Additional file 1 of RIPK1 or RIPK3 deletion prevents progressive neuronal cell death and improves memory function after traumatic brain injury

Additional File 1 Supplementary Fig. S1. Genotyping of RIPK1 and RIPK3 deficient mice and proof of neuronal specific RIPK1 knock-out in RIPK1flox/floxCamk2CreERT2 mice. a. Neuron specific RIPK1 deficient mice used for experiments were heterozygous for Camk2CreERT2 and homozygous for the floxed RIPK1 allele. Littermate controls were also homozygous for the floxed RIPK1 allele, but did not express the Cre recombinase. b. Global RIPK3 deficient mice were homozygous for disrupted allele, while control mice expressed only the wild type gene. c. and d. To demonstrate specific neuron specific RIPK1 deficiency in induced RIPK1flox/floxCamk2CreERT2 mice, we performed immunohistochemistry for RIPK1 and NeuN, a neuronal marker. In the cortex of control mice RIPK1 was almost exclusively expressed in neurons (upper panels), while in induced RIPK1flox/floxCamk2CreERT2 mice RIPK1 staining was significantly reduced (lower panels) to 20% of baseline (d). Additional File 1 Supplementary Fig. 2 Body weight and physical condition after experimental TBI. a. and b. Weight after TBI. Animals recovered from weight loss directly after trauma within one week after injury; in the following observation period weight constantly increased. No differences were detected between CCI and sham-operated animals in the RIPK1 (a) and RIPK3 (b) groups. c. and d. General health score to assess recovery. All animals’ general condition transiently worsened in the perioperative phase with a peak at day 1 after TBI, but returned to baseline within one week. There was no difference between groups, c. RIPK1, d. RIPK3. Data are presented as mean ± SD; n = 5 for sham, n = 8–10 for TBI. Additional File 1 Supplementary Fig. 3. Individual lesion volume progression for a. RIP1 and b. RIP3 deficient mice. Additional File 1 Supplementary Fig. 4. RIPK3 deficiency does not affect acute brain injury after TBI. No differences in lesion volume as assessed by histology was detected between RIPK3 knockout mice and C57BL/6 wild type controls at 24 h after TBI. Data are presented as mean ± SD; n = 10. Additional File 1 Supplementary Fig. 5. Lesion volumes by MRI and histology. a. T2-weighted MRI and Nissl stained coronal section three months after injury. Lesion volume was quantified in T2-weighted MRI images as well as Nissl stained sections obtained in the same animals three months post injury. b. Correlation analysis of lesion volumes assessed by histomorphometry or T2-weighted MRI revealed a strong positive correlation between both methods. Differences in measured lesion size are due to shrinking of tissue in the fixation process, with a shrinking factor of 0.77 ± 0.23. c.—f. Quantification of lesion volume (c. RIPK1, d. RIPK3) as well as hippocampal damage (e. RIPK1, f. RIPK3) using both methods. Data are presented as mean ± SD; n = 8–10 for TBI. Pearson correlation, Students t-test for parametric and Man-Whitney-Rank-Sum test for non-parametric data were used. *p < 0.05, ***p < 0,001.