Pharmacologic Toll-like receptor 4 inhibition skews toward a favorable A1/A2 astrocytic ratio improving neurocognitive outcomes following traumatic brain injury.

BACKGROUND: Astrocytes are critical neuroimmune cells that modulate the neuroinflammatory response following traumatic brain injury (TBI) because of their ability to acquire neurotoxic (A1) or neuroprotective (A2) phenotypes. Using C34, a novel pharmacologic Toll-like receptor (TLR) 4 inhibitor, we explored their respective polarization states after TBI. METHODS: A murine controlled cortical impact model was used, and the results were analyzed on postinjury days (PIDs) 1, 7, and 28. The experimental groups are as follows: (1) sham, (2) sham + C34, (3) TBI, and (4) TBI + C34. Quantitative real-time polymerase chain reaction was used to quantify gene expression associated with proinflammatory (A1) and anti-inflammatory (A2) phenotypes. Morris water maze was used to assess neurocognitive outcomes. Fixed frozen cortical samples were sectioned, stained for myelin basic protein and 4',6-diamidino-2-phenylindole, and then imaged. Student t test and one-way analysis of variance were used for statistical analysis with significance achieved when p < 0.05. RESULTS: On quantitative real-time polymerase chain reaction, C34-treated groups showed a significant decrease in the expression of A1 markers such as Gbp2 and a significant increase in the expression of A2 markers such as Emp1 when compared with untreated groups on PID 1. On PIDs 7 and 28, the expression of most A1 and A2 markers was also significantly decreased in the C34-treated groups. On immunohistochemistry, C34-treated groups demonstrated increased myelin basic protein staining into the lesion by PID 28. C34-treated groups showed more platform entries on Morris water maze when compared with untreated groups on PID 7 and PID 28. CONCLUSION: Following TBI, early TLR4 blockade modulates astrocytic function and shifts its polarization toward the anti-inflammatory A2-like phenotype. This is accompanied by an increase in myelin regeneration, providing better neuroprotection and improved neurocognitive outcomes. Targeting A1/A2 balance with TLR4 inhibition provides a potential therapeutic target to improve neurobehavioral outcomes in the setting of TBI.

Infiltrating anti-inflammatory monocytes modulate microglial activation through toll-like receptor 4/interferon-dependent pathways following traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) is the leading cause of morbidity and mortality in the pediatric population. Microglia and infiltrating monocyte-derived macrophages are crucial immune cells that modulate the neuroinflammatory response following TBI. Using C34, a novel pharmacologic toll-like receptor 4 inhibitor, we investigated the intricate interactions between these cells in a murine TBI model. METHODS: A murine controlled cortical impact model was used, and the results were analyzed on postinjury days 1, 7, 28, and 35. The experimental groups are as follows: (1) sham C57BL/6 wild-type (WT), (2) TBI WT, (3) sham WT + C34, and (4) TBI WT + C34. Quantitative real-time polymerase chain reaction was used to quantify gene expression associated with microglial activation, apoptotic pathways, and type 1 interferon pathway. Flow cytometry was used to isolate microglia and infiltrating monocytes. Brain lesion volumes were assessed using magnetic resonance imaging. Last, neurocognitive outcomes were evaluated using the Morris Water Maze test. Student's t test and one-way analysis of variance were used for statistical analysis with significance achieved when p < 0.05. RESULTS: Toll-like receptor 4 inhibition leads to improved neurological sequela post-TBI, possibly because of an increase in infiltrating anti-inflammatory monocytes and a decrease in IFN regulatory factor 7 during acute inflammation, followed by a reduction in apoptosis and M2 microglial expression during chronic inflammation. CONCLUSION: Toll-like receptor 4 inhibition with C34 skews infiltrating monocytes toward an anti-inflammatory phenotype, leading to enhanced neurocognitive outcomes. Moreover, although M2 microglia have been consistently shown as inducers of neuroprotection, our results clearly demonstrate their detrimental role during the chronic phases of healing post-TBI.