Indications and Coverage of Metabolic and Bariatric Surgery: A Worldwide IFSO Survey Comparing Different National Guidelines.

INTRODUCTION: Knowing how metabolic and bariatric surgery (MBS) is indicated in different countries is essential information for the International Federation for the Surgery of Obesity and Metabolic Disorders (IFSO). AIM: To analyze the indications for MBS recommended by each of the national societies that comprise the IFSO and how MBS is financed in their countries. METHODS: All IFSO societies were asked to fill out a survey asking whether they have, and which are their national guidelines, and if MBS is covered by their public health service. RESULTS: Sixty-three out of the 72 IFSO national societies answered the form (87.5%). Among them, 74.6% have some kind of guidelines regarding indications for MBS. Twenty-two percent are still based on the US National Institute of Health (NIH) 1991 recommendations, 43.5% possess guidelines midway the 1991s and ASMBS/IFSO 2022 ones, and 34% have already adopted the latest ASMBS/IFSO 2022 guidelines. MBS was financially covered in 65% of the countries. CONCLUSIONS: Most of the IFSO member societies have MBS guidelines. While more than a third of them have already shifted to the most updated ASMBS/IFSO 2022 ones, another significant number of countries are still following the NIH 1991 guidelines or even do not have any at all. Besides, there is a significant number of countries in which surgical treatment is not yet financially covered. More effort is needed to standardize indications worldwide and to influence insurers and health policymakers to increase the coverage of MBS.

Conversion of Adjustable Gastric Banding to Roux-en-Y Gastric Bypass in One or Two Steps: What Is the Best Approach? Analysis of a Multicenter Database Concerning 832 Patients.

BACKGROUND: Roux-en-Y gastric bypass (RYGB) is often the preferred conversion procedure for laparoscopic adjustable gastric banding (LAGB) poor responders. However, there is controversy whether it is better to convert in one or two stages. This study aims to compare the outcomes of one and two-stage conversions of LAGB to RYGB. METHODS: Retrospective review of a multicenter prospectively collected database. Data on conversion in one and two stages was compared. RESULTS: Eight hundred thirty-two patients underwent LAGB conversion to RYGB in seven specialized bariatric centers. Six hundred seventy-three (81%) were converted in one-stage. Patients in the two-stage group were more likely to have experienced technical complications, such as slippage or erosions (86% vs. 37%, p = 0.0001) and to have had a higher body mass index (BMI) (41.6 vs. 39.9 Kg/m2, p = 0.005). There were no differences in postoperative complications and mortality rates between the one-stage and two-stage groups (13.5% vs. 10.8%, and 0.7% vs. 0.0% respectively, p = ns). Mean final BMI and %total weight loss (%TWL) for the one-stage and the two-stage groups were 31.6 vs. 32.4 Kg/m2 (p = ns) and 30.4 vs. 26.8 (p = 0.017) after a mean follow-up of 33 months. Follow-up at 1, 3, and 5 years was 98%, 75%, and 54%, respectively. CONCLUSIONS: One-stage conversion of LAGB to RYGB is safe and effective. Two-stage conversion carries low morbidity and mortality in the case of band slippage, erosion, or higher BMI patients. These findings suggest the importance of patient selection when choosing the appropriate conversion approach.

Adjuvant therapies using normobaric oxygen with hypothermia or ethanol for reducing hyperglycolysis in thromboembolic cerebral ischemia.

