of inflammation, endoplasmic reticulum stress, and reactive oxygen species production and substantial improvement of neurological function. However, the effects of necrostatin-1 on intraventricular hemorrhage (IVH) remain unknown. In this study, we established a mouse model of IVH by injecting autologous blood into the lateral ventricle of the brain. We also injected necrostatin-1 into the lateral ventricle one hour prior to IVH induction. We found that necrostatin-1 effectively reduced the expression levels of the necroptosis markers receptor-interacting protein kinase (RIP)1, RIP3, mixed lineage kinase domain-like protein (MLKL), phosphorylated (p)-RIP3, and p-MLKL and the levels of interleukin-1β , interleukin-6, and tumor necrosis factor-α in the surrounding areas of the lateral ventricle. However, necrostatin-1 did not reduce ependymal ciliary injury or brain water content. These findings suggest that necrostatin-1 can prevent local inflammation and microglial activation induced by IVH but does not greatly improve prognosis.

Keywords: MLKL; RIP1; RIP3; ependymal cilia; hydrocephalus; inflammation; intraventricular hemorrhage; microglia; necroptosis; necrostatin-1.

Figure 1

Experimental design and establishment of the IVH model. (A) Experimental design and animal groups. (B) Brain tissue sections from mice in the sham and IVH groups 1 day after modeling. The ventricle size in the sham group was normal. In the IVH group, the obvious presence of blood was observed in the ventricular system at all levels. The arrows indicate the blood in the brain tissue. (C) T2-weighted MRI of brains from the sham and IVH groups 1 day after modeling. In the sham group, the ventricular system was filled with normal cerebrospinal fluid, and T2 images presented a high signal. In the IVH group, the T2 images of the ventricular system demonstrated a low signal, indicating blood retention in the ventricle. H&E: Hematoxylin-eosin staining; IVH: intraventricular hemorrhage; MRI: magnetic resonance imaging; Nec-1: necrostatin-1; SEM: scanning electron microscope.

Figure 2

Changes in levels of necroptosis markers within the periventricular tissue after IVH induction. (A) Representative western blots of RIP3 and MLKL. The box represents the tissue-extraction site for western blot assay. (B, C) Quantitative analyses of RIP3 (B) and MLKL (C) protein levels. (D, E) Representative images of immunostaining for p-RIP3 (D) and p-MLKL (E) in the sham and IVH groups on day 3 post-IVH induction. No significant positive results were observed for p-RIP3 or p-MLKL in the sham group, while p-RIP3 and p-MLKL were expressed in the IVH group. Scale bars: 50 μm. (F, G) Statistical analysis of p-RIP3 (F) and p-MLKL (G) expression in tissue from the sham and IVH groups on day 3. Data are expressed as the mean ± SEM (n = 6). *P < 0.05, **P < 0.01, vs. sham group (unpaired Student’s t-test). IVH: Intraventricular hemorrhage; MLKL: mixed lineage kinase domain-like protein; Nec-1: necrostatin-1; p-MLKL: phosphorylation mixed lineage kinase domain-like protein; p-RIP3: phosphorylation receptor-interacting protein 3; RIP3: receptor-interacting protein 3.

Figure 3

Immunofluorescent staining for the necroptosis markers p-RIP3 and p-MLKL in the periventricular tissue on day 3 after IVH. (A) Representative images after immunofluorescent staining for p-RIP3 (red, CoraLite594), NeuN (green, CoraLite488), Iba1 (green, CoraLite488), and GFAP (green, CoraLite488) in the periventricular tissue after IVH. Three days after IVH modeling, the necroptosis indicator p-RIP3 was mainly co-localized with neurons in the lateral ventricle tissue. (B) Representative images after immunofluorescent staining for p-MLKL (red, CoraLite594), NeuN (green, CoraLite488), Iba1 (green, CoraLite488), and GFAP (green, CoraLite488) in the periventricular tissue. Three days after IVH modeling, the necroptosis indicator p-MLKL in the lateral ventricle tissue was mainly co-localized with neurons. Scale bars: 20 μm. DAPI: 4’,6-Diamidino-2-phenylindole; GFAP: glial fibrillary acidic protein; Iba-1: ionized calcium binding adapter molecule 1; IVH: intraventricular hemorrhage; NeuN: neuronal nuclei antigen; p-MLKL: phosphorylation mixed lineage kinase domain-like protein; p-RIP3: phosphorylation receptor-interacting protein 3.

