Revisiting adult neurogenesis and the role of erythropoietin for neuronal and oligodendroglial differentiation in the hippocampus.

Recombinant human erythropoietin (EPO) improves cognitive performance in neuropsychiatric diseases ranging from schizophrenia and multiple sclerosis to major depression and bipolar disease. This consistent EPO effect on cognition is independent of its role in hematopoiesis. The cellular mechanisms of action in brain, however, have remained unclear. Here we studied healthy young mice and observed that 3-week EPO administration was associated with an increased number of pyramidal neurons and oligodendrocytes in the hippocampus of ~20%. Under constant cognitive challenge, neuron numbers remained elevated until >6 months of age. Surprisingly, this increase occurred in absence of altered cell proliferation or apoptosis. After feeding a 15N-leucine diet, we used nanoscopic secondary ion mass spectrometry, and found that in EPO-treated mice, an equivalent number of neurons was defined by elevated 15N-leucine incorporation. In EPO-treated NG2-Cre-ERT2 mice, we confirmed enhanced differentiation of preexisting oligodendrocyte precursors in the absence of elevated DNA synthesis. A corresponding analysis of the neuronal lineage awaits the identification of suitable neuronal markers. In cultured neurospheres, EPO reduced Sox9 and stimulated miR124, associated with advanced neuronal differentiation. We are discussing a resulting working model in which EPO drives the differentiation of non-dividing precursors in both (NG2+) oligodendroglial and neuronal lineages. As endogenous EPO expression is induced by brain injury, such a mechanism of adult neurogenesis may be relevant for central nervous system regeneration.

Neuropsychiatric disease relevance of circulating anti-NMDA receptor autoantibodies depends on blood-brain barrier integrity.

In 2007, a multifaceted syndrome, associated with anti-NMDA receptor autoantibodies (NMDAR-AB) of immunoglobulin-G isotype, has been described, which variably consists of psychosis, epilepsy, cognitive decline and extrapyramidal symptoms. Prevalence and significance of NMDAR-AB in complex neuropsychiatric disease versus health, however, have remained unclear. We tested sera of 2817 subjects (1325 healthy, 1081 schizophrenic, 263 Parkinson and 148 affective-disorder subjects) for presence of NMDAR-AB, conducted a genome-wide genetic association study, comparing AB carriers versus non-carriers, and assessed their influenza AB status. For mechanistic insight and documentation of AB functionality, in vivo experiments involving mice with deficient blood-brain barrier (ApoE(-/-)) and in vitro endocytosis assays in primary cortical neurons were performed. In 10.5% of subjects, NMDAR-AB (NR1 subunit) of any immunoglobulin isotype were detected, with no difference in seroprevalence, titer or in vitro functionality between patients and healthy controls. Administration of extracted human serum to mice influenced basal and MK-801-induced activity in the open field only in ApoE(-/-) mice injected with NMDAR-AB-positive serum but not in respective controls. Seropositive schizophrenic patients with a history of neurotrauma or birth complications, indicating an at least temporarily compromised blood-brain barrier, had more neurological abnormalities than seronegative patients with comparable history. A common genetic variant (rs524991, P=6.15E-08) as well as past influenza A (P=0.024) or B (P=0.006) infection were identified as predisposing factors for NMDAR-AB seropositivity. The >10% overall seroprevalence of NMDAR-AB of both healthy individuals and patients is unexpectedly high. Clinical significance, however, apparently depends on association with past or present perturbations of blood-brain barrier function.

Essential role for Stat5 in the neurotrophic but not in the neuroprotective effect of erythropoietin.

The transcription factors signal transducer and activator of transcription 5a and 5b (Stat5) are activated by the neuroprotective and neurotrophic cytokines, erythropoietin (EPO) and growth hormone (GH). Here, we show a dissociation of the intracellular pathway mediating the protective effect of EPO against glutamate toxicity from that needed for its neurotrophic activity using hippocampal neuronal cultures from Stat5a/b-knockout (Stat5(-/-)) mouse fetuses. Both pretreatment and post-treatment with EPO counteracted glutamate-induced cell death in Stat5(+/+) and Stat5(-/-) neurons. Acute pharmacological inhibition of Janus kinase 2 (JAK2)/Stat signalling had no effect on EPO neuroprotection, whereas inhibition of phosphatidylinositol-3' kinase (PI3K)/Akt pathway abolished the protective effect of EPO in both Stat5(+/+) and Stat5(-/-) neurons. GH effectively protected Stat5(+/+) cells against glutamate toxicity but had no effect in Stat5(-/-) neurons or in Stat5(+/+) neurons treated with JAK2/Stat or PI3K inhibitor. EPO and GH stimulated neurite outgrowth and branching of Stat5(+/+) neurons by activating PI3K/Akt signalling but had no trophic effect in Stat5(-/-) cells. We conclude that in hippocampal neurons, Stat5 is not required for neuroprotection by EPO but is together with Akt essential for its neurotrophic activity. Both Stat5 and Akt are needed for neuroprotective and neurotrophic signalling of GH in neurons.

