RIPK1 or RIPK3 deletion prevents progressive neuronal cell death and improves memory function after traumatic brain injury

Abstract

Traumatic brain injury (TBI) causes acute and subacute tissue damage, but is also associated with chronic inflammation and progressive loss of brain tissue months and years after the initial event. The trigger and the subsequent molecular mechanisms causing chronic brain injury after TBI are not well understood. The aim of the current study was therefore to investigate the hypothesis that necroptosis, a form a programmed cell death mediated by the interaction of Receptor Interacting Protein Kinases (RIPK) 1 and 3, is involved in this process. Neuron-specific RIPK1- or RIPK3-deficient mice and their wild-type littermates were subjected to experimental TBI by controlled cortical impact. Posttraumatic brain damage and functional outcome were assessed longitudinally by repetitive magnetic resonance imaging (MRI) and behavioral tests (beam walk, Barnes maze, and tail suspension), respectively, for up to three months after injury. Thereafter, brains were investigated by immunohistochemistry for the necroptotic marker phosphorylated mixed lineage kinase like protein(pMLKL) and activation of astrocytes and microglia. WT mice showed progressive chronic brain damage in cortex and hippocampus and increased levels of pMLKL after TBI. Chronic brain damage occurred almost exclusively in areas with iron deposits and was significantly reduced in RIPK1- or RIPK3-deficient mice by up to 80%. Neuroprotection was accompanied by a reduction of astrocyte and microglia activation and improved memory function. The data of the current study suggest that progressive chronic brain damage and cognitive decline after TBI depend on the expression of RIPK1/3 in neurons. Hence, inhibition of necroptosis signaling may represent a novel therapeutic target for the prevention of chronic post-traumatic brain damage.

Keywords: Chronic posttraumatic brain damage; Ferroptosis; Magnetic resonance imaging; Necroptosis; Neuroprotection; Traumatic brain injury.

Fig. 1

Experimental groups, methods, and time line

Fig. 2

Phosphorylated MLKL, a marker of necroptosis is reduced in RIP knockout animals. a. Exemplary staining of pMLKL and NeuN (upper panel) and a pMLKL positive neuron at higher magnification (lower panel) in the rim of a traumatic contusion in a wild type mouse. b. pMLKL is significantly reduced in a RIPK3 deficient mouse. c and d p-MLKL signal intensity was significantly increased after TBI in wild type animals (white bars) compared to sham animals indicating significant presence of necroptosis three months after TBI; this increase was significantly blunted in RIPK1 (c) as well as in RIPK3 d mice where there was no difference between TBI and sham animals. Data are presented as mean ± SD; n = 9–10 for RIPK1, n = 8–9 for RIPK3. Two-way RM ANOVA with Tukey’s multiple comparisons test was used. ***p < 0.001, n.s. indicates no significant statistical difference between groups

Fig. 3

RIPK1 and RIPK3 deficiency significantly reduces posttraumatic brain damage. a 3D reconstruction of lesion volume (blue) in relation to ipsi- and contralateral hippocampus (green) for a RIPK3 wild type and a RIPK3 deficient mouse 3 months after TBI. Scale bar = 5 mm. b and c Lesion volume over time quantified by repetitive T2-weighted MR imaging in RIPK1 (b) and RIPK3 (c) knockout animal. d and e. Hippocampal atrophy over time assessed in longitudinal T2-weighted MRI. RIPK1 (d) and RIPK3 (e) knockout mice show better preservation of hippocampal tissue over time. Mean ± SD; n = 9–10 for RIPK1, n = 8–9 for RIPK3. Two-way RM ANOVA with Sidak's multiple comparisons test was used. *p < 0.05, **p < 0.005, ***p < 0.001

Fig. 4

RIPK1 and RIPK3 deficiency reduces reactive astrogliosis three months after TBI. a Exemplary GFAP stainings in a wild type (left) and a RIPK1 knockout (right) animal at different distances from the traumatic contusion. Both images show the right hemisphere, but the left image has been mirrored along the midline for better visualization. Scale bar = 20 µm. Significant astrogliosis is present in the WT mouse, while it is heavily reduced in the neuronal RIPK1 deficient mouse. b and c Quantification of astrocyte coverage at different distances from the lesion. Astrocyte coverage was decreased in neuronal RIPK1 (b) and global RIPK3 (c) knockout animals compared to controls. Data are presented as mean ± SD; n = 9–10 for RIPK1, n = 8–9 for RIPK3. Students t-test for parametric and Man-Whitney-Rank-Sum test for non-parametric data were used. *p < 0.05, **p < 0.005, ***p < 0.001