BACKGROUND AND PURPOSE: Normobaric oxygen (NBO), ethanol (EtOH), and therapeutic hypothermia (TH) delivered alone or in combination have neuroprotective properties after acute stroke. We used an autologous thromboembolic rat stroke model to assess the additive effects of these treatments for reducing the deleterious effects of hyperglycolysis post-stroke in which reperfusion is induced with recombinant tissue plasminogen activator (rt-PA). METHODS: Sprague-Dawley rats were subjected to middle cerebral artery (MCA) occlusion with an autologous embolus. One hour after occlusion, rt-PA was administered alone or with NBO (60%), EtOH (1.0 g/kg), TH (33 °C), either singly or in combination. Infarct volume and neurological deficit were assessed at 24h after rt-PA-induced reperfusion with or without other treatments. The extent of hyperglycolysis, as determined by cerebral glucose and lactate levels was evaluated at 3 and 24h after rt-PA administration. At the same time points, expressions of glucose transporter 1 (Glut1), glucose transporter 3 (Glut3), phosphofructokinase1 (PFK-1), and lactate dehydrogenase were (LDH) measured by Western blotting. RESULTS: Following rt-PA in rats with thromboembolic stroke, NBO combined with TH or EtOH most effectively decreased infarct volume and neurological deficit. As compared to rt-PA alone, EtOH or TH but not NBO monotherapies significantly reduced post-stroke hyperglycolysis. The increased utilization of glucose and production of lactate post-stroke was prevented most effectively when NBO was combined with either EtOH or TH after reperfusion with rt-PA, as shown by the significantly decreased Glut1, Glut3, PFK-1, and LDH levels. CONCLUSIONS: In a rat thromboembolic stroke model, both EtOH and TH used individually offer neuroprotection after the administration of rt-PA. While NBO monotherapy does not appear to be effective, it significantly potentiates the efficacy of EtOH and TH. The similar neuroprotection and underlying mechanisms pertaining to the attenuation of hyperglycolysis provided by EtOH or TH in combination with NBO suggest a possibility of substituting EtOH for TH. Thus a combination of NBO and EtOH, which are widely available and easily used, could become a novel and effective neuroprotective strategy in the clinical setting.

Long-term effects of enriched environment on neurofunctional outcome and CNS lesion volume after traumatic brain injury in rats.

To determine whether the exposure to long term enriched environment (EE) would result in a continuous improvement of neurological recovery and ameliorate the loss of brain tissue after traumatic brain injury (TBI) vs. standard housing (SH). Male Sprague-Dawley rats (300-350 g, n=28) underwent lateral fluid percussion brain injury or SHAM operation. One TBI group was held under complex EE for 90 days, the other under SH. Neuromotor and sensorimotor dysfunction and recovery were assessed after injury and at days 7, 15, and 90 via Composite Neuroscore (NS), RotaRod test, and Barnes Circular Maze (BCM). Cortical tissue loss was assessed using serial brain sections. After day 7 EE animals showed similar latencies and errors as SHAM in the BCM. SH animals performed notably worse with differences still significant on day 90 (p<0.001). RotaRod test and NS revealed superior results for EE animals after day 7. The mean cortical volume was significantly higher in EE vs. SH animals (p=0.003). In summary, EE animals after lateral fluid percussion (LFP) brain injury performed significantly better than SH animals after 90 days of recovery. The window of opportunity may be wide and also lends further credibility to the importance of long term interventions in patients suffering from TBI.

Brain cellular localization of endothelin receptors A and B in a rodent model of diffuse traumatic brain injury.

Endothelin-1 exerts potent vasoconstrictor and vasodilatory effects through its actions on its receptors A (ETrA) and B (ETrB), respectively. While ETrA and B have classically been thought to be expressed on vascular cell types, more recent evidence suggests that, particularly following brain injury, their expression may be seen in other, non-vascular cell types. To date no studies have comprehensively studied the cellular location of endothelin receptors following traumatic brain injury (TBI). Therefore, this study investigates the cellular localization of ETrA and B in normal and traumatized brains using an impact acceleration device. Adult male Sprague-Dawley rats were subjected to TBI by weight drop (450 g) from either 1.5, a distance known to elicit mild TBI in the absence of changed in cerebral blood flow (CBF) or 2 m, a distance shown to cause a significant reduction in CBF. One set of impacted brains were processed for Western determination of ETrA and B expression. Another set were processed for immunofluorescence (IF). For IF, ETrA and ETrB antibodies were combined with cell markers for neurons, astrocytes, microglia, oligodendrocytes, smooth muscle cells and endothelial cells of blood vessels. While ETrA and B was upregulated after more moderate to severe injury (2 m) overall receptor expression was unchanged in response to mild trauma (1.5 m). Double labeling IF confirmed prominent ETrA and ETrB labeling in NeuN labeled pyramidal neurons and interneurons in sensorymotor cortex (smCx) and hippocampus (hipp) post TBI. ETrA rather than ETrB was preferentially co-localized in vascular smooth muscle cells. After injury, a subpopulation of astrocytes in white matter co-localized ETrA but not ETrB. Localization of either receptor in endothelial cells was sparse. No prominent IF was detected in microglia and oligodendrocytes. Taken together with previous findings in other pathological states that show an apparent shift in the localization of ETrA and B, the observed receptor shifts in this work may underlie the ET-1-mediated pathotrajectory of TBI including hypoperfusion.

