Partial Ablation of Astrocytes Exacerbates Cerebral Infiltration of Monocytes and Neuronal Loss After Brain Stab Injury in Mice

Abstract

In traumatic brain injury (TBI), mechanical injury results in instantaneous tissue damages accompanied by subsequent pro-inflammatory cascades composed of microgliosis and astrogliosis. However, the interactive roles between microglia and astrocytes during the pathogenesis of TBI remain unclear and sometimes debatable. In this study, we used a forebrain stab injury mouse model to investigate the pathological role of reactive astrocytes in cellular and molecular changes of inflammatory response following TBI. In the ipsilateral hemisphere of stab-injured brain, monocyte infiltration and neuronal loss, as well as increased elevated astrogliosis, microglia activation and inflammatory cytokines were observed. To verify the role of reactive astrocytes in TBI, local and partial ablation of astrocytes was achieved by stereotactic injection of diphtheria toxin in the forebrain of Aldh1l1-CreER^T2::Ai9::iDTR transgenic mice which expressed diphtheria toxin receptor (DTR) in astrocytes after tamoxifen induction. This strategy achieved about 20% of astrocytes reduction at the stab site as validated by immunofluorescence co-staining of GFAP with tdTomato-positive astrocytes. Interestingly, reduction of astrocytes showed increased microglia activation and monocyte infiltration, accompanied with increased severity in stab injury-induced neuronal loss when compared with DTR^-/- mice, together with elevation of inflammatory chemokines such as CCL2, CCL5 and CXCL10 in astrogliosis-reduced mice. Collectively, our data verified the interactive role of astrocytes as an immune modulator in suppressing inflammatory responses in the injured brain. Schematic diagram shows monocyte infiltration and neuronal loss, as well as increased elevated astrogliosis, microglia activation and chemokines were observed in the injured site after stab injury. Local and partial ablation of astrocytes led to increased microglia activation and monocyte infiltration, accompanied with increased severity in neuronal loss together with elevation of inflammatory chemokines as compared with control mice subjected stab injury.

Keywords: Astrocytes; Microglia; Monocytes; Neuroinflammation; Stab injury; Traumatic brain injury.

Fig. 1

Forebrain stab injury induces astrogliosis and neuronal loss. a Schematic of breeding strategy for Aldh1l1-CreER^T2::Ai9 mice. b A diagram illustrates the timeline of tamoxifen administration (75 mg/kg daily for 3 consecutive days, i.p), surgery and tissue analysis. c The representative image of hematoxylin and eosin-stained coronal section at 7 days after injury (Lecia DM6B, magnification, ×200). d Representative confocal images of GFAP staining in the contralateral and ipsilateral cortex from Aldh1l1-CreER^T2::Ai9 mice 7 days after stab injury. Scale bar = 100 μm (Nikon C2, magnification, ×200 and ×600). e The quantification of GFAP immunoreactivity in (d). f The quantitative analysis of tdTomato⁺ cell density in (d) (Nikon C2, magnification, ×200). g–i NeuN, IBA1 and DAPI staining in the contralateral and ipsilateral cortex at 7 days post-stab injury (g) and the quantification of IBA1⁺ cell density (h) and NeuN⁺ cell density (i) in the injured cortex (ipsilateral) and non-injured control (contralateral). Scale bar = 100 μm. Data were presented as mean ± SD. N = 5 mice per group. The results are representatives of three different experiments. **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 2

