Reproductive senescence blunts response of estrogen receptor-α expression to estrogen treatment in rat post-ischemic cerebral microvessels.

BACKGROUND: Several studies demonstrate that estrogen treatment improves cerebral blood flow in ischemic brain regions of young ovariectomized (OVX) rats. Estrogen receptor-α (ER-α) may mediate estrogen's beneficial actions via its effects on the cerebral microvasculature. However, estrogen-derived benefit may be attenuated in aged, reproductively senescent (RS) rats. Our goal was to determine the effects of aging, estrogen deprivation and estrogen repletion with oral conjugated estrogens (CE) on postischemic cerebral microvascular protein expression of ER-α and ER-ß. METHODS: Fisher-344 (n = 37) female rats were randomly divided into the following groups: OVX, OVX CE-treated, RS untreated, and RS CE-treated. After 30 days pretreatment with CE (0.01 mg/kg) rats were subjected to 15 min. transient global cerebral ischemia. Non-ischemic naïve, OVX and RS rats were used as controls. Expression of ER-α and ER-ß in isolated cortical cerebral microvessels (20 to 100 µm in diameter) was assessed using Western blot and immunohistochemistry techniques. RESULTS: Age and reproductive status blunted nonischemic ER-α expression in microvessels of OVX rats (0.31 ± 0.05) and RS rats (0.33 ± 0.06) compared to naïve rats (0.45 ± 0.02). Postischemic microvascular expression of ER-α in OVX rats (0.01 ± 0.0) was increased by CE treatment (0.04 ± 0.01). Expression of ER-α in microvessels of RS rats (0.03 ± 0.02) was unaffected by CE treatment (0.01 ± 0.02). Western blot data are presented as a ratio of ER-α or ER-ß proteins to ß-actin and. Oral CE treatment had no effect on ER-ß expression in postischemic microvessels of OVX and RS rats. Statistical analysis was performed by One-Way ANOVA and a Newman-Keuls or Student's post-hoc test. CONCLUSION: Chronic treatment with CE increases ER-α but not ER-ß expression in cerebral microvessels of OVX rats. Aging appears to reduce the normal ability of estrogen to increase ER-α expression in postischemic cerebral microvessels.

The role of free radicals in traumatic brain injury.

Traumatic brain injury (TBI) is a significant cause of death and disability in both the civilian and the military populations. The primary impact causes initial tissue damage, which initiates biochemical cascades, known as secondary injury, that expand the damage. Free radicals are implicated as major contributors to the secondary injury. Our review of recent rodent and human research reveals the prominent role of the free radicals superoxide anion, nitric oxide, and peroxynitrite in secondary brain injury. Much of our current knowledge is based on rodent studies, and the authors identified a gap in the translation of findings from rodent to human TBI. Rodent models are an effective method for elucidating specific mechanisms of free radical-induced injury at the cellular level in a well-controlled environment. However, human TBI does not occur in a vacuum, and variables controlled in the laboratory may affect the injury progression. Additionally, multiple experimental TBI models are accepted in rodent research, and no one model fully reproduces the heterogeneous injury seen in humans. Free radical levels are measured indirectly in human studies based on assumptions from the findings from rodent studies that use direct free radical measurements. Further study in humans should be directed toward large samples to validate the findings in rodent studies. Data obtained from these studies may lead to more targeted treatment to interrupt the secondary injury cascades.

Evaluating the Joint Theater Trauma Registry as a data source to benchmark casualty care.

Just as data from civilian trauma registries have been used to benchmark and evaluate civilian trauma care, data contained within the Joint Theater Trauma Registry (JTTR) present a unique opportunity to benchmark combat care. Using the iterative steps of the benchmarking process, we evaluated data in the JTTR for suitability and established benchmarks for 24-hour mortality in casualties with polytrauma and a moderate or severe blunt traumatic brain injury (TBI). Mortality at 24 hours was greatest in those with polytrauma and a severe blunt TBI. No mortality was seen in casualties with polytrauma and a moderate blunt TBI. Secondary insults after TBI, especially hypothermia and hypoxemia, increased the odds of 24-hour mortality. Data contained in the JTTR were found to be suitable for establishing benchmarks. JTTR data may be useful in establishing benchmarks for other outcomes and types of combat injuries.