Chronophin is a glial tumor modifier involved in the regulation of glioblastoma growth and invasiveness.

Glioblastoma is the most aggressive primary brain tumor in adults. Although the rapid recurrence of glioblastomas after treatment is a major clinical challenge, the relationships between tumor growth and intracerebral spread remain poorly understood. We have identified the cofilin phosphatase chronophin (gene name: pyridoxal phosphatase, PDXP) as a glial tumor modifier. Monoallelic PDXP loss was frequent in four independent human astrocytic tumor cohorts and increased with tumor grade. We found that aberrant PDXP promoter methylation can be a mechanism leading to further chronophin downregulation in glioblastomas, which correlated with shorter glioblastoma patient survival. Moreover, we observed an inverse association between chronophin protein expression and cofilin phosphorylation levels in glioma tissue samples. Chronophin-deficient glioblastoma cells showed elevated cofilin phosphorylation, an increase in polymerized actin, a higher directionality of cell migration, and elevated in vitro invasiveness. Tumor growth of chronophin-depleted glioblastoma cells xenografted into the immunodeficient mouse brain was strongly impaired. Our study suggests a mechanism whereby the genetic and epigenetic alterations of PDXP resulting in altered chronophin expression may regulate the interplay between glioma cell proliferation and invasion.

Impaired sodium excretion, decreased glomerular filtration rate and elevated blood pressure in endothelin receptor type B deficient rats.

The renal endothelin (ET) system, particularly the ET type B receptor, has been implicated in the regulation of sodium excretion and glomerular filtration rate (GFR). We analyzed kidney morphology and function in a rat strain characterized by complete absence of a functional ETB receptor. Due to Hirschsprung's disease limiting lifetime in these rats, studies were performed in 23-day-old rats. Kidney size and morphology (glomerular and interstitial matrix content, glomerular size and cell density and intrarenal vascular morphology) were normal in ETB-deficient rats. There were also no evidence of altered kidney cell cycle regulation in these rats. GFR was significantly lower, by 72% (P<0.001), in homozygous ETB-deficient rats than in wild-type rats. Fractional sodium excretion was likewise markedly reduced by 84% in homozygous ETB-deficient rats (P<0.001 versus wild-type rats). Treatment with the specific epithelial sodium channel blocker amiloride led to a much higher increase in fractional sodium excretion in ETB-deficient rats (934.2+/-73% in ETB-deficient rats versus 297+/-20% in wild-type rats, expressed as percentage of corresponding placebo treated control; P<0.001). Mean arterial blood pressure was elevated by 7.9 mmHg in homozygous ETB-deficient rats (P<0.05 versus wild-type rats). Our study demonstrates that ETB-deficiency causes early onset kidney dysfunction characterized by a markedly reduced sodium excretion, decreased GFR, and slightly elevated blood pressure. The complete absence of the ETB receptor causes in the kidney--in contrast to the colon--a functional rather than a developmental, neural crest cell dependent disease, since kidney morphology was normal in ETB-deficient rats. The much higher increase in the fractional sodium excretion in ETB-deficient rats after pharmacological blockade of the epithelial sodium channel indicates that the decreased fractional sodium excretion in ETB-deficient rats is most probably due to a lack of the inhibitory property of the ETB receptor on the epithelial sodium channel activity.

The opioid peptides. A role in hypertension?

This review is an attempt to highlight evidence that may implicate the endogenous opioid system in the pathogenesis of hypertension in humans. The evidence raised includes biochemical, physiological, pharmacological, and behavioral studies conducted in in vitro and in vivo systems, experimental models of hypertension, and humans with essential hypertension. While the compelling biochemical and pharmacological evidence in experimental animals clearly shows the presence of opioid peptides and their receptors in strategic sites of cardiovascular control and potent cardiovascular response to opioid peptides, opioid antagonists show no consistent blockade or reversal of hypertension in experimental animals or humans. One possible explanation for this phenomenon could be the vast redundancy in systems regulating blood pressure (i.e., the blockade of one system still leaves many other systems fully able to rapidly offset the eliminated system). Regarding the opioid system, the situation is much more complex, since some opioid receptors (mu-type) mediate pressor responses, while other receptors (kappa-type) mediate depressor responses. Therefore, nonselective opioid receptor antagonists (e.g., naloxone), which block both types of receptors, can be devoid of any cardiovascular activity, while a selective mu-receptor antagonist or a selective and potent kappa-receptor agonist may produce the desired antihypertensive effect. A combination of both actions (i.e., a drug that is both a mu-antagonist and a kappa-agonist) might be even more advantageous. Until such compounds are developed, this hypothesis will be hard to prove.