Fig. 5

RIPK1 and RIPK3 deficiency reduces microglia activation. a. Exemplary stainings for the microglia marker iba1 in WT (upper inserts) and neuronal RIPK1 deficient mice (lower inserts) 100 µm (left inserts) and 300 µm (right inserts) from the rim of the lesion. b-i. Coverage and fractal analysis of microglia. In areas closer to the lesion site (100 µm, left side of each panel), knockout animals of both lines showed a decrease in microglia coverage (b. RIP 1, d. RIPK3) compared to their wild type littermates. Fractal analysis revealed that microglia of knockout animals in proximity to the lesion have less processes (c. RIP 1, e. RIPK3) are less circular (f. RIP 1, h. RIPK3), and overall smaller (g. RIP 1, i. RIPK3). In the more distal region, cells resembled those in sham operated animals, with no differences between genotypes. j-m. Sholl analysis also shows increased ramification, i. e. more active cells, close to the lesion (j. RIP 1, l. RIPK3), but not further away from the lesion site (k. RIP 1, m. RIPK3). Data are presented as mean ± SD; n = 9–10 for RIPK1, n = 8–9 for RIPK3. Student t-test for normalized and Man-Whitney-Rank-Sum-test for non-normalized data was used. *p < 0.05, **p < 0.005, ***p < 0.001. n.s. indicates no significant statistical difference between groups

Fig. 6

RIPK1 and RIPK3 deficiency improves neurocognitive performance three months after TBI. a and b Motor impairment after TBI. Beam Walk Test revealed long term impaired motor function of the left hind limb in TBI animals (missteps compared to respective baseline, # for WT, * for KO), but there were no differences between a RIPK1 or b RIPK3 knockout animals and their respective controls. c and d Depression-like behavior after TBI. Mice of both strains showed an increase of total immobility time throughout the time course of three months, however no significant differences could be detected between RIPK1 (c) and RIPK3 (d) knockout animals compared to wild type. e and f Learning and memory dysfunction after TBI. CCI induces severe long-term memory deficits in WT mice (open circles) while RIPK1 or RIPK3 knockout animals (grey circles) show similar long-term memory function as uninjured littermate controls. Data are presented as mean ± SD; n = 9–10 for RIPK1, n = 8–9 for RIPK3. Two-way RM ANOVA with Tukey’s multiple comparisons test was used. *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001

Fig. 7

Lesion progression occurs in areas with iron deposits. a T1-weighted MRI (upper panel) and Prussian blue staining (lower panel) three months after injury. There is a close spatial correlation between the T1-hyperintense signal and iron staining (arrowheads). b There is high spatial correlation between the area of T1 hyperintensities and iron deposits as assessed by Prussian blue staining. Pearson product-moment correlation analysis. c and d Iron deposits in pericontusional brain tissue assessed by longitudinal MRI. Extent of hemorrhage is comparable in RIPK1 (c) and RIPK3 (d) knockout animals and controls, indicating no differences in hemorrhage size after TBI between groups. T1 hyperintensities decreased over time in all groups, suggesting a very slow resorption of iron over time. e. Co-localization of iron deposits (red) observed at 1 month after TBI (upper panels) and lesion size assessed at the end of the observation period (3 months, middle row, green) suggests a progressive expansion of the lesion towards the regions with iron deposits. f and g Quantification of overlap between iron deposits and lesion. The higher the overlap of iron deposits and lesion size at three months, the higher the rate of tissue loss/ cell death in iron containing tissue. Co-localization is significantly less pronounced in RIPK1 (f) or RIPK3 (g) deficient mice, suggesting a reduced lesion growth in RIP knockouts due to toxic iron residues. Data are presented as mean ± SD; n = 9–10 for RIPK1, n = 8–9 for RIPK3. Two-way RM ANOVA with Tukey’s multiple comparisons test was used. **p < 0.005, ***p < 0.001