Imaging of estrogen receptor-α in rat pial arterioles using a digital immunofluorescent microscope.

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors (1, 2, 3). Although two sub-types exist (estrogen receptor -α and ß),estrogen receptor-α has been identified in both the smooth muscle and in endothelial cells of pial arterial segments using fluorescent staining combined with confocal laser scanning microscopy (4). Furthermore, ER-α is located in the nuclei and in the cytoplasm of rat basilar arteries (5). The receptors are abundant and fluoresce brightly, but clear visualization of discrete groups of receptors is difficult likely due to the numbers located in many cell layers of pial vessel segments. Additionally, many reports using immunohistochemical techniques paired with confocal microscopy poorly detail the requirements critical for reproduction of experiments (6). Our purpose for this article is to describe a simple technique to optimize the staining and visualization of ER-α using cross-sectional slices of pial arterioles obtain from female rat brains. We first perfuse rats with Evans blue dye to easily identify surface pial arteries which we isolate under a dissecting microscope. Use of a cryostat to slice 8 µm cross sections of the arteries allows us to obtain thin vessel sections so that different vessel planes are more clearly visualized. Cutting across the vessel rather than use of a small vessel segment has the advantage of easier viewing of the endothelial and smooth muscle layers. In addition, use of a digital immunofluorescent microscope with extended depth software produces clear images of ten to twelve different vessel planes and is less costly than use of a confocal laser scanning microscope.

Effects of estrogen on postischemic pial artery reactivity to ADP.

OBJECTIVE: The aims of this work were to determine if 1) ischemia alters pial artery responsiveness to the partially nitric oxide (NO)-dependent dilator, ADP, 2) the alteration depends on 17beta-estradial (E2), and 3) NO contributes to E2 protective effects. MATERIALS AND METHODS: Response to ADP and the non-NO-dependent dilator, PGE(2), were examined through closed cranial windows. Ovariectomized (OVX) and E2-replaced (E25, 0.025 mg; or E50, 0.05 mg) rats were subjected to 15-minute forebrain ischemia and one-hour reperfusion. Endothelial NO synthase (eNOS) expression was determined in pre- and postischemic isolated cortical microvessels. RESULTS: In OVX rats, ischemia depressed pial responses to ADP, but not to PGE(2). Both doses of E2 maintained responses to ADP and had no effect on the response to PGE(2). eNOS inhibition decreased the ADP response by 60% in the E25 rats and 50% in the E50 rats, but had no effect in the OVX rats. Compared to the OVX group, microvessel expression of eNOS was increased by E2, but postischemic eNOS was unchanged in both groups. CONCLUSIONS: The nearly complete loss of postischemic dilation to ADP suggests that normal non-NO-mediated dilatory mechanisms may be acutely impaired after ischemic injury. Estrogen's protective action on ADP dilation may involve both NO- and non-NO-mediated mechanisms.

Interventions to improve patient education regarding multifactorial genetic conditions: a systematic review.