Infiltration of peripheral monocytes in the injured brain tissues. a IBA1 and CD169 staining in the contralateral and ipsilateral cortex at 7 days post-stab injury. Scale bar = 100 μm (Nikon C2, magnification, ×200 and ×600). b Representative confocal images of IBA1 and TMEM119 immunofluorescence in the contralateral and ipsilateral cortex at 7 days post-stab injury. Scale bar = 100 μm (Nikon C2, magnification, ×200 and ×600). c Schematic of breeding strategy for CX3CR1-CreER::Ai9 mice. d A diagram illustrates the experimental timeline of tamoxifen administration (75 mg/kg daily for 3 consecutive days, i.p), surgery, and tissue analysis. e–g Representative CD169 and IBA1 staining in the contralateral and ipsilateral cortex of CX3CR1-CreER::Ai9 mice at 7 days post-stab injury (e) (Zeiss LSM 800, magnification, ×200), the ratio of monocytes (IBA1⁺ tdTomato⁻) to total IBA1⁺ cell (f), and the ratio of activated monocytes (IBA1⁺ tdTomato⁻ CD169⁺) to total monocyte (IBA1⁺ tdTomato⁻) (g). Scale bar = 100 μm. Data were presented as mean ± SD. N = 5 mice per group. The results are representatives of three different experiments. *p < 0.05, ***p < 0.001

Fig. 3

Stab injury induces elevation of inflammatory cytokines in the forebrain cortex. Results of quantitative PCR analysis of mRNA level of Ccl2, Ccl8, Ccl5, Tnf-a, Cxcl10, Il-6, Il-1b, and Il-10 in the cortex of injured hemisphere (ipsi) and control hemisphere (contra). Data were presented as mean ± SD. N = 3–6 mice per group. The results are representatives of three different experiments. *p < 0.05

Fig. 4

Local ablation of astrocytes exacerbates microglia activation, monocyte infiltration and neuronal loss in the ipsilateral cortex after stab injury. a Schematic of breeding strategy for Aldh1l1-CreER^T2::Ai9::iDTR mice. b The experimental timeline of TAM administration (75 mg/kg daily for 3 consecutive days), DTX (6 ng daily, every other day), surgery, and tissues analysis. c–e Representative images of GFAP staining in the ipsilateral cortex from Aldh1l1-CreER^T2::Ai9::iDTR and Aldh1l1-CreER^T2::Ai9 mice at 7 days post-stab injury and DTX treatment (c) (magnification, ×200), the quantification of GFAP immunoreactivity (d), and tdTomato⁺ DAPI⁺ cell density (e) in the ipsilateral cortex. Scale bar = 100 μm. f–h Representative images of NeuN, IBA1 and DAPI staining in the ipsilateral cortex of Aldh1l1-CreER^T2::Ai9::iDTR and Aldh1l1-CreER^T2::Ai9 mice at 7 days post-stab injury and DTX treatment (f) (magnification, ×200), quantification of NeuN⁺ cell density (g), and IBA1⁺ cell density (h). Scale bar = 100 μm. i and j Representative images of CD169 and IBA1 staining in the ipsilateral cortex from Aldh1l1-CreER^T2::Ai9::iDTR and Aldh1l1-CreER^T2::Ai9 mice at 7 days post-stab injury and DTX treatment (i) (magnification, ×200), the quantification of CD169⁺ cells (j). Scale bar = 100 μm. Data were presented as mean ± SD. N = 5 mice per group. The results are representatives of three different experiments. **p < 0.01 and ****p < 0.0001. Abbreviations: AA, Aldh1l1-CreER^T2::Ai9 mice; AAI, Aldh1l1-CreER^T2::Ai9::iDTR mice

Fig. 5

Astrocytes repress the production of inflammatory cytokines after stab brain injury. The quantitative PCR analysis of mRNA levels of Ccl2 (a), Ccl5 (b), Cxcl10 (c), Ccl8 (d), Il-6 (e), Il-10 (f), Tnf-a (g), Cox2 (h) and Il-1b (i) in the ipsilateral cortex of Aldh1l1-CreER^T2::Ai9 (AA) and Aldh1l1-CreER^T2::Ai9::iDTR (AAI) mice at 7 days post-stab injury and DTX treatment. Data were presented as mean ± SD. N = 3–6 mice per group. The results are representatives of three different experiments. *p < 0.05