Role of lipocalin 2 in intraventricular haemoglobin-induced brain injury

Abstract

Objective: Our recent studies have shown that blood components, including haemoglobin and iron, contribute to hydrocephalus development and brain injury after intraventricular haemorrhage (IVH). The current study investigated the role of lipocalin 2 (LCN2), a protein involved in iron handling, in the ventricular dilation and neuroinflammation caused by brain injury in a mouse model of IVH.

Design: Female wild-type (WT) C57BL/6 mice and LCN2-deficient (LCN2^-/-) mice had an intraventricular injection of haemoglobin, and control mice received an equivalent amount of saline. MRI was performed presurgery and postsurgery to measure ventricular volume and the brains were used for either immunohistochemistry or western blot.

Results: Ventricular dilation was observed in WT mice at 24 h after haemoglobin (25 mg/mL, 20 µL) injection (12.5±2.4 vs 8.6±1.5 mm³ in the control, p<0.01). Western blotting showed that LCN2 was significantly upregulated in the periventricular area (p<0.01). LCN2 was mainly expressed in astrocytes, whereas the LCN2 receptor was detected in astrocytes, microglia/macrophages and neurons. Haemoglobin-induced ventricle dilation and glia activation were less in LCN2^-/- mice (p<0.01). Injection of high-dose haemoglobin (50 mg/mL) resulted in lower mortality in LCN2^-/- mice (27% vs 86% in WT; p<0.05).

Conclusions: Intraventricular haemoglobin caused LCN2 upregulation and ventricular dilation. Haemoglobin resulted in lower mortality and less ventricular dilation in LCN2^-/- mice. These results suggest that LCN2 has a role in haemoglobin-induced brain injury and may be a therapeutic target for IVH.

Keywords: Hemorrhage; brain injury; hemoglobin; lipocalin 2; mouse.

Figure 1

Intraventricular injection of haemoglobin caused ventricular dilation. T2-weighted MRI and frozen coronal brain sections (A) and the measurement of ventricular volume with T2-weighted MRI (B) from mice 24 h after injection of 20 µL of saline or haemoglobin (25 mg/mL) into the right lateral ventricle. The data points represent means±SD, n=6–8, **p<0.01 versus saline group.

Figure 2

Lipocalin 2 (LCN2) protein was markedly upregulated in the brain 24 h after intraventricular haemoglobin injection compared with saline controls. Immunohistochemistry staining demonstrated that LCN2 immunoreactivity was expressed in the periventricular area and cortex 24 h after intraventricular haemoglobin injection (A) compared with saline controls (B). Boxes show areas examined at higher magnification in the higher power micrographs on the right (scale bars=100 and 20 µm). LCN2 protein levels were upregulated in the periventricular area (C) and cortex (D) 24 h after intraventricular haemoglobin injection, as shown by western blot. The data points represent means±SD, n=3–5, ^**p<0.01 versus saline group.

Figure 3

Immunoreactivity for lipocalin 2 (LCN2), glial fibrillary acidic protein (GFAP), Iba-1 and neuronal nuclei (NeuN) 24 h after intraventricular haemoglobin injection. Immunofluorescent double labelling showed that LCN2-positive cells were mainly astrocytes. Scale bar=50 µm.

Figure 4

Immunoreactivity for the lipocalin 2 (LCN2) receptor (SLC22A17), glial fibrillary acidic protein (GFAP), Iba-1 and neuronal nuclei (NeuN) 24 h after intraventricular haemoglobin injection. Co-localisation of the LCN2 receptor with GFAP, Iba-1 and NeuN by double labelling is shown. Scale bar=50 µm.

Figure 5

Ventricular volumes were measured before and after (24 h) the injection of haemoglobin (25 mg/mL) or saline into wild-type (WT) and in lipocalin 2-deficient (LCN2^−/−) mice. Ventricular dilation was calculated as a per cent of presurgery values. Haemoglobin caused marked ventricular dilation in WT mice compared with saline controls, but not LCN2^−/− mice. The data represent means±SD, n=5 for each, ^**p<0.01 versus WT saline group, ^##p<0.01 versus LCN2^−/− haemoglobin group.

Figure 6

In wild-type (WT) mice, intraventricular injection of haemoglobin (25 mg/mL) resulted in periventricular activation of astrocytes (glial fibrillary acidic protein (GFAP) staining) and microglia (amoeboid Iba-1 staining) at 24 h compared with saline-injected controls (A). The activation by haemoglobin was attenuated in lipocalin 2-deficient (LCN2^−/−) mice (A). The numbers of periventricular cells expressing GFAP (B) and Iba-1 (C) were counted in the WT and LCN2^−/− mice. The data points represent the mean±SD, scale bar=50 µm, n=5 per group; ^**p<0.01 versus WT saline group, ^##p<0.01 versus LCN2^−/− haemoglobin group.