Tumor necrosis factor α antagonism improves neurological recovery in murine intracerebral hemorrhage

Abstract

Background: Intracerebral hemorrhage (ICH) is a devastating stroke subtype characterized by a prominent neuroinflammatory response. Antagonism of pro-inflammatory cytokines by specific antibodies represents a compelling therapeutic strategy to improve neurological outcome in patients after ICH. To test this hypothesis, the tumor necrosis factor alpha (TNF-α) antibody CNTO5048 was administered to mice after ICH induction, and histological and functional endpoints were assessed.

Methods: Using 10 to 12-week-old C57BL/6J male mice, ICH was induced by collagenase injection into the left basal ganglia. Brain TNF-α concentration, microglia activation/macrophage recruitment, hematoma volume, cerebral edema, and rotorod latency were assessed in mice treated with the TNF-α antibody, CNTO5048, or vehicle.

Results: After ICH induction, mice treated with CNTO5048 demonstrated reduction in microglial activation/macrophage recruitment compared to vehicle-treated animals, as assessed by unbiased stereology (P = 0.049). This reduction in F4/80-positive cells was associated with a reduction in cleaved caspase-3 (P = 0.046) and cerebral edema (P = 0.026) despite similar hematoma volumes, when compared to mice treated with vehicle control. Treatment with CNTO5048 after ICH induction was associated with a reduction in functional deficit when compared to mice treated with vehicle control, as assessed by rotorod latencies (P = 0.024).

Conclusions: Post-injury treatment with the TNF-α antibody CNTO5048 results in less neuroinflammation and improved functional outcomes in a murine model of ICH.

Figure 1

Timeline of experiments. Separate cohorts of animals were used to assess brain TNF-α and cleaved caspase-3 levels, F4/80 cell positivity, brain water content, hematoma volume, and rotorod latencies.

Figure 2

Cleaved caspase-3 expression after intracerebral hemorrhage. Representative cleaved caspase-3 western blots (A) and band density measurements (B) demonstrate reduction at 4, 24, and 72 hours after intrastriatal collagenase injection in mice given 7 mg/kg CNTO5048 or an equivalent volume of phosphate-buffered saline (PBS) via tail vein injection at 30 minutes after injury. (ANOVA; *P = 0.045 compared to PBS at 4 hours, ^#P = 0.046 compared to PBS at 72 hours; n = 3/group) CNTO4h, CNTO5048-treated mice at 4 hours after injury; CNTO24h, CNTO5048-treated mice at 24 hours after injury; CNTO72h, CNTO5048-treated mice at 72 hours after injury; GAPHD, glyceraldehyde-3-phosphate dehydrogenase; h, hours; PBS4h, PBS-treated mice at 4 hours after injury; PBS24h, PBS-treated mice at 24 hours after injury; PBS 72h, PBS-treated mice at 72 hours after injury.

Figure 3

Macrophage recruitment/microglial activation after intracerebral hemorrhage. Representative photos of F4/80 positive cells in ipsilateral hippocampus are shown from CNTO5048-treated (A) and phosphate-buffered saline-treated (B) mice. As a measure of microglial activation/macrophage recruitment, F4/80-positive cells were reduced in the ipsilateral hippocampus 7 days after intrastriatal collagenase injection in mice given 7 mg/kg CNTO5048 compared to those treated with an equivalent volume of phosphate-buffered saline via tail vein injection at 30 minutes after injury. (CNTO5048 versus vehicle: 3,880 ± 949 versus 4,953 ± 691 cells/mm³; t-test; *P = 0.049; n = 6/group).

Figure 4

Brain water content and hemorrhage volume after intracerebral hemorrhage. Brain water content (A) was decreased, while hemorrhage size (B) was unaffected at 24 hours after intrastriatal collagenase injection in mice given 7 mg/kg CNTO5048 compared to those treated with an equivalent volume of phosphate-buffered saline via tail vein injection at 30 minutes after injury. (CNTO5048 versus vehicle: 78.82% ± 0.64 versus 80.16% ± 0.89; t-test; *P = 0.026; n = 5/group).

Figure 5

Rotorod latencies after intracerebral hemorrhage. Over the first 7 days after intrastriatal collagenase injection, rotorod latencies were longer in mice given 7 mg/kg CNTO5048 compared to those treated with an equivalent volume of phosphate-buffered saline via tail vein injection at 30 minutes after injury. Due to differences in pre-injury latencies, post-injury latencies are reported as percentages of the baseline. Shams are not shown as they behave like uninjured animals, as previously demonstrated. (ANOVA; *P = 0.0243; n = 14/group; error bars represent standard deviation).