| Backgroud Germinal matrix hemorrhage(GMH) refers to bleeding that arises from the subependymal(or periventricular) germinal region of the immature brain. It occurs in up to 20% of infants delivered at <32 weeks gestation. In the United States, >12 000 premature infants develop GMH every year. Clinical studies have shown that infants who experience germinal matrix hemorrhage can develop hydrocephalus or suffer from long-term neurologic dysfunction, including cerebral palsy, seizures, and learni ng disabilities. Therefore, investigative studies are needed to assess novel therapeutic modalities. Intraventricular hemorrhage(IVH) is a cause of significant morbidity and mortality and is an independent predictor of a worse outcome in intracerebral hem orrhage(ICH) and GMH. IVH can lead to immediate obstructive hydrocephalus, delayed communicating hydrocephalus and direct brain injury. The mechanisms by which ventricular hemorrhage causes brain injury and hydrocephalus is unclear. Our preliminary study in an adult rat model of IVH have demonstrated that iron played a role in ventricular dilatation and neuronal death following IVH. In addition, it is well known that iron overload causes perihematomal brain edema, neuronal death, brain atrophy, and neurological deficits after brain hemorrhage. Therefore, we hypothesized that iron may play a role in hydrocephalus and brain damage after GMH. As a second-generation tetracycline, minocycline(MC) is a potent inhibitor of microglia and astrocyte activation. It is a highly lipophilic compound and can penetrate the blood-brain barrier easily. It has a clear neurovascular protective effect in animal models of ICH and cerebral ischemia, and it is in current clinical trial for patients with ischemic stroke. In neonates, beneficial brain injury outcomes using minocycline have mainly been described for models of hypoxia-ischemia(HI). However, besides its anti-inflammatory properties, minocycline has also attracted tremendous attention in brain hemorrhage injury for itsiron-chelating activity, which has been of some clinical relevance. In addition, a recent study found that minocycline reduces iron overload after ICH and iron-induced brain injury. Therefore, the aim of the present study was to investigate iron accumulation and iron-handling proteins after GMH and examined whether minocycline can attenuate iron overload and brain injury after GMH.Part Ⅰ Iron and iron-handling proteins in the brain after Germinal matrix hemorrhageObjective To investigate iron accumulation and iron-handling proteins(heme oxygenase-1 and ferritin) after germinal matrix hemorrhage.Methods Forty P7 SD rat pups were randomized into sham group and experimental group. GMH was induced by intraparenchymal infusion of bacterial collagenase into the right basal ganglia. Equivalent saline was injected into the sham group. The brain water content was measured using the wet-weight/dry-weight method. Enhanced Perl’s reaction was used for iron staining. Brain heme oxygenase-1 and ferritin were examined by Western blot analysis and immunohistochemistry.Results 1. At 24 hours after GMH onset, edema was increased in the ipsilateral hemisphere compared with sham-operated controls 2. 1, 3,7and 28 days after GMH, Enhanced Perl’s reaction revealed iron overload in the perihematomal tissue. 3. GMH upregulated heme oxygenase-1 and ferritin levels compared with sham-operated controls 1 day after injection. HO-1 was increased on day 1, peaked on day 3 and was still detectable on day 7 after GMH. Both ferritin-L-chain and ferritin-H-chain protein in the perihematomal zone were low on day 1, increased significantly on day 3, and stayed at high levels on day 7.Conclusion The iron overload and upregulation of iron-handling proteins, including heme oxygenase-1 and ferritin, in the brain after GMH suggest that iron could be a target forGMH therapy.Part II Minocycline-Induced Attenuation of Iron Overload and Brain Injury after Experimental Germinal matrix hemorrhageObjective To investigate whether minocycline reduces iron overload after GMH and ironinduced brain injury in vivo.Methods To select a better time points for the brain edema measurements, the rats were treated with minocycline at 15 min, 1 hour and 2 hours after the operation, respectively. Base d on the brain edema measurements, we use the following time points :the first dose of minocycline and its vehicle(saline) were administered i.p. 15 min after the operation(45 mg/kg), and then every 24 hours from P8 to P13(45 mg/kg, i.p.). Rats were divided into 3 groups. There were saline injection(Sham) group, GMH with vehicle treatment(GMH+Veh) group, and GMH with minocycline treatment(GMH+MC) group. Rats were euthanized at 24 hours and the brains were used for brain cell death measurements. MRI scans were obtained on days 1, 7 and 28 after GMH, and the rats were then euthanized for brain histology. Behavioral tests were performed between days 21 and 28 after surgery.Results 1. Minocycline treatment significantly reduced GMH-induced brain edema, hydrocephalus and brain damage. 2. Both ferritin-L-chain and ferritin-H-chain protein levels were lower in the minocycline-treated group on day 7.Conclusion Mminocycline was found to reduce iron overload after GMH and iron-induced brain injury. These effects of minocycline suggest a potential therapeutic approach for treating infants with GMH. |
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