Convergence of stress granules and protein aggregates in hippocampal cornu ammonis 1 at later reperfusion following global brain ischemia.

The delayed and selective vulnerability of post-ischemic hippocampal cornu ammonis (CA) 1 pyramidal neurons correlates with a lack of recovery of normal protein synthesis. Recent evidence implicates sequestration of translational machinery into protein aggregates and stress granules as factors underlying persistent translation arrest in CA1 neurons. However, the relationship between protein aggregates and stress granules during brain reperfusion is unknown. Here we investigated the colocalization of protein aggregates and stress granules using immunofluorescence microscopy and pair-wise double labeling for ubiquitin/T cell internal antigen (TIA-1), ubiquitin/small ribosomal subunit protein 6 (S6), and TIA-1/S6. We evaluated the rat dorsal hippocampus at 1, 2 or 3 days of reperfusion following a 10 min global brain ischemic insult. At 1 day of reperfusion, ubiquitin-containing aggregates (ubi-protein clusters) occurred in neurons but did not colocalize with stress granules. At 2 days' reperfusion, only in CA1, cytoplasmic protein aggregates colocalized with stress granules, and ubiquitin-containing inclusions accumulated in the nuclei of CA1 pyramidal neurons. Functionally, a convergence of stress granules and protein aggregates would be expected to sustain translation arrest and inhibit clearance of ubiquitinated proteins, both factors expected to contribute to CA1 pyramidal neuron vulnerability.

Exercise preconditioning upregulates cerebral integrins and enhances cerebrovascular integrity in ischemic rats.

We hypothesized that exercise preconditioning strengthens brain microvascular integrity against ischemia/reperfusion injury through the tumor necrosis factor (TNF)-integrin signaling pathway. Adult male Sprague Dawley rats (n = 24) were studied in: (1) exercise (the animals run on a treadmill 30 min each day) for 3 weeks, (2) non-exercise. Six animals from each group (n = 12) were subjected to stroke, the remaining animals served as controls (n = 6 x 2). Brain infarction and edema were determined by Nissl staining. Cerebral integrin expression was detected by immunochemistry and stereological methods. In addition, we used flow cytometry to address the causal role of TNF-alpha in inducing the expression of integrins in the human umbilical vein endothelial cells under TNF-alpha or vascular endothelial growth factor (VEGF) pretreatment. Exercise reduces brain infarction and brain edema in stroke. Expressions of integrin subunit alpha(1), alpha(6), beta(1), and beta(4) were increased after exercise. Exercise preconditioning reversed stroke-reduced integrin expression. An in vitro study revealed a causal link between the gradual upregulation of TNF-alpha (rather than VEGF) and cellular expression of integrins. These results demonstrated an increase in cerebral expression of integrins and a decrease in brain injury from stroke after exercise preconditioning. The study suggests that upregulation of integrins during exercise enhances neurovascular integrity after stroke. The changes in integrins might be altered by TNF-alpha.

Immunohistochemical mapping of total and phosphorylated eukaryotic initiation factor 4G in rat hippocampus following global brain ischemia and reperfusion.

Partial proteolysis and phosphorylation of the translation initiation factor eukaryotic initiation factor 4G (eIF4G) occur in reperfused brain, but the contribution of eIF4G alterations to brain injury has not been established. A component of the complex delivering mRNA to the small ribosomal subunit, eIF4G is also found in stress granules. Stress granules sequester inactive 48S preinitiation complexes during stress-induced translation arrest. We performed double-labeling immunofluorescence histochemistry for total or ser 1108 phosphorylated eIF4G and the stress granule component T-cell internal antigen following normothermic, 10 min cardiac arrest-induced global brain ischemia and up to 4 h reperfusion in the rat. In cornu ammonis (Ammon's horn; CA) 1 at 90 min and 4 h reperfusion, eIF4G staining transformed from a homogeneous to an aggregated distribution. The number of eIF4G-containing stress granules differed between CA1 and CA3 during reperfusion. In hippocampal pyramidal neurons, phosphorylated eIF4G appeared exclusively in stress granules. Supragranular interneurons of the dentate gyrus showed a large increase in cytoplasmic eIF4G(P) following reperfusion. Immunoblot analysis with antisera against different portions of eIF4G showed a large increase in phosphorylated C-terminal eIF4G fragments, suggesting these accumulate in the cytoplasm of dentate gyrus interneurons. Thus, altered eIF4G subcellular compartmentalization may contribute to prolonged translation arrest in CA1 pyramidal neurons. Accumulation of phosphorylated eIF4G fragments may contribute to the vulnerability of dentate interneurons. Ischemia and reperfusion invoke different translational control responses in distinct hippocampal neuron populations, which may contribute to the differential ischemic vulnerabilities of these cells.

