Anti-Inflammatory Signaling Inhibits Neuroprotective Pro-Inflammatory Function Post-Traumatic Brain Injury in Berkeley Sickle Cell Mice: A Pilot Study
Authors: Rao Sohan , Parchuri Ektha , Carlson Shaun W 
Published: December 2024

Introduction:The pathophysiology and clinical outcomes of traumatic brain injury (TBI) in patients with sickle cell disease (SCD) is grossly understudied. TBI triggers a complex neuroinflammatory response involving a balance between pro- and anti-inflammatory mechanisms that is critical for optimal recovery. In the acute phase, the initial response is primarily pro-inflammatory, characterized by the activation of resident microglia, release of pro-inflammatory cytokines such as IL-6 and TNF-alpha, and recruitment of immune cells to the injury site. In the chronic phase, there is a shift towards an anti-inflammatory response, involving the production of anti-inflammatory cytokines such as IL-10 and the promotion of tissue repair and regeneration. Given that SCD is characterized by a chronic baseline pro-inflammatory state, we examine whether TBI in mice with SCD reflects a new homeostatic setpoint for neuroinflammatory response. To our knowledge, this is the first preclinical pilot study of the inflammatory response to TBI in SCD.

Methods:6 Berkeley (SCD) ɑ-/-, β-/-, Tg(HBɑ-HBBs)41Paz)/J hemizygote and 6 C57BL/6J (wild type) controls aged 12-18 weeks were split into 2 cohorts (n=3/group) for TBI and control (sham). Following a midline scalp incision, a 5mm craniotomy was performed via dental drill over the left parietal cortex-2.7mm lateral to midline and 2.5mm posterior to Bregma. Using a pneumatic impactor (Bimba, Monee, IL) with a 3 mm rounded-tipped impounder, a controlled cortical impact (CCI) model was performed over the craniotomy site (3.5 m/sec velocity; 1.0 mm depth) for the TBI groups. Sham controls were both anesthetized and surgically prepared without cortical impact. Plasma samples were collected at baseline, 24 hours, and 72 hours post-injury/sham. A multiplex assay (MesoScale Discovery, Rockville MD) quantified markers of inflammation in plasma samples. Two-way ANOVAs and post-hoc t-tests identified significant group mean differences in inflammatory marker concentrations. Tukey's honestly significant difference test was used to correct for multiple comparisons.

Results:During the study, between 24-28 hours post-injury, one Berkeley-CCI mice experienced mortality while another was prematurely sacrificed due to significant weight loss and frailty. Causes of death and weight loss were unknown. IL-10 plasma concentrations at baseline were significantly higher in SCD mice (mean [SEM]: 2.559 [0.150] pg/mL; p=0.0008) compared to wild type (WT) mice (1.337 [0.178] pg/mL). Conversely, IL-1ß levels 24 hours post-injury/sham were significantly lower in SCD mice (33.18 [8.923] pg/mL; p=0.0320) in comparison to WT mice (105.80 [30.48] pg/mL). Similarly, IFN-y concentrations in SCD mice 24 hours post-CCI/sham (0.163 [0.0293] pg/mL) were significantly lower when compared to WT mice (0.308 [0.0394] pg/mL; p=0.0264). IL-2 levels showed significant difference between SCD-sham (0.210 [0.0156] pg/mL) and SCD-CCI (0.382 [0.0142] pg/mL; p=0.0276) groups. Analyses of IL-4, IL-5, IL-6, KC/GRO and TNF-a did not yield statistical significance. The two SCD-CCI mice with mortality showed the highest levels of IL-10 (36.255 and 10.021 pg/mL) and lowest levels of IL-1ß (7.179 and 10.482 pg/mL) at 24 hours post-injury/sham across all animals.

Conclusion:This pilot study suggests an altered inflammatory response following TBI in SCD, with dampening of key pro-inflammatory markers such as IL-1ß and IFN-y. Baseline elevations in IL-10 suggests priming of anti-inflammatory signaling as a response to the basal inflammatory state present in SCD. However, this may inhibit critical neuroprotective effects of inflammation in the acute phase post-TBI, predisposing individuals with SCD to serious complications and mortality. Further exploration and larger studies are needed to fully understand this balance and characterize TBI responses and outcomes in SCD.