Hemoglobin and iron handling in brain after subarachnoid hemorrhage and the effect of deferoxamine on early brain injury

Abstract

The purpose of this study was to investigate hemoglobin and iron handling after subarachnoid hemorrhage (SAH), examine the relationship between iron and neuroglial cell changes, and determine whether deferoxamine (DFX) can reduce SAH-induced injury. The SAH was induced in Sprague-Dawley rats (n=110) using an endovascular perforation technique. Animals were treated with DFX (100 mg/kg) or vehicle 2 and 6 hours after SAH induction followed by every 12 hours for 3 days. Rats were killed at 6 hours, Days 1 and 3 to determine nonheme iron and examine iron-handling proteins using Western blot and immunohistochemistry. 8-Hydroxyl-2'-deoxyguanosine and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining were performed to assess oxidative DNA damage and neuronal cell death. After SAH, marked heme-oxygenase-1 (HO-1) upregulation at Day 3 (P<0.01) was accompanied by elevated nonheme iron (P<0.01), transferrin (Tf) (P<0.01), Tf receptor (P<0.05), and ferritin levels (P<0.01). Deferoxamine treatment reduced SAH-induced mortality (12% versus 29%, P<0.05), brain nonheme iron concentration, iron-handling protein expression, oxidative stress, and neuronal cell death at Day 3 (P<0.01) after SAH. These results suggest that iron overload in the acute phase of SAH causes oxidative injury leading to neuronal cell death. Deferoxamine effectively reduced oxidative stress and neuronal cell death, and may be a potential therapeutic agent for SAH.

Figure 1

(A) Time course of the subarachnoid hemorrhage (SAH) caused by endovascular perforation of the internal carotid artery. Severe SAH was seen around the Circle of Willis and along the ventral brainstem 6 hours after hemorrhage (B). Then, SAH diminished slowly over 3 days after hemorrhage (D). The extent of SAH was assessed 24 hours after hemorrhage (C). The basal surface of the brain was divided into six segments and each segment rated for hemorrhage (3: massive hemorrhage covering the cerebral arteries; 2: moderate blood clot with recognizable arteries; 1: minimal subarachnoid blood; 0: no blood).

Figure 2

(A) Western blot analysis showing significant increase in heme-oxygenase-1 (HO-1) expression 24 hours (lanes 5 and 6) and 72 hours after hemorrhage (lanes 7 and 8) compared with sham-operated control group (lanes 1 and 2). Deferoxamine (DFX) treatment resulted in significant reduced HO-1 expression 6 hours (lanes 9 and 10), 24 hours (lanes 11 and 12), and 72 hours (lanes 13 and 14) after subarachnoid hemorrhage (SAH) induction. MW of HO-1 is ∼32 kDa. Values are mean±s.d. ^**P<0.01 versus sham. ^# and ^##P<0.05 and P<0.01 versus vehicle-treated SAH group, respectively. The HO-1 immunoreactivities in the left basal brain after a sham operation (B) or 3 days after SAH induction without (C) and with DFX treatment (D). Scale bar=50 μm.

Figure 3

Nonheme iron concentration in left basal brain tissue after subarachnoid hemorrhage (SAH) in vehicle-treated and deferoxamine (DFX)-treated rats and in sham-operated rats. Values are mean±s.d. ^**P<0.01 versus sham; ^#P<0.05 versus vehicle-treated SAH group.

Figure 4

Western blot analyses showing time course of transferrin (Tf) (left) and transferrin receptor (TfR) (right) protein levels in the left basal brain tissue at 6 hours (lanes 2 and 3), Day 1 (lanes 4 and 5), and Day 3 (lanes 6 and 7) after subarachnoid hemorrhage (SAH) induction without (lanes 2, 4, and 6) and with deferoxamine (DFX) treatment (lanes 3, 5, and 7). Lane 1: sham-operated control group. Values are mean±s.d. ^*P<0.05 and ^**P<0.01 versus sham. ^#P<0.05 versus vehicle-treated SAH group.

Figure 5

Representative Western blot showing ferritin expression at 6 hours (lanes 2 and 3), Day 1 (lanes 4 and 5), and Day 3 (lanes 6 and 7) after subarachnoid hemorrhage (SAH) induction without (lanes 2, 4, and 6) and with deferoxamine (DFX) treatment (lanes 3, 5, and 7). Lane 1: sham-operated control group. Values are mean±s.d. ^**P<0.01 versus sham. ^##P<0.01 versus vehicle-treated SAH group.

Figure 6

Double immunofluorescent labeling for ferritin and NeuN (A–C), glial fibrillary protein (GFAP; D–F), and OX-42 (G–I), showing that ferritin-positive cells are neurons and microglia. Moreover, ferritin-positive cells exhibit DNA damage, indicating apoptotic cell death (J–L). Scale bar=50 μm.

Figure 7

Photomicrographs showing terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL; A–D) and 8-hydroxyl-2′-deoxyguanosine (8-OHdG; E–H) immunoreactivity in the ipsilateral basal brain on Days 1 (A, B, E, F) and 3 (C, D, G, H) after subarachnoid hemorrhage (SAH) without (A, C, E, G) and with deferoxamine (DFX; B, D, F, H) treatment. Quantification of the TUNEL (I) and 8-OHdG (J) results. Values are mean±s.d., n=5. ^**P<0.01.