Prolonged translation arrest in reperfused hippocampal cornu Ammonis 1 is mediated by stress granules.

Global brain ischemia and reperfusion cause phosphorylation of the alpha subunit of eukaryotic initiation factor 2alpha, a reversible event associated with neuronal translation inhibition. However, the selective vulnerability of cornu Ammonis (CA) 1 pyramidal neurons correlates with irreversible translation inhibition. Phosphorylation of eukaryotic initiation factor 2alpha also leads to the formation of stress granules, cytoplasmic foci containing, in part, components of the 48S pre-initiation complex and the RNA binding protein T cell internal antigen-1 (TIA-1). Stress granules are sites of translationally inactive protein synthesis machinery. Here we evaluated stress granules in rat hippocampal formation neurons after 10 min global brain ischemia and 10 min, 90 min or 4 h of reperfusion by double-labeling immunofluorescence for two stress granule components: small ribosomal subunit protein 6 and TIA-1. Stress granules in CA3, hilus and dentate gyrus, but not CA1, increased at 10 min reperfusion and returned to control levels by 90 min reperfusion. Dynamic changes in the nuclear distribution of TIA-1 occurred in resistant neurons. At 4 h reperfusion, small ribosomal subunit protein 6 was solely localized within stress granules only in CA1 pyramidal neurons. Both TIA-1 and small ribosomal subunit protein 6 levels decreased approximately 50% in hippocampus homogenates. Electron microscopy showed stress granules to be composed of electron dense bodies 100-200 nm in diameter, that were not membrane bound, but were associated with endoplasmic reticulum. Alterations in stress granule behavior in CA1 pyramidal neurons provide a definitive mechanism for the continued inhibition of protein synthesis in reperfused CA1 pyramidal neurons following dephosphorylation of eukaryotic initiation factor 2alpha.

Intracellular coexpression of endothelin-1 and inducible nitric oxide synthase underlies hypoperfusion after traumatic brain injury in the rat.

We used Marmarou's rat model of traumatic brain injury to demonstrate colocalization of mRNAs for endothelin-1 (ET-1, a powerful vasoconstrictor) and inducible nitric oxide synthase (iNOS, generator of NO, a vasodilator) in individual cells that form the brain's microvascular wall. The results were confirmed with double immunocytochemistry. After trauma endothelial, smooth muscle cells and macrophages contributed to the abnormal synthesis of ET-1 and iNOS which may underlie a dysfunctional brain microcirculation. This is the first in vivo single cell demonstration of ET-1 and iNOS colocalization, suggesting reciprocal regulation of each other's expression both at the transcriptional and translational levels. The results further indicate that interaction between ET-1 and iNOS occurs at the cytosol and possibly the nuclear membranes, implicating mediation via endothelin receptors.

Attenuation of iNOS mRNA exacerbates hypoperfusion and upregulates endothelin-1 expression in hippocampus and cortex after brain trauma.

Nitric oxide (NO, a vasodilator) and endothelin-1 (ET-1, a powerful vasoconstrictor) participate in the regulation of brain's microcirculation influencing each other's expression and synthesis. Following injury to the brain, NO is derived largely from the inducible form of nitric oxide synthase (iNOS). We used Marmarou's model of traumatic brain injury (TBI) to study the cerebral blood flow and expression (mRNA) of ET-1 in rats that were pretreated with antisense iNOS oligodeoxynucleotides (ODNs). Intracerebroventricular application of iNOS ODNs resulted in reduced synthesis of iNOS as detected by Western blot analysis. The cerebral blood flow (measured by laser Doppler flowmetry), generally decreased after TBI, was further markedly reduced in the treated animals and remained at low levels up to 48 h post-TBI. The expression of ET-1 (detected by in situ hybridization in cortex and hippocampus) was increased 2-3-fold following TBI alone and this increase reached 5-6-fold in animals pretreated with antisense iNOS ODNs. The results indicate that most likely, NO, generated primarily by iNOS, suppresses ET-1 production and that a decrease of NO results in upregulation of ET-1 via transcriptional and translational mechanisms. Increased availability of ET-1 at the vascular bed and the neuropil may contribute to the altered microvascular reactivity and reduced perfusion of the brain following TBI.

