Practice Patterns for Acute Ischemic Stroke Workup: A Longitudinal Population-Based Study.

BACKGROUND: We examined practice patterns of inpatient testing to identify stroke etiologies and treatable risk factors for acute ischemic stroke recurrence. METHODS AND RESULTS: We identified stroke cases and related diagnostic testing from four 1-year study periods (July 1993 to June 1994, 1999, 2005, and 2010) of the Greater Cincinnati/Northern Kentucky Stroke Study. Patients aged ≥18 years were included. We focused on evaluation of extracranial arteries for carotid stenosis and assessment of atrial fibrillation because randomized controlled trials supported treatment of these conditions for stroke prevention across all 4 study periods. In each study period, we also recorded stroke etiology, as determined by diagnostic testing and physician adjudication. An increasing proportion of stroke patients received assessment of both extracranial arteries and the heart over time (50%, 58%, 74%, and 78% in the 1993-1994, 1999, 2005, and 2010 periods, respectively; P<0.0001 for trend), with the most dramatic individual increases in echocardiography (57%, 63%, 77%, and 83%, respectively). Concurrently, we observed a decrease in strokes of unknown etiology (47%, 48%, 41%, and 38%, respectively; P<0.0001 for trend). We also found a significant increase in strokes of other known causes (32%, 25%, 45% and 59%, respectively; P<0.0001 for trend). CONCLUSIONS: Stroke workup for treatable causes of stroke are being used more frequently over time, and this is associated with a decrease in cryptogenic strokes. Future study of whether better determination of treatable stroke etiologies translates to a decrease in stroke recurrence at the population level will be essential.

GABAB Encephalitis: A Fifty-Two-Year-Old Man with Seizures, Dysautonomia, and Acute Heart Failure.

Autoantibodies to the γ-aminobutyric acid receptor, subtype B (GABAB), are a known cause of limbic encephalitis. The spectrum of clinical manifestations attributable to this antibody is not well defined at the present time. Here we present a case of GABAB encephalitis presenting with encephalopathy, status epilepticus, dysautonomia, and acute heart failure. To our knowledge, heart failure and dysautonomia have not yet been reported with this syndrome.

Neuroendocrine Function After Hypothalamic Depletion of Glucocorticoid Receptors in Male and Female Mice.

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.

Depletion of GR-1-Positive Cells Is Associated with Reduced Neutrophil Inflammation and Astrocyte Reactivity after Experimental Intracerebral Hemorrhage.

Intracerebral hemorrhage (ICH) is the stroke subtype with the highest mortality and morbidity with 25% of patients dying within the first 48 h and a high incidence of poor outcomes. Because of high early mortality rates, an understanding of acute brain injury mechanisms is essential. In this study, we have investigated the putative role of acute inflammation in brain injury after experimental ICH. We depleted GR-1(+) cells in mice by intraperitoneal administration of anti-GR-1 antibody or normal rat serum (control). We then induced ICH by infusion of autologous whole blood into the striatum and compared functional outcome and brain injury markers between the two groups. We found that administration of anti-GR-1 antibody led to a profound decrease in circulating GR-1(+) cells (1.5 ± 0.34% vs. 50.3 ± 8.3% of CD45(+) cells, p ≤ 0.01) and that brain neutrophils decreased by approximately 50% (p ≤ 0.05). We observed a reduction in astrocyte immunoreactivity in the GR-1(+) cell-depleted group (p ≤ 0.05). Conversely, we did not find attenuation of brain edema or differences in behavioral deficits between the two groups. In summary, our results are promising and suggest that larger studies or different neutrophil manipulations may produce greater attenuation of injury after ICH.

Unconjugated bilirubin contributes to early inflammation and edema after intracerebral hemorrhage.

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.

Iron and early brain injury after subarachnoid hemorrhage.

Aneurysmal subarachnoid hemorrhage (SAH) affects approximately 27,000 Americans per year. Although delayed cerebral vasospasm is of high clinical significance, mortality within the first 2 days may approach 30%. In this issue of the Journal of Cerebral Blood Flow and Metabolism, Lee et al have studied the role of iron in early brain injury after experimental SAH. They found that iron chelation with deferoxamine reduced mortality and oxidative DNA damage, and lessened the induction of iron-handling proteins. Taken together, these results highlight the deleterious potential of blood breakdown products and provide an insight into future intervention.

