Effect of Hypoxia-Ischemia on the Expression of Iron-Related Proteins in Neonatal Rat Brains

Abstract

Hypoxic-ischemic white matter injury (WMI) pathogenesis in preterm infants is not well established, and iron-related proteins in the brain may play an important role in imbalanced iron metabolism. We aimed to investigate the iron-related protein changes in neonatal rats after hypoxia-ischemia (HI), clarify the role of iron-related proteins in hypoxic-ischemic WMI, and potentially provide a new target for the clinical treatment of hypoxic-ischemic WMI in preterm infants. We adopted a WMI animal model of bilateral common carotid artery electrocoagulation combined with hypoxia in neonatal 3-day-old Sprague-Dawley rats. We observed basic myelin protein (MBP) and iron-related protein expression in the brain (ferritin, transferrin receptor [TfR], and membrane iron transporter 1 [FPN1]) via Western blot and double immunofluorescence staining. The expression of MBP in the WMI group was significantly downregulated on postoperative days (PODs) 14, 28, and 56. Ferritin levels were significantly increased on PODs 3, 7, 14, and 28 and were most significant on POD 28, returning to the sham group level on POD 56. FPN1 levels were significantly increased on PODs 7, 28, and 56 and were still higher than those in the sham group on POD 56. TfR expression was significantly upregulated on PODs 1, 7, and 28 and returned to the sham group level on POD 56. Immunofluorescence staining showed that ferritin, TfR, and FPN1 were expressed in neurons, blood vessels, and oligodendrocytes in the cortex and corpus callosum on POD 28. Compared with the sham group, the immune-positive markers of three proteins in the WMI group were significantly increased. The expression of iron-related proteins in the brain (ferritin, FPN1, and TfR) showed spatiotemporal dynamic changes and may play an important role in hypoxic-ischemic WMI.

Figure 1

Effect of hypoxia-ischemia on the expression of MBP in rat brains. (a) Representative Western blot images of MBP bands and PODs (postoperative days). (b) Quantitative analysis of MBP protein expression (^##p < 0.01 and ^###p < 0.001 vs. sham group, n = 4). The results showed that the MBP level was clearly reduced in the WMI group compared with the sham group. (c) Double immunofluorescence staining of MBP (red) and DAPI (blue). Among them, a3, c3, and e3 are the high-magnification images in the white line square areas of b3, d3, and f3. CC: corpus callosum; a1–a3, b1–b3, c1–c3, d1–d3, e1–e3, f1–f3, bar = 50 μm; a4, b4, c4, d4, e4, f4, bar = 25 μm.

Figure 2

Effect of hypoxia-ischemia on the expression of ferritin, FPN1, and TfR in brain tissue on PODs 1, 3, and 7. (a) Bands of Western blots from brain tissue. (b–d) Relative protein expression of ferritin, FPN1, and TfR (^#p < 0.05 and ^##p < 0.01 vs. sham group, n = 4).

Figure 3

Effect of hypoxia-ischemia on the expression of ferritin, FPN1, and TfR in brain tissue on PODs 14, 28, and 56. (a) Bands of Western blots from brain tissue. (b–d) Relative protein expression of ferritin, FPN1, and TfR (^#p < 0.05, ^##p < 0.01, and ^###p < 0.01 vs. sham group, n = 4).

Figure 4

Changes in the expression of ferritin, FPN1, and TfR in brain tissue over time (^#p < 0.05, ^##p < 0.01, and ^###p < 0.01 vs. sham group, n = 4).

Figure 5

Representative images of immunofluorescence staining for ferritin (green), NeuN (neuronal marker; red)/CD31 (vascular marker; red)/Olig2 (oligodendrocyte marker; red), and DAPI (nuclear marker; blue) in rat brains on POD 28. Ferritin-positive cells were colocalized with CD31- or Olig2-positive cells; however, they were largely observed in the WMI group than in the sham group. White arrows indicate ferritin⁺CD31⁺ cells in c5 and d5 and ferritin⁺Olig2⁺ cells in f5. Scale bar, 50 μm.

Figure 6

Representative images of immunofluorescence staining for FPN1 (green), NeuN (neuronal marker; red)/CD31 (vascular marker; red)/Olig2 (oligodendrocyte marker; red), and DAPI (nuclear marker; blue) in rat brains on POD 28. FPN1-positive cells were colocalized with NeuN- or CD31- or Olig2-positive cells; however, they were largely observed in the WMI group than in the sham group. White arrows indicate FPN1⁺NeuN⁺ cells in a5 and b5, FPN1⁺CD31⁺ cells in c5 and d5, and FPN1⁺Olig2⁺ cells in e5 and f5. Scale bar, 50 μm.

Figure 7

Representative images of immunofluorescence staining for TfR (green), NeuN (neuronal marker; red)/CD31 (vascular marker; red)/Olig2 (oligodendrocyte marker; red), and DAPI (nuclear marker; blue) in rat brains on POD 28. TfR-positive cells were colocalized with NeuN- or CD31- or Olig2-positive cells; however, they were largely observed in the WMI group than in the sham group. White arrows indicate TfR⁺NeuN⁺ cells in b5, TfR⁺CD31⁺ cells in c5 and d5, and TfR⁺Olig2⁺ cells in e5 and f5. Scale bar, 50 μm.