Global forebrain ischemia results in differential cellular expression of interleukin-1beta (IL-1beta) and its receptor at mRNA and protein level.

The mRNA expression of the proinflammatory cytokine interleukin-1beta (IL-1beta) has been shown to be induced in neural elements during ischemia. It is not clear which cells generate the IL-1beta mRNA and eventually synthesize IL-1 protein and which cells respond to this signaling by producing IL-1 receptors during ischemia. To clarify this question, rats were subjected to global ischemia by bilateral carotid occlusion and hypotension for 20 minutes, followed by reperfusion for 2 hours (n = 7), 8 hours (n = 7), or 24 hours (n = 7). Cryostat sections were hybridized using antisense oligonucleotide probes (30 dimer). Multiple cell markers were used in immunohistochemical staining to identify the cells expressing IL-1beta and IL-1R protein. The sham animals (n = 5) showed no or only a weak expression of IL-1R or IL-1beta mRNA. The number of IL-1beta mRNA-expressing cells was significantly increased by 2 hours of reperfusion in several brain areas including cortex (12-fold compared with sham) and caudate-putamen (14-fold), and was maximally increased in most hippocampal regions by 8 hours of reperfusion (mean +/- SD of positive cells/field versus sham equivalent being 37.9 +/- 12.3 versus 4.0 +/- 3.3; 30.6 +/- 9.0 versus 3.1 +/- 2.3; 41.3 +/- 17.5 versus 2.9 +/- 1.9; in CA1; CA2; CA3/CA4 regions of the hippocampus, respectively). IL-1beta mRNA signal was also intensified in the white matter areas. Changes in IL-1R mRNA were seen in the hippocampus (after 2 hours CA1: 16-fold; CA2: 17-fold; DG: 24-fold increase; and CA3/CA4: 10-fold increase after 8 hours), and the expression was prolonged especially in CA1 and CA2 regions up to 24 hours of reperfusion. The major cellular source of IL-1beta protein was glia (astrocytes, oligodendrocytes, microglia, and scattered perivascular macrophages/monocytes), while neurons and sporadic microvascular endothelia showed IL-1R immunoreactivity. The data suggest that neurons in discrete areas vulnerable for selective neuronal death, and possibly the vascular endothelium, are target cells for ischemia-induced glial IL-1beta production.

Differential expression of HSC73 and HSP72 mRNA and proteins between young and adult gerbils after transient cerebral ischemia: relation to neuronal vulnerability.

This study presents a quantitative comparison of the time courses and regional distribution of both constitutive HSC73 and inducible HSP72 mRNA expression and their respective encoded proteins between young (3-week-old) and adult (3-month-old) gerbil hippocampus after transient global ischemia. The constitutive expression of HSC73 mRNA and protein in the hippocampus of the young sham-operated gerbils was significantly higher than in the adults. The HSC73 mRNA expression after ischemia in the CA1 layer of young gerbils was greater than in adult gerbils. HSC73 immunoreactivity was not significantly changed after ischemia-reperfusion in adult hippocampus, whereas it decreased in young gerbils. Ischemia-reperfusion led to induction of HSP72 mRNA expression throughout the hippocampus of both young and adult gerbils. HSP72 mRNA induction was more intense and sustained in the CA1 subfield of young gerbils; this was associated with a marked induction of HSP72 proteins and neuronal survival. The transient expression of HSP72 mRNA in the CA1 layer of adult gerbils was not associated with a subsequent synthesis of HSP72 protein but was linked to neuronal loss. Expression of HSP72 mRNA was shifted to an earlier period of reflow in CA3 and dentate gyrus (DG) subfields of young animals. These findings suggest that the induction of both HSP72 mRNA and proteins in the CA1 pyramidal neurons of young gerbils, as well as the higher constitutive expression of HSC73, may partially contribute to higher neuronal resistance of young animals to transient cerebral ischemia.

Heat-shock protein and C-fos expression in focal microvascular brain damage.