The careful education of patients with complex genetic disease is essential. However, healthcare providers often have limited time to spend providing thorough genetic education. Furthermore, the number of healthcare professionals possessing strong genetics training may be inadequate to meet increasing patient demands. Due to such constraints, several interventions have been investigated over the past decade to identify potential resources for the facilitation of this specific type of patient education. This systematic literature review of these interventions for patient education attempts to elucidate the answer to the question: is there sufficient evidence for best practice for delivering genetic information to patients with multifactorial conditions? The various interventions (CD-ROM, group counseling, video/decision aid, and miscellaneous) were analyzed in terms of quality criteria and achievement of specific outcomes and were rated according to the Stetler model for evidence-based practice. Seven main outcomes were evaluated: (1) objective and subjective knowledge assessment, (2) psychological measures (general anxiety, depression, stress, cancer worry), (3) satisfaction/effectiveness of intervention, (4) time spent in counseling (time spent on basic genetic information vs. specific concerns), (5) decision-making/intent to undergo genetic testing, (6) treatment choice and value of that choice, and, finally (7) risk perception. Overall, the computer interventions resulted in more significant findings that were beneficial than any other category followed by the video category, although the group and miscellaneous categories did not measure all of the outcomes reported by the other two categories. Nevertheless, while these groups had neutral or negative findings in some of the outcomes, the computer intervention group showed significant improvement in genetics knowledge, psychological measures, satisfaction/effectiveness, time spent with counselor, and decision/intent to undergo testing.

Directions for disaster nursing education in the United States.

Because of their diverse education, experience, and practice settings, nurses are uniquely qualified to be first receivers, care givers, and leaders in any large-scale public health emergency. Many nurses, however, continue to feel inadequately prepared to function effectively in these types of situations. Great strides have been made since 2001, but much work remains to be accomplished. This article focuses on newer approaches used to teach nurses the principles of disaster preparedness. It also addresses the need to incorporate mass casualty care and disaster management skills into undergraduate curricula, continuing nurse education, and advanced degree programs for nurses in the United States.

Male--female differences in the impact of beta-adrenoceptor stimulation on resistance to experimental metastasis: exploring the effects of age and gonadal hormone involvement.

We studied the development of sexual dimorphism in resistance to NK-sensitive experimental metastasis under baseline conditions and following adrenoceptor stimulation. With increasing age, baseline resistance to MADB106 lung tumor retention (LTR) increased in both sexes, but also the susceptibility to the tumor-enhancing effects of a beta-adrenergic agonist, metaproterenol. Beginning at 13 weeks, males exhibited a 2- to 3-fold greater increase in LTR than females following adrenoceptor stimulation. This adult dimorphism was robust to ovariectomy, and questionably related to androgens. The findings are consistent with reduced female responsiveness to sympathetic activation, and substantiate the importance of including both sexes when studying neuroimmunomodulation.

Effects of estrogen on platelet reactivity after transient forebrain ischemia in rats.

Estrogen's prothrombotic effects are of increasing concern, particularly in stroke risk and recovery. Using an ischemic rodent model, the authors sought to determine (a) if estrogen replacement increases postischemic platelet reactivity, (b) if changes in estrogen status alter intraplatelet endothelial nitric oxide synthase (eNOS) synthesis, and (c) if estrogen-mediated effects on platelets alter cerebral blood flow during reperfusion. Intact (I), ovariectomized (OVX), and OVX + 17 beta-estradiol (E50) rats were subjected to 30 min of forebrain ischemia and 60 min of reperfusion. Using the platelet activation marker P-selectin, postischemic platelet reactivity was quantified by flow cytometry. In a separate cohort (I, OVX, E50), the authors quantified platelet eNOS by Western blot. Another cohort (OVX, E50) was subjected to ischemia/reperfusion, and cerebral blood flow was determined using the iodoantipyrine technique. Collagen-stimulated platelet P-selectin expression was increased in the OVX rats at 60 min of reperfusion, and this effect was reversed by estrogen treatment. No differences in platelet eNOS expression were detected among groups. Cerebral blood flow at 60 min reperfusion was comparable between the OVX and the E50 rats. The authors conclude that during reper-fusion, estrogen deficiency increases postischemic platelet sensitivity to stimuli in estrogen-deficient rats. Estrogen treatment mitigates effects of estrogen loss on platelets, but this early effect is apparently not caused by intraplatelet eNOS depression. Neither estrogen deficiency nor estrogen treatment changes early postischemic regional brain blood flow. In this rodent global cerebral ischemic model, physiologic doses of estrogen are not deleterious to platelet reactivity and may initially reduce postischemic platelet reactivity.