Exercise pre-conditioning reduces brain damage in ischemic rats that may be associated with regional angiogenesis and cellular overexpression of neurotrophin.

There is increasing evidence that physical activity is associated with a decreased stroke risk. The purpose of this study was to determine if exercise could also reduce brain damage in rats subjected to transient middle cerebral artery (MCA) occlusion, and if the reduced brain injury is associated with angiogenesis as well as cellular expression of the nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in regions supplied by the MCA. Adult male Sprague Dawley rats (n=36) exercised 30 min each day for 3 weeks on a treadmill on which repetitive locomotor movement was required. Then, stroke was induced by a 2-h MCA occlusion using an intraluminal filament, followed by 48 h of reperfusion. In addition to the two exercised groups of animals with or without MCA occlusion, there were two other groups of animals, with or without MCA occlusion, housed for the same duration and used as non-exercised controls. Brain damage in ischemic rats was evaluated by neurologic deficits and infarct volume. Exercise preconditioned and non-exercised brains were processed for immunocytochemistry to quantify the number of microvessels or NGF- and BDNF-labeled cells. Pre-ischemic motor activity significantly (P<0.01) reduced neurologic deficits and infarct volume in the frontoparietal cortex and dorsolateral striatum. Cellular expressions of NGF and BDNF were significantly (P<0.01) increased in cortex (neuron) and striatum (glia) of rats under the exercise condition. Significant (P<0.01) increases in microvessel density were found in striatum. Physical activity reduced stroke damage. The reduced brain damage may be attributable to angiogenesis and neurotrophin overexpression in brain regions supplied by the MCA following exercise.

Motor balance and coordination training enhances functional outcome in rat with transient middle cerebral artery occlusion.

The goal of this study was to determine if relatively complex motor training on Rota-rod involving balance and coordination plays an essential role in improving motor function in ischemic rats, as compared with simple locomotor exercise on treadmill. Adult male Sprague-Dawley rats with (n=40) or without (n=40) ischemia were trained under each of three conditions: (1) motor balance and coordination training on Rota-rod; (2) simple exercise on treadmill; and (3) non-trained controls. Motor function was evaluated by a series of tests (foot fault placing, parallel bar crossing, rope and ladder climbing) before and at 14 or 28 days after training procedures in both ischemic and normal animals. Infarct volume in ischemic animals was determined with Nissl staining. Compared with both treadmill exercised and non-trained animals, Rota-rod-trained animals with or without ischemia significantly (P<0.01) improved motor performance of all tasks except for foot fault placing after 14 days of training, with normal rats having better performance. Animals trained for up to 28 days on the treadmill did not show significantly improved function. With regard to foot fault placing task, performance on foot placing was improved in ischemic rats across the three measurements at 0, 14 and 28 days regardless of training condition, while the normal group reached their best performance at the beginning of measurement. No significant differences in infarct volume were found in rats trained either with Rota-rod (47+/-4%; mean+/-S.E.), treadmill (45+/-5%) or non-exercised control (45+/-3%). In addition, no obvious difference could be detected in the location of the damage which included the dorso-lateral portion of the neostriatum and the frontoparietal cortex, the main regions supplied by the middle cerebral artery. The data suggest that complex motor training rather than simple exercise effectively improves functional outcome.

Sources of endothelin-1 in hippocampus and cortex following traumatic brain injury.

