Genetic analysis of the role of tumor necrosis factor receptors in functional outcome after traumatic brain injury in mice

Abstract

We previously reported that tumor necrosis factor-alpha (TNF-alpha) and Fas receptor induce acute cellular injury, tissue damage, and motor and cognitive deficits after controlled cortical impact (CCI) in mice (Bermpohl et al. 2007 ); however, the TNF receptors (TNFR) involved are unknown. Using a CCI model and novel mutant mice deficient in TNFR1/Fas, TNFR2/Fas, or TNFR1/TNFR2/Fas, we tested the hypothesis that the combination of TNFR2/Fas is protective, whereas TNFR1/Fas is detrimental after CCI. Uninjured knockout (KO) mice showed no differences in baseline physiological variables or motor or cognitive function. Following CCI, mice deficient in TNFR2/Fas had worse post-injury motor and Morris water maze (MWM) performance than wild-type (WT) mice (p < 0.05 group effect for wire grip score and MWM performance by repeated measures ANOVA). No differences in motor or cognitive outcome were observed in TNFR1/Fas KO, or in TNFR2 or TNFR1 single KO mice, versus WT mice. Additionally, no differences in propidium iodide (PI)-positive cells (at 6 h) or lesion size (at 14 days) were observed between WT and TNFR1/Fas or TNFR2/Fas KO mice. Somewhat surprisingly, mice deficient in TNFR1/TNFR2/Fas also had PI-positive cells, lesion size, and motor and MWM deficits similar to those of WT mice. These data suggest a protective role for TNFR2/Fas in the pathogenesis of TBI. Further studies are needed to determine whether direct or indirect effects of TNFR1 deletion in TNFR2/Fas KO mice mediate improved functional outcome in TNFR1/TNFR2/Fas KO mice after CCI.

FIG. 1.

Genotype analyses of knockout (KO) mice by polymerase chain reaction of tail DNA. Wild-type TNFR1 allele (470 bp) and neomycin resistance (Neo) cassette (300 bp) DNA sequences were amplified from tail DNA of mice using Jackson Laboratories protocol #002818. Wild-type TNFR2 allele (200 bp) and Neo cassette (400 bp) DNA sequences were amplified from tail DNA of mice using Jackson Laboratories protocol #002620. Wild-type Fas allele (700 bp) and Neo cassette (170 bp) DNA sequences were amplified from tail DNA of mice using Jackson Laboratories protocol #003233. (A) Wild-type versus R1/Fas KO mice (lanes 1, 3, 5, and 7: wild-type mice; lanes 2, 4, 6, and 8: R1/Fas KO mice). (B) Wild-type versus R2/Fas KO mice (lanes 1, 3, 5, and 7: wild-type mice; lanes 2, 4, 6, and 8: R2/Fas KO mice). (C) Wild-type versus triple KO mice (lanes 1, 3, 5, 7, 9, and 11: wild-type mice; lanes 2, 4, 6, 8, 10, and 12: triple KO mice; M, molecular-weight markers [100 bp]; TNFR, tumor necrosis factor receptor).

FIG. 2.

Vestibulomotor function as assessed by the wire-grip test before and up to 7 days after controlled cortical impact (CCI). (A) Wild-type (WT, n = 16) and TNFR1/Fas KO (n = 17, p = ns). (B) WT (n = 10) and TNFR2/Fas KO (n = 10, p = 0.016 group effect by repeated measures ANOVA). (C) WT (n = 9) and TNFR1 KO (n = 10, p = ns). (D) WT (n = 10) and TNFR2 KO (n = 10, p = ns). (E) WT (n = 8) and triple KO (TNFR1/TNFR2/Fas KO, n = 6, p = ns; KO, knockout; ANOVA, analysis of variance; TNFR, tumor necrosis factor receptor).

FIG. 3.

Morris water maze performance in naïve animals. No difference in performance was observed between uninjured (naïve) wild-type (WT) mice and knockout (KO) mice in hidden or visible platform trials. (A) Wild-type (WT, n = 10) and TNFR1 KO (n = 10) mice. (B) WT (n = 10) and TNFR2 KO (n = 10) mice. (C) WT (n = 10) and TNFR1/Fas KO (n = 7) mice. (D) WT (n = 10) and TNFR2/Fas KO (n = 8) mice. (E) Naïve animals (n = 10 TKO and n = 4 WT; TNFR, tumor necrosis factor receptor; TKO, triple knockout).

FIG. 4.

Morris water maze performance in injured mice. (A) Wild-type (WT, n = 10) and TNFR1 KO (n = 17) mice. (B) WT (n = 10) and TNFR2 KO (n = 16) mice. (C) WT (n = 10) and TNFR1/Fas KO (n = 10) mice. (D) WT (n = 20) and TNFR2/Fas KO mice (n = 15, p = 0.003 group effect by repeated measures ANOVA). (E) TNFR1/TNFR2/Fas KO (n = 5) and WT (n = 6) mice (ANOVA, analysis of variance; KO, knockout; TNFR, tumor necrosis factor receptor).

FIG. 5.

Lesion volume and propidium iodide (PI)-positive cell counts of wild-type (WT) versus knockout mice after controlled cortical impact (CCI). (A) Representative images of brain sections at 14 days after CCI, showing cavitary lesions. (B) Lesion volume at 14 days after injury for WT (n = 10) versus TNFR1 KO (n = 18) mice, WT (n = 10) versus TNFR1/Fas KO (n = 10) mice, WT (n = 10) versus TNFR2 KO (n = 16) mice, WT (n = 20) versus TNFR2/Fas KO (n = 15) mice, and WT (n = 10) versus TNFR1/TNRF2/Fas KO (n = 9) mice. (C) Representative images of PI labeling in vivo. (D) PI-positive cell counts at 6 h after injury. The y axis is the number of PI-positive cells per 200 × field for WT (n = 4) versus TNFR1/Fas KO (n = 4) mice, WT (n = 4) versus TNFR2/Fas KO (n = 4) mice, and WT (n = 10) versus TNFR1/TNFR2/Fas KO (n = 8) mice (p = ns for all between-group comparisons; TNFR, tumor necrosis factor receptor; KO, knockout).