Glucocorticoids act rapidly at the paraventricular nucleus (PVN) to inhibit stress-excitatory neurons and limit excessive glucocorticoid secretion. The signaling mechanism underlying rapid feedback inhibition remains to be determined. The present study was designed to test the hypothesis that the canonical glucocorticoid receptors (GRs) is required for appropriate hypothalamic-pituitary-adrenal (HPA) axis regulation. Local PVN GR knockdown (KD) was achieved by breeding homozygous floxed GR mice with Sim1-cre recombinase transgenic mice. This genetic approach created mice with a KD of GR primarily confined to hypothalamic cell groups, including the PVN, sparing GR expression in other HPA axis limbic regulatory regions, and the pituitary. There were no differences in circadian nadir and peak corticosterone concentrations between male PVN GR KD mice and male littermate controls. However, reduction of PVN GR increased ACTH and corticosterone responses to acute, but not chronic stress, indicating that PVN GR is critical for limiting neuroendocrine responses to acute stress in males. Loss of PVN GR induced an opposite neuroendocrine phenotype in females, characterized by increased circadian nadir corticosterone levels and suppressed ACTH responses to acute restraint stress, without a concomitant change in corticosterone responses under acute or chronic stress conditions. PVN GR deletion had no effect on depression-like behavior in either sex in the forced swim test. Overall, these findings reveal pronounced sex differences in the PVN GR dependence of acute stress feedback regulation of HPA axis function. In addition, these data further indicate that glucocorticoid control of HPA axis responses after chronic stress operates via a PVN-independent mechanism.

Intracerebral hemorrhage (ICH) is a stroke subtype with high rates of mortality and morbidity. The immune system, particularly complement and cytokine signaling, has been implicated in brain injury after ICH. However, the cellular immunology associated with ICH has been understudied. In this report, we use flow cytometry to quantitatively profile immune cell populations that infiltrate the brain 1 and 4 days post-ICH. At 1 day CD45 hi GR-1 + cells were increased 2.0-fold compared with saline controls (Pp0.05); however, we did not observe changes in any other cell populations analyzed. At 4 days ICH mice presented with a 2.4-fold increase in CD45 hi cells, a 1.9-fold increase in CD45 hi GR-1 À cells, a 3.4-fold increase in CD45 hi GR-1 + cells, and most notably, a 1.7-fold increase in CD4 + cells (Pp0.05 for all groups), compared with control mice. We did not observe changes in the numbers of CD8 + cells or CD45 lo GR-1 À cells (P = 0.43 and 0.49, respectively). Thus, we have shown the first use of flow cytometry to analyze leukocyte infiltration in response to ICH. Our finding of a CD4 T-cell infiltrate is novel and suggests a role for the adaptive immune system in the response to ICH.

Hematoma and perihematomal regions after intracerebral hemorrhage (ICH) are biochemically active environments known to undergo potent oxidizing reactions. We report facile production of bilirubin oxidation products (BOXes) via hemoglobin/Fenton reaction under conditions approximating putative in vivo conditions seen following ICH. Using a mixture of human hemoglobin, physiological buffers, unconjugated solubilized bilirubin, and molecular oxygen and/or hydrogen peroxide, we generated BOXes, confirmed by spectral signature consistent with known BOXes mixtures produced by independent chemical synthesis, as well as HPLC-MS of BOX A and BOX B. Kinetics are straightforward and uncomplicated, having initial rates around 0.002 microM bilirubin per microM hemoglobin per second under normal experimental conditions. In hematomas from porcine ICH model, we observed significant production of BOXes, malondialdehyde, and superoxide dismutase, indicating a potent oxidizing environment. BOX concentrations increased from 0.084 +/- 0.01 in fresh blood to 22.24 +/- 4.28 in hematoma at 72h, and were 11.22 +/- 1.90 in adjacent white matter (nmol/g). Similar chemical and analytical results are seen in ICH in vivo, indicating the hematoma is undergoing similar potent oxidations. This is the first report of BOXes production using a well-defined biological reaction and in vivo model of same. Following ICH, amounts of unconjugated bilirubin in hematoma can be substantial, as can levels of iron and hemoglobin. Oxidation of unconjugated bilirubin to yield bioactive molecules, such as BOXes, is an important discovery, expanding the role of bilirubin in pathological processes seen after ICH.

Aneurysmal subarachnoid hemorrhage is a stroke subtype with high rates of mortality and morbidity. Cerebral vasospasm can lead to ischemic injury or death and is a common complication of aneurysmal subarachnoid hemorrhage, usually occurring 3-9 days afterwards. The cause of vasospasm is not known. Recently, there has been strong evidence that vasoactive oxidation products of bilirubin may be involved. Currently, the factors that lead to bilirubin oxidation are poorly characterized. In this study, we have designed an in vitro model of hemorrhagic stroke in order to investigate conditions that promote the oxidation of bilirubin to form vasoactive compounds. Using our model, we created a basic hematoma system of blood, CSF, and hemeoxygenase-1. We manipulated this system in various ways, incubated it and determined the concentration of vasoactive bilirubin oxidation products that resulted. Conditions where cytochrome oxidase was stimulated caused an increase bilirubin oxidation products (292.6 +/- 39.9 micromol/L respectively, vs. 79.3 +/- 1.3 micromol/L for the basic reaction, p < 0.05), which was attenuated by cyanide. Our data suggest that bilirubin oxidation products may be produced by oxidation(s) requiring an oxygen-utilizing enzyme like cytochrome oxidase.

Intracerebral hemorrhage (ICH) is a stroke subtype with significant mortality and morbidity. The role of unconjugated bilirubin (UBR) in ICH brain injury is not well understood. Therefore, we studied the effects of UBR on brain injury markers and inflammation, as well as mechanisms involved therein. We induced ICH in mice by infusion of autologous whole blood with vehicle (dimethyl sulfoxide) or UBR. We found that UBR led to an increase in edema (P≤0.05), but a decrease in nitrate/nitrite formation (7.0±0.40  nmol/mg versus 5.2±0.70  nmol/mg protein, P≤0.05) and no change in protein carbonyls. Unconjugated bilirubin was also associated with an increase in neutrophil infiltration compared with ICH alone, as determined by both immunofluorescence and flow cytometry (36%±3.2% versus 53%±1.3% of CD45(+) cells, P≤0.05). In contrast, we observed reduced perihematomal microglia immunoreactivity in animals receiving UBR (P≤0.05). Using in vitro techniques, we show neutrophil activation by UBR and also show that protein kinase C participates in this signaling pathway. Finally, we found that UBR was associated with an increased expression of the leukocyte adhesion molecule intercellular adhesion molecule-1. Our results suggest that UBR possesses complex immune-modulatory and antioxidant effects.