Endothelin 1 (ET-1) exerts normally a powerful vasoconstrictor role in the control of the brain microcirculation. In altered states, such as following traumatic brain injury (TBI), it may contribute to the development of ischemia and/or secondary cell injury. Because little is known of ET-1's cellular compartmentalization and its association to vulnerable neurons after TBI, we assessed its expression (both mRNA and protein) in cerebral cortex and hippocampus using correlative in situ hybridization and immunocytochemical techniques.Sprague-Dawley male rats were killed at 4, 24 or 48 h after TBI (450 g from 2 m, Marmarou's model). Semiquantitative analysis of our in situ hybridization results indicated a 2.5- and a 2.0-fold increase in ET-1 mRNA content in the hippocampus and cortex respectively which persisted up to 48 h post TBI. At 4 and 24 h after TBI enzyme-linked immunosorbent assay showed a tendency for increased ET-1 synthesis. In animals subjected to TBI, qualitative immunocytochemical analysis revealed a shift in ET-1 expression from astrocytes (in control animals) to endothelial cells, macrophages and neurons. Astrocytes and macrophages were identified unequivocally by using double immunofluorescence revealing ET-1 and glial fibrillary acidic protein or ED-1, respectively, the markers being specific for these cellular types. While this redistribution was most prominent at 4 and 24 h post TBI, at 48 h the endothelial cells remained strongly ET-1 immunopositive. The results suggest that cellular types which in the intact animal synthesize little or no ET-1 provide novel sources of the peptide after TBI. These sources may contribute to the sustained cerebrovascular hypoperfusion observed post TBI.

Upregulation of iNOS expression and phosphorylation of eIF-2alpha are paralleled by suppression of protein synthesis in rat hypothalamus in a closed head trauma model.

When the inducible form of nitric oxide synthase (iNOS) is expressed after challenge to the nervous system, it results in abnormally high concentrations of nitric oxide (NO). Under such conditions, NO could phosphorylate the eukaryotic translation initiation factor (eIF)-2alpha, thus suppressing protein synthesis in neurons that play a role in endocrine and autonomic functions. Using the Marmarou model of traumatic brain injury (TBI), we observed a rapid increase (at 4 h after TBI) of iNOS mRNA in magno- and parvocellular supraoptic and paraventricular neurons, declining gradually by approximately 30% at 24 h and by approximately 80% at 48 h. Western analysis indicated a trend towards increased iNOS protein synthesis at 4 h, which peaked at 8 h, and tended to decrease at the later time points. At the same time points, we detected immunocytochemically the phosphorylated form of eIF-2alpha (eIF-2alpha[P]) as cytoplasmic and more often as nuclear labeling. The incidence of double-labeled [iNOS and eIF-2alpha(P)] neuronal profiles, particularly at 24 h and 48 h after TBI, was high. De novo protein synthesis assessed quantitatively after infusion of 35S methionine/cysteine was reduced by approximately 20% at 4 h, remained depressed at 24 h, and did not return to control levels up to 48 h following the trauma. The results suggest that iNOS may trigger phosphorylation of eIF-2alpha, which in turn interferes with protein synthesis at the translational (ribosomal complex) and transcriptional (chromatin) levels. The depression in protein synthesis may include downregulation of iNOS itself, which could be an autoregulatory inhibitory feedback mechanism for NO synthesis. Excessive amounts of NO may also participate in dysfunction of hypothalamic circuits that underlie endocrine and autonomic alterations following TBI.

GnRH and gonadotropin release is decreased in chronic nitric oxide deficiency.

Nitric oxide synthetase (NOS), the conversion enzyme for nitric oxide (NO) is localized in the anterior pituitary of female rats, particularly in gonadotrophs and folliculo-stellate cells, suggesting that NO regulates the release of luteinizing hormone (LH) and follicle stimulating hormone (FSH) from the anterior pituitary. The focus of this study was to determine the effect of chronic NO deficiency on the subsequent pituitary release of LH and FSH in vitro and the hypothalamic immunoexpression of GnRH in vivo. NO deficiency was induced by adding the NOS inhibitor, N-nitro-L-arginine (L-NNA, 0.6 g/L) to the drinking water of female Wistar rats. After 8 weeks, the animals were euthanized, the pituitaries were removed, and they were incubated in vitro. Pituitaries were perfused for 4 hr in the presence of pulsatile gonadotropin release hormone (GnRH, 500 ng/pulse) every 30 min. S-Nitroso-L-acetyl penicillamine (SNAP, an NO donor, 0.1 mM) or L-nitro-argine methyl ester (L-NAME, a NOS inhibitor, 0.1 mM) was added to the media and perfusate samples were collected at 10-min intervals. LH and FSH levels in the perfusate were measured by double antibody radioimmunoassays. Pituitaries from the NO-deficient rats had a significantly smaller GnRH-stimulated release of LH and FSH compared with proestrous control rats. The addition of S-NAP to the perfusate resulted in decreased LH and FSH secretion in the control group, but increased LH secretion in the NO-deficient group. The addition of L-NAME to the perfusate suppressed LH secretion from control pituitaries, but not in pituitaries from NO-deficient animals. Immunohistochemistry of brain slices demonstrated that NO-deficient rats had a large qualitative decrease of GnRH in the median eminence compared with their controls. This decrease was particularly evident in the external capillary plexus of the median eminence. We concluded that chronic NO deficiency is associated with a decreased GnRH in neurosecretory terminals in the external capillary layer of the median eminence, accompanied by a decrease in LH and FSH release from the pituitaries.