A novel brain injury mechanism after intracerebral hemorrhage: the interaction between heme products and the immune system.

In this paper we hypothesize a novel mechanism by which brain injury occurs after intracerebral hemorrhage. We propose the following mechanism: (1) heme that is derived from extravasated erythrocytes is degraded into bilirubin and bilirubin oxidation products (BOXes). (2) Bilirubin and BOXes activate microglia and astrocytes, which are cells with immune functions in the brain. This activation leads to release of cytokines and activation of leukocyte adhesion molecules on the luminal surface of cerebral endothelial cells. (3) Leukocytes then traffic into the brain. (4) Leukocytes, activated glial cells and cytokines contribute to injury processes. We present preliminary data in support of our hypothesis that BOXes activate glia.

Intracerebral hemorrhage leads to infiltration of several leukocyte populations with concomitant pathophysiological changes.

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 (P

Chemical and biochemical oxidations in spinal fluid after subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH) is a stroke with high rates of mortality and morbidity. SAH-induced cerebral vasospasm can lead to ischemic injury or death and is a common complication of SAH. Recently there has been an accumulation of emerging evidence that oxidation of heme-derived bilirubin into bilirubin oxidation products (BOXes) may be involved in cerebral vasospasm. BOXes are produced by the oxidation of bilirubin yielding a mixture of isomers: 4-methyl-5-oxo-3-vinyl-(1,5-dihydropyrrol-2-ylidene)acetamide (BOX A) and 3-methyl-5-oxo-4-vinyl- (1,5-dihydropyrrol-2-ylidene)acetamide (BOX B). BOXes have been a subject of interest in the neurosurgical and neurological fields for several years because of their purported correlation with and or role in subarachnoid hemorrhage induced cerebral vasospasm. We believe that it is critical to understand the chemical and biochemical environment in the hemorrhagic spinal fluid after SAH that leads to the oxidation of bilirubin. There is a growing body of information concerning their putative role in vasospasm; however, there is a dearth of information concerning the chemical and biochemical characteristics of BOXes.

A novel duplex ELISA method for quantitation of plasma proteins in areas of brain edema.

We have developed a novel type of duplex enzyme-linked immunosorbent assay (ELISA) for the quantitation of the major plasma proteins, IgG and albumin, in edematous brain tissue. We test this duplex ELISA on our porcine intracerebral hemorrhage (ICH) model and show that it is as accurate and sensitive as independent single ELISAs. This method is useful as a marker of edema in brain tissue and the same design can be applied to other proteins and sample types.

An in vitro model of aneurysmal subarachnoid hemorrhage: oxidation of unconjugated bilirubin by cytochrome oxidase.

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.

Plasma proteins in edematous white matter after intracerebral hemorrhage confound immunoblots: an ELISA to quantify contamination.

Intracerebral hemorrhage (ICH) and traumatic brain injury can induce brain tissue edema (i.e., interstitial and/or vasogenic), containing high concentrations of plasma proteins. To understand biochemical processes in edema development following these insults, it would be useful to examine alterations in various proteins (e.g., transcription factors, signaling). However, determining altered protein responses in edematous brain tissue using standard immunoblotting techniques is problematic due to contaminating plasma proteins. To solve this problem, we developed an enzyme-linked immunosorbent assay (ELISA) method to quantify the two major plasma proteins, albumin and immunoglobulin G (IgG), that comprise about 80% of the total plasma proteins. We tested our method on edematous white matter samples from our porcine ICH model. To induce ICH, we infused autologous arterial whole blood (3 mL) into frontal hemispheric white matter of pentobarbital- anesthetized pigs ( approximately 20 kg) over 15 min. We froze brains in situ at various times up to 24 h post- ICH and sampled white matter adjacent and contralateral to hematomas. We prepared cytoplasmic extracts that we subjected to ELISA and immunoblotting analyses. Our results demonstrate that this ELISA method is accurate, reproducible, and enables the concentrations of albumin and IgG in edematous brain tissue samples to be accurately determined. By using this correction method, equal amounts of cellular protein can be loaded onto gels during immunoblotting procedures. This method is applicable to edematous tissue samples in brain injury models in which high plasma protein concentrations result from interstitial or vasogenic edema development.