Figure 4

Effects of necrostatin-1 on the expression of necroptosis markers in periventricular tissue 3 days after IVH induction. (A) Representative western blot of RIP1. (B) Quantitative analysis of RIP1 protein expression. (C, D) Representative images of p-RIP3 (C; red, CoraLite594) and p-MLKL (D; red, CoraLite594) immunofluorescent staining in the periventricular tissue after IVH. Compared with that in the sham group, p-RIP3 and p-MLKL were significantly increased in the IVH group. Nec-1 attenuated the IVH-mediated increases in p-RIP3 and p-MLKL. Scale bars: 20 μm. (E, F) Quantitative analyses of p-RIP3 (E) and p-MLKL (F) relative fluorescent intensities in the periventricular tissue. (G, H) qRT-PCR analyses of Rip3 (G) and Mlkl (H) mRNA expression. Data are expressed as the mean ± SEM (n = 3–6). *P < 0.05, **P < 0.01, vs. sham group; #P < 0.05, ##P < 0.01, vs. IVH group (unpaired Student’s t-test). DAPI: 4′,6-Diamidino-2-phenylindole; IVH: intraventricular hemorrhage; MLKL: mixed lineage kinase domain-like protein; Nec-1: necrostatin-1; p-MLKL: phosphorylation mixed lineage kinase domain-like protein; p-RIP3: phosphorylation receptor-interacting protein 3; qRT-PCR: quantitative reverse transcription-polymerase chain reaction; RIP3: receptor-interacting protein 3.

Figure 5

Effects of necrostatin-1 on necrotic cell death and pathological damage in the periventricular tissue on day 3 after IVH induction. (A) Representative images of PI (red) staining. Compared with that in the sham group, the numbers of PI-positive cells in the IVH group were increased. Compared with that in the IVH group, the numbers of PI-positive cells in the IVH + Nec-1 group were decreased. Scale bars: 50 μm. (B) The changes in the number of PI-positive cells in the periventricular tissue. Data are expressed as the mean ± SEM (n = 6). **P < 0.01, vs. sham group; #P < 0.05, vs. IVH group (unpaired Student’s t-test). (C) Representative HE-stained images of the periventricular tissue. In the sham group, the morphology of the periventricular tissue was normal, and the ependymal membrane and cilia were intact. The IVH group showed damage of the periventricular tissue and destruction of the ependymal cilia, while the IVH + Nec-1 group showed no significant improvement in periventricular tissue damage or repair of ependymal cilia. Scale bars: 50 μm. DAPI: 4′,6-Diamidino-2-phenylindole; IVH: intraventricular hemorrhage; Nec-1: necrostatin-1; PI: propidium lodide.

Figure 6

Effects of necrostatin-1 on microglial activation and inflammation around the lateral ventricle after IVH induction. (A) Representative images of Iba-1 (red, CoraLite594) immunofluorescent staining in the periventricular tissue. Compared with that in the sham group, the expression of Iba-1 was increased in the IVH group. Compared with that in the IVH group, Iba-1 expression decreased in the IVH + Nec-1 group. Scale bars: 20 μm. (B) Quantitative analyses of Iba-1 relative fluorescent intensities in the periventricular tissue. (C) Representative images of IL-6 (green, CoraLite488) immunofluorescent staining in the periventricular tissue. Compared with that in the sham group, the expression of IL-6 was increased in the IVH group. Compared with that in the IVH group, IL-6 expression decreased in the IVH + Nec-1 group. Scale bars: 20 μm. (D) Quantitative analyses of IL-6 relative fluorescent intensities in the periventricular tissue. (E) Representative western blots showing the protein expression of IL-1β, IL-6, and TNF-α. (F–H) Quantitative analyses of the changes in protein levels of IL-1β (F), IL-6 (G), and TNF-α (H). Data are expressed as the mean ± SEM (n = 6). **P < 0.01, vs. sham group; #P < 0.05, ##P < 0.01, vs. IVH group (unpaired Student’s t-test). DAPI: 4′,6-Diamidino-2-phenylindole; Iba-1: ionized calcium binding adapter molecule 1; IL: interleukin; IVH: intraventricular hemorrhage; Nec-1: necrostatin-1; TNF-α: anti-tumor necrosis factor-α.

Figure 7

Effects of necrostatin-1 on hydrocephalus after IVH induction. (A) Representative images of T2 MRI scans. The sham group showed no hydrocephalus, while the IVH and IVH + Nec-1 groups showed significant hydrocephalus. (B) On day 3 after IVH induction, Nec-1 pretreatment did not significantly reduce the brain water content that was observed in the IVH only group. Data are expressed as the mean ± SEM (n = 3–6). **P < 0.01, vs. sham group (unpaired Student’s t-test). (C) Scanning electron microscopy images showing the ependymal cilia. The ependymal cilia in the sham group were dense, while the ependymal cilia in IVH and IVH + Nec-1 groups were sparse and irregular. Scale bars: 10 μm. IVH: Intraventricular hemorrhage; MRI: magnetic resonance imaging; Nec-1: necrostatin-1.