Endothelial cell activation following moderate traumatic brain injury.

Traumatic brain injury (TBI) initiates a cascade of acute and chronic injury responses which include disturbances in the cerebrovasculature that may result in the activation of the microvascular endothelial development of a dysfunction endothelium. The present study examines endothelial cell (EC) activation in a percussion model of moderate TBI. The criteria for endothelial activation used in these studies was surface expression of a number of markers collectively termed endothelial activation antigens. Temporal induction of the major histocompatibility (MHC) class II molecules, E-selectin (CD62E), vascular cell adhesion molecule (VACM-1) (CD106) as well as altered expression of constitutively expressed intercellular adhesion molecule-1 (ICAM-1) (CD54), the glucose transporter protein (glut-1), the transferrin receptor (tfR) (CD71), and MHC class I molecules was examined at various times following impact. Induction of E-selectin and increased expression of ICAM-1 was seen by 2 h post-impact (PI) and was sustained through 24 h PI. Decreased expression of immunologically reactive glut-1 and tfR was observed by 2-4 h PI and remained low up to 24 h PI. No induction of VCAM-1, MHC class II molecules or altered constitutive expression or MHC class I molecules was seen. Changes in EC activation were observed predominantly at the site of impact and were diminished temporarily. These results indicate that mild concussive injury to the brain results in activation of the endothelium.

Acute alterations of endothelin-1 and iNOS expression and control of the brain microcirculation after head trauma.

The biosynthetic equilibrium between endothelin-1 (ET-1, a vasoconstricting agent) and nitric oxide (NO, a gas with vasodilating effects) is thought to play a role in the autoregulation of microvessel contractility and maintenance of adequate perfusion after traumatic brain injury. ET-1 is a constitutively expressed peptide, while the gene that encodes for the inducible nitric oxide synthase (iNOS, an enzyme responsible for the synthesis of excessive and toxic amounts of NO) is solely activated after brain injury. We employed the Marmarou acceleration impact model of brain injury (400 g from 2 m) to study the effect of closed head trauma on the rat brain microcirculation. Following head trauma we analyzed changes of cerebral cortex perfusion using laser Doppler flowmetry and ultrastructural alterations of endothelial cells. We temporally correlated these changes with the expression of ET-1 (immunocytochemistry) and iNOS (in situ hybridization) to assess the role of these vasoactive agents in vascular contractility and cortical perfusion. Cortical perfusion was reduced by approximately 50% during the second hour as compared to values during preceding time points after TBI, reached a peak minutes before 3 h, and subsequently showed a trend towards normalization. A significant reduction in the lumen of microvessels and severe distortion of their shape were observed after the fourth hour post-trauma. At the same time period ET-1 expression in endothelial cells was stronger than in microvessels of control animals. ET-1 expression was further increased at 24 h after TBI. iNOS mRNA synthesis was strongly upregulated in the same cells at 4 h but was undetectable at 24 h post trauma. Our combined functional, cellular and molecular approach supports the notion that ET-1 and iNOS are expressed differentially in time within individual endothelial cells of cortical microvessels for the control of cortical blood flow following closed head trauma. This differential expression further indicates a reciprocal interaction in the synthesis of these two molecules which may underlie the control of microvascular autoregulation.

Brain ischemia and reperfusion: molecular mechanisms of neuronal injury.