Characterization of heme oxygenase in adult rodent platelets.

Thromboembolism--and its involvement with tissue infarction and ischemic necrosis--continues to be of major importance in the area of vascular biology that affects all areas of clinical medicine. Activated platelets and their aggregations are key initiators in the formation of the thrombus. Several mechanisms have been described to modulate thrombus formation in the circulation, such as prostacyclins and endothelium-derived relaxing factors (the most studied being nitric oxide). Similar to nitrous oxide (NO), carbon monoxide (CO) can modulate guanylate cyclase and has been associated with anti-inflammatory and anti-apoptotic activities. Heme oxygenase (HO), in addition to being the rate-limiting enzyme of CO generation, degrades heme, which is a pro-oxidant/pro-inflammatory and generates the antioxidant molecules biliverdin and bilirubin. HO-2 is generally considered to be enriched in the brain. Here, by studying mouse platelets, we showed that it is highly present in wildtype (WT) animals and not detectable in HO-2 knockout mice. A similar finding was observed in female rats. We also investigated whether modification of estrogen levels (naturally occurring, with age, or surgically) and estrogen replacement would affect intraplatelet HO levels. Under these chronic conditions, HO-1 was barely detectable, while HO-2 was consistently stably expressed at high levels. Further investigation into the functional properties of HO itself, heme degradation, and heme bioactive metabolites remains to be conducted to determine the role of HO on platelet dynamics and on microvasculature.

Estrogen restores postischemic sensitivity to the thromboxane mimetic U46619 in rat pial artery.

The objectives of the study were to (1) characterize the dose-response relationship to the TXA2 analog, U46619 (0.01, 0.1, and 1 micromol/L) after global cerebral ischemia, (2) determine whether chronic 17beta-estradiol (E2) replacement alters this relationship, and (3) determine if E2's mechanisms are transduced through cognate estrogen receptors. Rats were assigned to five groups (n=6): placebo-implanted ovariectomized (OVX) females, OVX plus chronic E2 (CE), OVX plus acute E2 (AE), OVX plus chronic E2 plus the estrogen receptor inhibitor ICI 182,780 (CEI), and OVX plus acute E2 plus ICI 182,780 (AEI). Rats were anesthetized, intubated, cannulated (femoral artery and vein), fitted with a closed cranial window, and subjected to 15-min reversible forebrain ischemia (4-vessel occlusion, 4-VO) and 60 mins of reperfusion. Arterial blood gases, intrawindow pressure, and temperature were controlled. Vessel diameter was measured before and 5 mins after superfusion of each concentration of U46619. Compared with preischemic responses, contractile response to U46619 was depressed at all concentrations after ischemia in the OVX group. In the chronic E2 and acute E2 groups, contractile response to 1 micromol/L of U46619 was normalized to near baseline values. However, in the CEI and the AEI groups, postischemic vasoconstriction was similar to that observed in the OVX rats. We conclude that E2 targets the cerebral microvasculature to preserve postischemic pial artery reactivity and that the effect is receptor mediated. Restoration of normal constriction to vascular agonists may be an important mechanism by which E2 protects the vasculature and diminishes tissue damage after ischemia.

Effects of combined oral conjugated estrogens and medroxyprogesterone acetate on brain infarction size after experimental stroke in rat.

The reason that estrogen is strongly protective in various estrogen-deficient animal models while seemingly detrimental in postmenopausal women remains unclear. It hypothesized that prolonged oral medroxyprogesterone (MPA) plus oral conjugated equine estrogens (CEE) diminishes estrogen ability to reduce stroke damage in the rodent stroke model. To test the hypothesis, we fed ovariectomized rats CEE or MPA, or a combination of CEE and MPA (CEP), before inducing 120 min of reversible focal stroke, using the intraluminal filament model. After 22 h reperfusion, the brains were harvested and infarction volumes were quantified. Treatment with CEE alone or with CEP reduced cortical infarction volume. However, CEP failed to provide ischemic protection in subcortical regions. It was concluded that CEE alone, or with CEP, is neuroprotective in the cortex, but interactive effects between the hormones may counteract CEE beneficial effects in subcortical brain regions.

