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 ironhandling proteins using Western blot and immunohistochemistry. 8-Hydroxyl-2 0 -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 hemeoxygenase-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.

Background and Purpose Brain iron overload plays a detrimental role in brain injury after intracerebral hemorrhage (ICH). A recent study found that minocycline acts as an iron chelator and reduces iron-induced neuronal death in vitro. The present study investigated if minocycline reduces iron overload after ICH and iron-induced brain injury in vivo. Methods This study was divided into four parts. (1) Rats with different sizes of ICH were euthanized 3 days later for serum total iron and brain edema determination. (2) Rats had an ICH treated with minocycline or vehicle. Serum iron, brain iron, and brain iron handling proteins were measured. (3) Rats had an intracaudate injection of either saline, iron, iron+minocycline or iron+macrophage/microglia inhibitory factor and were used for brain edema and neuronal death measurements. (4) Rats had an intracaudate injection of iron and were treated with minocycline. The brains were used for edema measurement. Results After ICH, serum total iron and brain non-heme iron increased and these changes were reduced by minocycline treatment. Minocycline also reduced ICH-induced upregulation of brain iron handling proteins and neuronal death. Intracaudate injection of iron caused brain edema, blood-brain barrier leakage and brain cell death, all of which were significantly reduced by co-injection with minocycline. Conclusions The current study found that minocycline reduces iron overload after ICH and iron-induced brain injury. It is also well known minocycline is an inhibitor of microglial activation. Minocycline may be very useful for ICH patients because both iron accumulation and microglia activation contribute to brain damage following ICH.

Autophagy contributes to ischemic brain injury, but it is not clear if autophagy occurs after intracerebral hemorrhage (ICH). This study examined whether ICH-induced cell death is partly autophagic. It then examined the role of iron in inducing this form of cell death after ICH. Male, adult Sprague-Dawley rats received an infusion of autologous whole blood or ferrous iron into the right basal ganglia. Control rats (sham) had a needle insertion. The rats were killed at 1, 3, 7, or 28 days later. Some rats were treated with either deferoxamine or vehicle after ICH. Microtubule-associated protein light chain-3 (LC3), a biomarker of autophagosome, and cathepsin D, a lysosomal biomarker, were measured by Western blot analysis and immunohistochemistry. Immunofluorescent double-labeling was used to identify the cell types expressing cathepsin D. Electron microscopy was performed to examine the cellular ultrastructure changes after ICH. We found that conversion of LC3-I to LC3-II, cathepsin D expression, and vacuole formation are increased in the ipsilateral basal ganglia after ICH. Intracerebral infusion of iron also resulted in enhanced conversion of LC3-I to LC3-II and increased cathepsin D levels. Deferoxamine (an iron chelator) treatment significantly reduced the conversion of LC3-I to LC3-II and cathepsin D levels after ICH. Our results demonstrated that autophagy occurs after ICH, and iron has a key role in ICH-induced autophagy. This also suggests that iron-induced autophagy may play a role in brain injury in other diseases associated with iron overload.

Background and Purpose-Mineralocorticoid receptor (MR) antagonists have protective effects in rodent models of ischemic stroke, but the cell type-specific actions of these drugs are unknown. In the present study, we examined the contribution of myeloid cell MR during focal cerebral ischemia using myeloid-specific MR knockout mice. Methods-Myeloid-specific MR knockout mice were subjected to transient (90 minutes) middle cerebral artery occlusion followed by 24 hours reperfusion (nϭ5 to 7 per group). Ischemic cerebral infarcts were identified by hematoxylin and eosin staining and quantified with image analysis software. Immunohistochemical localization of microglia and macrophages was performed using Iba1 staining, and the expression of inflammatory markers was measured after 24 hours of reperfusion by quantitative reverse transcription-polymerase chain reaction. Results-Myeloid-specific MR knockout resulted in a 65% reduction in infarct volume (Pϭ0.005) after middle cerebral artery occlusion. This was accompanied by a significant reduction in activated microglia and macrophages in the ischemic core. Furthermore, myeloid-specific MR knockout suppressed classically activated M1 macrophage markers tumor necrosis factor-␣, interleukin-1␤, monocyte chemoattractant protein-1, macrophage inflammatory protein-1␣, and interleukin-6 at the same time as partially preserving the induction of alternatively activated, M2, markers Arg1, and Ym1. Conclusions-These data demonstrate that myeloid MR activation exacerbates stroke and identify myeloid MR as a critical target for MR antagonists. Furthermore, these data indicate that MR activation has an important role in controlling immune cell function during the inflammatory response to stroke. (Stroke. 2011;42:179-185.)

Hypoxia inducible factors (HIFs) are activated in many tumors and show either promoter or suppressor activity depending on the tumor cell biology and background. However, the role of HIF member HIF-2α remains unclear in hepatocellular carcinoma (HCC). Here, HIF-2α expression was measured in HCC and paired peritumoral tissues by qRT-PCR, western blot analysis, and immunofluorescence assays, and the clinical significance was explored in 246 HCC patients. In cell culture, HIF-2α levels were over-expressed or knocked-down by use of expression or short hairpin RNA recombinant plasmid respectively. Cells were analyzed by immunoblot, chromatin immunoprecipitation coupled with microarray, co-immunoprecipitation, and histochemical staining. In vivo tumor growth was analyzed in nude mice. We found that the average expression of HIF-2α was relatively low in HCC tissues, and the decreased level was associated with lower overall survival (p=0.006). High HIF-2α expression in HCC cells induced higher levels of apoptosis and expression of pro-apoptotic proteins, and it inhibited cell and tumor growth. Furthermore, HIF-2α inhibited expression of the novel target gene transcription factor dimerization partner 3 (TFDP3). TFDP3 protein was found to bind with E2F transcription factor 1 (E2F1) and inhibit its transcriptional activity through both p53-dependent and -independent pathways. Re-introduction of TFDP3 expression reversed HIF-2α-induced apoptosis. Conclusions Data gathered from cell lines, tumorigenicity studies, and primary HCC samples demonstrate a negative role of HIF-2α in tumors, which is mediated by the TFDP3/E2F1 pathway. Our study provides evidence supporting a possible tumor suppressor role for HIF-2α and has uncovered a mechanism that links HIF-2α to a fundamental biological regulator, E2F1.