Brain ischemia and reperfusion engage multiple independently-fatal terminal pathways involving loss of membrane integrity in partitioning ions, progressive proteolysis, and inability to check these processes because of loss of general translation competence and reduced survival signal-transduction. Ischemia results in rapid loss of high-energy phosphate compounds and generalized depolarization, which induces release of glutamate and, in selectively vulnerable neurons (SVNs), opening of both voltage-dependent and glutamate-regulated calcium channels. This allows a large increase in cytosolic Ca(2+) associated with activation of mu-calpain, calcineurin, and phospholipases with consequent proteolysis of calpain substrates (including spectrin and eIF4G), activation of NOS and potentially of Bad, and accumulation of free arachidonic acid, which can induce depletion of Ca(2+) from the ER lumen. A kinase that shuts off translation initiation by phosphorylating the alpha-subunit of eukaryotic initiation factor-2 (eIF2alpha) is activated either by adenosine degradation products or depletion of ER lumenal Ca(2+). Early during reperfusion, oxidative metabolism of arachidonate causes a burst of excess oxygen radicals, iron is released from storage proteins by superoxide-mediated reduction, and NO is generated. These events result in peroxynitrite generation, inappropriate protein nitrosylation, and lipid peroxidation, which ultrastructurally appears to principally damage the plasmalemma of SVNs. The initial recovery of ATP supports very rapid eIF2alpha phosphorylation that in SVNs is prolonged and associated with a major reduction in protein synthesis. High catecholamine levels induced by the ischemic episode itself and/or drug administration down-regulate insulin secretion and induce inhibition of growth-factor receptor tyrosine kinase activity, effects associated with down-regulation of survival signal-transduction through the Ras pathway. Caspase activation occurs during the early hours of reperfusion following mitochondrial release of caspase 9 and cytochrome c. The SVNs find themselves with substantial membrane damage, calpain-mediated proteolytic degradation of eIF4G and cytoskeletal proteins, altered translation initiation mechanisms that substantially reduce total protein synthesis and impose major alterations in message selection, down-regulated survival signal-transduction, and caspase activation. This picture argues powerfully that, for therapy of brain ischemia and reperfusion, the concept of single drug intervention (which has characterized the approaches of basic research, the pharmaceutical industry, and clinical trials) cannot be effective. Although rigorous study of multi-drug protocols is very demanding, effective therapy is likely to require (1) peptide growth factors for early activation of survival-signaling pathways and recovery of translation competence, (2) inhibition of lipid peroxidation, (3) inhibition of calpain, and (4) caspase inhibition. Examination of such protocols will require not only characterization of functional and histopathologic outcome, but also study of biochemical markers of the injury processes to establish the role of each drug.

Expression of the inducible nitric oxide synthase in distinct cellular types after traumatic brain injury: an in situ hybridization and immunocytochemical study.

The Marmarou's acceleration traumatic brain injury (TBI) model, in situ hybridization and immunocytochemistry were utilized to study the temporal expression of the inducible form of nitric oxide synthase (iNOS) mRNA and protein in different cellular compartments of the rat brain. Four hours following TBI, expression of iNOS was observed in the endothelial cells of cerebral blood vessels, macrophages and many cortical and hippocampal neurons. In the cortex labeled neuronal and nonneuronal cells were primarily found in the superficial layers. In the hippocampus the strongest neuronal labeling was observed in the CAI and CA3 (lateral part) regions. By 24 h post TBI endothelial cells no longer expressed iNOS mRNA, and the macrophage and neuronal iNOS expression was reduced by 30-50%. The reduction was assessed by automated quantitation of the silver grains that occupy individual cellular profiles using an image analysis system. Immunocytochemistry revealed de novo iNOS synthesis in non-neuronal cells at the different time points, thus paralleling the changes in iNOS mRNA expression. In contrast, iNOS immunoreactivity in neurons was not observed before 24 h post TBI, suggesting failure of iNOS protein translation at 4 h after trauma. The results demonstrate complex spatial and temporal patterns of iNOS expression in discrete cellular populations, indicating different times of nitric oxide synthesis (and release) following TBI. Uncoupling of iNOS mRNA and protein synthesis in neurons suggests differential synthesis of nitric oxide in these cells as compared to non-neuronal cellular populations after trauma.