Genetic ablation of soluble tumor necrosis factor with preservation of membrane tumor necrosis factor is associated with neuroprotection after focal cerebral ischemia

Abstract

Microglia respond to focal cerebral ischemia by increasing their production of the neuromodulatory cytokine tumor necrosis factor, which exists both as membrane-anchored tumor necrosis factor and as cleaved soluble tumor necrosis factor forms. We previously demonstrated that tumor necrosis factor knockout mice display increased lesion volume after focal cerebral ischemia, suggesting that tumor necrosis factor is neuroprotective in experimental stroke. Here, we extend our studies to show that mice with intact membrane-anchored tumor necrosis factor, but no soluble tumor necrosis factor, display reduced infarct volumes at one and five days after stroke. This was associated with improved functional outcome after experimental stroke. No changes were found in the mRNA levels of tumor necrosis factor and tumor necrosis factor-related genes (TNFR1, TNFR2, TACE), pro-inflammatory cytokines (IL-1β, IL-6) or chemokines (CXCL1, CXCL10, CCL2); however, protein expression of TNF, IL-1β, IL-6 and CXCL1 was reduced in membrane-anchored tumor necrosis factor(Δ/Δ) compared to membrane-anchored tumor necrosis factor(wt/wt) mice one day after experimental stroke. This was paralleled by reduced MHCII expression and a reduction in macrophage infiltration in the ipsilateral cortex of membrane-anchored tumor necrosis factor(Δ/Δ) mice. Collectively, these findings indicate that membrane-anchored tumor necrosis factor mediates the protective effects of tumor necrosis factor signaling in experimental stroke, and therapeutic strategies specifically targeting soluble tumor necrosis factor could be beneficial in clinical stroke therapy.

Keywords: Tumor necrosis factor; behavior; chemokines; cytokines; macrophages; microglia; neuroprotection.

Figure 1.

Genetic ablation of solTNF does not change microglial cell density and morphology or behavioral phenotype under naive conditions. (a, b) To assess exploratory behavior, the total number of arm entries in the Y-maze (a) and spontaneous alternation (b) were measured. (c–e) To assess spontaneous locomotor activity and anxiety-related behavior, total distance travelled (c), center/perimeter ratio (d) and number of rearings (vertical activity) (e) were measured in the open field. (f, g) Motor coordination was assessed with the rotarod test, (f) and strength was measured by grip strength analysis (g). No differences were observed between groups (Student's t test, n = 5–15 mice/group). (h) Representative immunohistochemical photomicrographs of Iba1⁺ cells in the cingulate gyrus of the neocortex (upper panel) and magnifications of Iba1⁺ microglia in the neocortex (lower panel). (i) Quantification of the number of Iba1⁺ microglial cells/area in the neocortex of naive mTNF^wt/wt and mTNF^Δ/Δ mice (Student's t test, n = 6 mice/group). (j) Quantification of the neocortical volume of naive mTNF^wt/wt and mTNF^Δ/Δ mice (Student's t test, n = 6 mice/group). Scale bars: 10 μm. Results are expressed as mean ± SEM.

Figure 2.

Genetic ablation of solTNF reduces infarct volumes after pMCAO with no changes in locomotor activity and anxiety-related behaviors assessed in the open field test. (a) Toluidine blue staining of frontal brain sections from mTNF^wt/wt and mTNF^Δ/Δ one and five days after pMCAO. (b) Schematic drawing of frontal sections of the mouse brain showing the location of motor (M) and sensory (S) areas in the cortex affected by pMCAO. (c) Estimation of cortical infarct volume in mTNF^Δ/Δ mice and mTNF^wt/wt littermates at one and five days after surgery (*P < 0.05, one-tailed Student's t test, n = 7–18 mice/group). (d,e) Rostrocaudal distributions of the infarcts at one day (d) and five days (e) after pMCAO. (f–h) Open field test on mTNF^Δ/Δ mice and TNF^wt/wt littermates two days after pMCAO. Locomotion was measured as total distance travelled (c), and stress-related behavior as center/perimeter ratio (d), and number of rearings (e). Results are expressed as mean ± SEM (n = 8–9 mice/group). No significant differences were measured between groups, Student's t test. Ctx: cortex; IF: infarct; Str: striatum.

Figure 3.

Rung walk, rotarod and grip strength assessments of mTNF^Δ/Δ and mTNF^wt/wt mice after pMCAO. (a) Motor coordination and asymmetry by rung walk analysis was assessed two days after pMCAO and expressed as total number of missteps of the contralateral, right front limb compared to the unaffected left limb (paired Student's t test). (b) Motor coordination with the rotarod test was assessed three and five days after pMCAO as time spent on the rod (Student's t test). (c) Neuromuscular function was measured by grip strength analysis and expressed as Δ grip strength (change from baseline grip strength) (paired Student's t test). Results are expressed as mean ± SEM (n = 6–15 mice/group). *P < 0.05.

Figure 4.

Gene expression profiling one day following pCMAO. Differential gene expression was evaluated one day after pMCAO in mTNF^wt/wt and mTNF^Δ/Δ mice, and compared to corresponding sham and naive mice brain tissue expression. For each gene, results are expressed as percent of WT ± SEM after normalization to β-tubulin gene expression (n = 5–6 mice/group). *P < 0.05 one-way ANOVA, Tukey test; P < 0.05, Student's t test.

Figure 5.

Cytokine protein expression profiling following pMCAO. (a) Cytokines and chemokines were quantified by multiplex technology in naive mice and 3 h, one day and five days after pMCAO in mTNF^wt/wt and mTNF^Δ/Δ mice. For each protein, results are expressed as mean ± SEM (n = 5–6 mice/group). ***P < 0.001 one-way ANOVA with Bonferroni multiple comparison test. (b) Western blot quantification of TNFR1 in the brain of naive, sham and pMCAO mice with one day survival. Results are expressed as mean ± SEM (n = 6 mice/group). *P < 0.05 and **P < 0.005, Student's t test.

Figure 6.

Flow cytometry analysis of microglia and infiltrating leukocytes following pMCAO. (a, b) Number and percentage of infiltrating macrophages [CD45^highCD11b⁺Gr1⁻], granulocytes [CD45^highCD11b⁺Gr1⁺] and T cells [CD45^highCD3⁺] in mTNF^wt/wt and mTNF^Δ/Δ mice one day after pMCAO. (C) Representative flow cytometry plots comparing macrophage and granulocyte infliltration in mTNF^wt/wt and mTNF^Δ/Δ mice. (d, e) Number and percentage of microglia [CD45^dimCD11b⁺] in mTNF^wt/wt and mTNF^Δ/Δ mice one day after pMCAO. (f) Western blot quantification of MHCII in the brain of naive and pMCAO mice with one day survival. Results are expressed as mean ± SEM (n = 5–6 mice/group). *P < 0.05, Student's t test.