Inhibition of Colony Stimulating Factor 1 Receptor Suppresses Neuroinflammation and Neonatal Hypoxic-Ischemic Brain Injury

Abstract

Hypoxic-ischemic (HI) brain injury is a major cause of neonatal death or lifetime disability without widely accepted effective pharmacological treatments. It has been shown that the survival of microglia requires colony-stimulating factor 1 receptor (CSF1R) signaling and microglia participate in neonatal HI brain injury. We therefore hypothesize that microglia depletion during a HI insult period could reduce immature brain injury. In this study, CD1 mouse pups were treated with a CSF1R inhibitor (PLX3397, 25 mg/kg/daily) or a vehicle from postnatal day 4 to day 11 (P4-11), and over 90% of total brain microglia were deleted at P9. Unilateral hemisphere HI injury was induced at P9 by permanently ligating the left common carotid arteries and exposing the pups to 10% oxygen for 30 min to produce moderate left hemisphere injury. We found that the PLX3397 treatment reduced HI brain injury by 46.4%, as evaluated by the percentage of brain infarction at 48 h after HI. Furthermore, CSF1R inhibition suppressed the infiltration of neutrophils (69.7% reduction, p = 0.038), macrophages (77.4% reduction, p = 0.009), and T cells (72.9% reduction, p = 0.008) to the brain, the production of cytokines and chemokines (such as CCL12, CCL6, CCL21, CCL22, CCL19, IL7, CD14, and WISP-1), and reduced neuronal apoptosis as indicated by active caspase-3 labeled cells at 48 h after HI (615.20 ± 156.84/mm² vs. 1,205.00 ± 99.15/mm², p = 0.013). Our results suggest that CSF1R inhibition suppresses neuroinflammation and neonatal brain injury after acute cerebral hypoxia-ischemia in neonatal mice.

Keywords: PLX3397; colony stimulating factor 1 receptor; microglia; neonatal hypoxic-ischemic brain injury; neuroinflammation.

Figure 1

PLX3397 reduces brain microglia in neonatal CD1 mice. (A) Experimental schematic design of PLX3397 administration in neonatal CD1 mice. (B) Expression of CSF1R on brain microglia (CD45^int CD11b⁺ cells) of neonatal CD1 mice at 4 days after birth. FMO stands for Fluorescence Minus One Control. (C) Flow cytometry plots and statistics of brain microglia (CD11b⁺CD45^int cells) at 9 days after birth in neonatal CD1 mice treated with PLX3397 (n = 6, 3 males and 3 females) or the vehicle (n = 6, 3 males and 3 females). Unpaired two-tailed t-test. Data are presented as mean ± SEM, **p < 0.01.

Figure 2

PLX3397 attenuates neonatal hypoxic-ischemic brain injury in mice. (A) 2,3,5-Tripenyltetrazolium chloride (TTC) staining shows brain injury at 48 h after hypoxic-ischemic brain injury induction in neonatal CD1 mice treated with PLX3397 or the vehicle. (B) Statistics of infarct size measured by TTC staining in HI mice treated with PLX3397 (n = 9, 6 males and 3 females) or vehicle (n = 11, 5 males and 6 females) Unpaired two-tailed t-test. (C) Corner turn test at 48 h after HI induction in mice treated with PLX3397 (n = 6, 3 males and 3 females) or vehicle (n = 6, 3 males and 3 females). Unpaired two-tailed t-test. Data are presented as mean ± SEM, **p < 0.01.

Figure 3

PLX3397 reduces immune cell recruitment into the brains of HI mice. (A) Flow cytometry gating strategy for neutrophils (CD45^highCD11b⁺Ly6G⁺), macrophages (CD45^highCD11b⁺F4/80⁺), T cells (CD45^highCD3⁺), and B cells (CD45^highCD3⁻CD19⁺) in the brains of neonatal HI mice. (B) The quantitation of brain neutrophils, macrophages, T cells, and B cells at 48 h after HI in mice treated with PLX3397 (n = 6, 4 males and 2 females) or the vehicle (n = 6, 4 males and 2 females). Unpaired two-tailed t-test. Data are presented as mean ± SEM, *p < 0.05, **p < 0.01.

Figure 4

PLX3397 changes brain cytokine/chemokine profile in HI mice. (A) The total differentially expressed cytokines/chemokines in the brains of HI mice treated with PLX3397 or the vehicle. Brain tissues were collected at 48 h after HI induction and the protein levels were measured with a cytokine/chemokine profile ELISA array kit in which 111 factors were detected. n = 3 independent experiments. Each independent experiment needed 10 mice per group (5 males and 5 females). Unpaired two-tailed t-test. (B) The mRNA levels of representative differentially expressed factors related to regulation of chemotaxis (CCL2, CCL12), immune response (CD14, M-CSF, IL-7), and BBB integrity (ICAM-1, VEGF, and WISP-1). CX3CL1 was as a negative control that was not significantly changed in protein level between HI mice treated with PLX3397 or the vehicle (data not shown). Brain tissues were collected at 48 h after HI from HI mice treated with PLX3397 or the vehicle. n = 5 mice per group. Unpaired two-tailed t-test. Data are presented as mean ± SEM, *p < 0.05, **p < 0.01.

Figure 5

PLX3397 attenuates cell death in injured brain tissue of HI mice. (A) immunostaining of activated caspase 3 (red) and neuron (green) in injured brain tissue at 48 h after HI induction in mice treated with PLX3397 or the vehicle. Scale bar = 20 μm. (B) Quantification of neuron and cell apoptosis at 48 h after HI induction in injured brain tissue of HI mice treated with PLX3397 (n = 6, 1 male and 5 females) or vehicle (n = 6, 3 males and 3 females). Unpaired two-tailed t-test. Data are presented as mean ± SEM, *p < 0.05.