Transferrin, Transferrin Receptor, Ferritin Expression And Functional Behavior After Intracerebral Hemorrhage In The Rats With Iron Deficiency Anemia

IntroductionIntracerebral hemorrhage (ICH) is a common and often fatal subtype of stroke, aswell as a common neurological disease in newborns, especially in preterm infants. If the patient survives the ictus, the resulting hematoma within brain parenchyma triggers a series of events leading to severe neurological deficits, even causes long-term brain damage, such as cerebral palsy, mental retardation and epilepsy for newborns. Iron, a hemoglobin degradation product, plays a key role in neurodegeneration in many disease states, and an increase in brain iron can result in lipid peroxidation and free radical formation. Evidence suggests that iron and oxidative stress contribute to delayed edema formation after ICH. Proteins involved in iron transport and storage, such as ferritin, transferrin (Tf) and Transferrin receptor (TfR), might modulate iron-induced cellular and neuronal toxicity. Thus, Our understanding of the mechanisms of iron management, the effects on brain functioning is essential for generating insight into related diseases.ICH also occurs in the context of iron deficiency anemia (IDA), especially in developing brains. Examples include premature birth, medical interventions. The purpose of present study was therefore to compare brain edema, expression of Tf, TfR and ferritin levels, and functional behavior after ICH in developing rats with or without iron deficiency anemia, in order to examine the effect of IDA on expression of Tf, TfR and ferritin and the potential relationship among iron handling proteins, brain edema and behavioral deficits after ICH in developing rats.MethodsPart one: The effect of IDA on brain edema after ICH in developing ratsTwelve SD male rats were randomized to two experimental diet groups. Control group (non-IDA) received an iron sufficient diet (40mg iron/kg) and IDA group rats received an iron deficient diet(3-6mg iron/kg). Once hematocrit was reduced by 40% for the IDA group, the ICH model was set using steriotaxic methods for the two experimental diet groups, 100ul autologous blood was infused into the right basal ganglia by a microfusion pump. Regional brain edema was measured at day 3 after ICH as follows: A 3mm thick coronal brain slice, 4mm from the frontal pole, was cut. The slice was divided into four samples: ipsilateral (injured) and contralateral (non-injured) basal ganglia and ipsilateral (injured) and contralateral (non-injured) cortex. Cerebellum was obtained as a reference brain area. Water content was determined as (wet-dry weight)/wet weight.Part two: Transferrin, Transferrin receptor expression and changes over time after ICH32 SD male rats were randomized to two experimental diet groups. IDA and ICH model were established as described in part one. Right and left basal ganglia and cortex were prepared as described for brain water content. Western blots were run using standard methods for transferrin and transferrin receptor at days 1, 3, 7, and 28 after experimental ICH for four animals per experimental group each day. The relative densities of Tf and TfR protein bands were analyzed using NIH image software and described as pixels.Part three: Ferritin expression after ICH24 SD male rats were randomized to two experimental diet groups. IDA and ICH model were established as described in part one. Whole brains were reserved to take 18 'urn frozen sections for immunochernistry and immunofluorescent double labeling studies at days 1, 3, 7, and 28 after ICH for three animals per experimental group each day. Immunohistochemistry were applied for ferritin measurement, and immuno-fluorescent double-labeling to document ferritin-positive cells type. Part four: Behavioral Assessments Upper extremity functional behavior was assessed prior to ICH surgery and at days3, 7, 14 and 28 post- ICH for per experimental group animals, which included the animals for part one, part two, part three and IDA sham group (8 SD male rats). Two different sensorimotor behavioral tests were applied to detect behavioral deficits after ICH in d...