Effects of combined estrogen and progesterone on brain infarction in reproductively senescent female rats.

Recent data from the Women's Health Initiative have highlighted many fundamental issues about the utility and safety of long-term estrogen use in women. Current hormone replacement therapy for postmenopausal women incorporates progestin with estrogen, but it is uncertain if combined therapy provides major cerebrovascular risks or benefits to these women. No experimental animal stroke studies have examined combined hormone administration. The authors tested the hypothesis that combined hormone treatment reduces ischemic injury in middle-aged female rat brain. Reproductively senescent female rats underwent 2-hour middle cerebral artery occlusion (MCAO) followed by 22 hours reperfusion. Estrogen implants were placed subcutaneously at least 7 days before MCAO, and progesterone intraperitoneal injections were given 30 minutes before MCAO, at initiation, and at 6 hours of reperfusion. Rats received no hormone, a 25-microg estrogen implant, a 25-microg estrogen implant plus 5 mg/kg intraperitoneal progesterone, or 5 mg/kg intraperitoneal progesterone. Cortical, caudoputamen, and total infarct volumes were assessed by 2,3,5-triphenyltetrazolium chloride staining and digital image analysis at 22 hours reperfusion. Cortical and total infarct volumes, except in the acute progesterone-treated group, were significantly attenuated in all estrogen-alone and combined hormone-treated groups. There were no significant differences in caudoputamen infarct volumes in all hormone-treated groups as compared with untreated rats. These data have potential clinical implications relative to stroke for postmenopausal women taking combined hormone replacement therapy.

Progesterone administration during reperfusion, but not preischemia alone, reduces injury in ovariectomized rats.

Although progesterone is neuroprotective in traumatic brain injury, its efficacy in stroke is unclear. The authors determined whether there are infarction differences after middle cerebral artery occlusion (MCAO) in ovariectomized rats treated acutely with progesterone before MCAO or both pre- and postischemia. Rats received vehicle, 5 (P5), 10 (P10), or 20 (P20) mg/kg progesterone intraperitoneally 30 minutes before MCAO. In another cohort, animals received vehicle or 5 (P5R) mg/kg progesterone intraperitoneally 30 minutes before MCAO, at reperfusion initiation, and at 6-hour reperfusion. Animals underwent 2-hour MCAO by the intraluminal filament technique, followed by 22-hour reperfusion. Cortical (CTX) and caudate-putamen (CP) infarctions were determined by 2,3,5-triphenyltetrazolium chloride staining and digital image analysis. End-ischemic and early reperfusion regional cerebral blood flow (CBF) was measured by [ C]-iodoantipyrine quantitative autoradiography in vehicle- or progesterone (5 mg/kg)-treated rats. Cortical infarction (% contralateral CTX) was 31 +/- 30% (vehicle), 39 +/- 23% (P5), 41 +/- 14% (P10), and 28 +/- 20% (P20). Caudate-putamen infarction (% contralateral CP) was 45 +/- 37% (vehicle), 62 +/- 34% (P5), 75 +/- 17% (P10), and 52 +/- 30% (P20). In vehicle and P5R groups, CTX infarction was 37 +/- 20% and *20 +/- 17%, respectively (* < 0.05 from vehicle). In vehicle and P5R groups, CP infarction was 63 +/- 26% and 43 +/- 29%, respectively. End-ischemic regional CBF and CBF recovery during initial reperfusion was unaffected by progesterone treatment. These data suggest that progesterone administration both before MCAO and during reperfusion decreases ischemic brain injury.