Cortical brain damage was produced in rats by a focal pulse from a Nd-YAG laser, and evolution of the lesion was evaluated at 30 min, and 2, 8, and 24 h with respect to microvascular perfusion, blood-brain barrier (BBB) permeability, and expression of both the heat-shock/stress protein, hsp72, and the c-fos proto-oncogene transcription factor. A double-labeling fluorescence technique employing intravenously injected Evans blue albumin (EBA) and fluorescein-labeled dextran was used to map and measure BBB damage and microvascular perfusion in fresh frozen brain sections. Hsp72 and c-fos mRNAs were localized by in situ hybridization, and the respective proteins were identified by immunocytochemistry. Parallel sections were stained for glial fibrillary acidic protein and for routine histologic examination. Striking hsp72 mRNA expression was evident by 2 h in an approximately 300 microns wide rim surrounding an area of expanding BBB damage. Increased hsp72 mRNA was observed only in regions of preserved microcirculation, where the hsp72 protein was subsequently localized exclusively in the vasculature at 24 h after the insult. Hsp72-positive endothelial cells spanned the narrow margin between the lesion and histologically normal, glial fibrillary acidic protein (GFAP)-positive cortical tissue. There was no hsp72 expression in the area of subcortically migrating edema fluid. Inductions of c-fos mRNA and Fos protein were not strikingly evident around the focal brain lesion, but were observed transiently throughout the injured hemisphere at 30 min and 2.5 h, respectively, indicating that spreading depression was triggered by the focal injury. These results are in striking contrast to those previously obtained from studies of models of focal ischemic or traumatic brain injury, which are characterized by a complex pattern of glial and neuronal hsp72 expression in the periphery of an infarct, and which suggest that the tightly demarcated lesion produced by the Nd-YAG laser lacks these components of graded injury that are evident following other types of focal brain damage.

Quantitation of perivascular monocytes and macrophages around cerebral blood vessels of hypertensive and aged rats.

The numbers of monocytes and macrophages in the walls of cerebral blood vessels were counted on perfusion-fixed frozen brain sections (16 microns) of spontaneously hypertensive rats (SHR), stroke-prone SHR (SHR-SP), normotensive Wistar-Kyoto (WKY) rats, and young (16-week-old) and old (2-year-old) normotensive Sprague-Dawley rats (SD-16w and SD-2y, respectively) using monoclonal antibodies against rat macrophages (ED2). The staining was visualized with fluorescein-labeled second antibodies. The ED2-specific staining in brain sections was restricted to macrophages in a perivascular location. The number of perivascular cells per square millimeter of high-power field was significantly greater in SHR-SP (8.6 +/- 2.1; n = 4) and SHR (6.7 +/- 0.9; n = 6) than in normotensive WKY (4.0 +/- 0.5; n = 6; p < 0.01). The number of perivascular macrophages was also greater in SD-2y (7.5 +/- 2.7; n = 9) than in SD-16w (2.9 +/- 1.8; n = 8; p < 0.01). No ED2 staining was found in the resident microglia or in the endothelial cells, which were identified by double staining with rhodamine-labeled anti-factor VIII-related antigen antibodies. The results suggest that the stroke risk factors hypertension and advanced age are associated with increased subendothelial accumulation of monocytes and macrophages. This accumulation could increase the tendency for the endothelium to convert from an anticoagulant to a procoagulant surface in response to mediators released from these subendothelial cells.

Delayed loss of ETB receptor-mediated vasorelaxation after cold lesion of the rat parietal cortex.

The aim of this study was to investigate the involvement of endothelins (ET) in brain injury. The effect of ET was studied in the isolated basilar artery (BA) taken from control, sham-operated, and cold-lesioned rats. Cold lesion was induced by application of a precooled (-78 degrees C) copper cylinder (outer diameter 5 mm) for 60 seconds to the intact dura over the parietal cortex. After precontraction with prostaglandin (PG) F2alpha, ET-3 (10(-10) to 10(-8) mol/L) dilated BA with a pD2 (negative log of the half-maximal concentration) of 9.06+/-0.031 (mean +/- SD) and a maximal effect (Emax) of 1.64+/-1.0 mN at 3 x 10(-9) mol/L in sham-operated animals. This dilation was reduced 24 and 48 hours after cold lesion by 33% and 73%, respectively, at 3 x 10(-9) mol/L. The effects of acetylcholine (10(-8) to 10(-4) mol/L) and sodium nitroprusside (10(-3) mol/L) were unaltered. Activation of the ETB receptor in thoracic aorta by the specific agonist IRL 1620 also resulted in a reduced dilation (51% by 48 hours after cold lesion). Reverse transcriptase-polymerase chain reaction of the BA showed unaltered expression of mRNA for the ETB receptor after cold lesion whereas ETB immunoreactivity in BA and in its intraparenchymal arteries was reduced at 24 and 48 hours. In contrast to the reduction of ET-3-induced dilation, the constrictor effects of ET-1 and ET-3 were retained after cold lesion. Endothelin-1 (10(-12) to 10(-6) mol/L) dose-dependently contracted segments of untreated control BA segments under resting conditions with a pD2 of 8.03+/-0.22 and an Emax of 6.35+/-0.70 mN. Further evidence that the constrictor ability of BA was not influenced by cold lesion is given by the unaltered response to 124 mmol/L K+ and 10(-6) mol/L serotonin. We conclude that the ETB receptor of BA after cold lesion is downregulated specifically, apparently at the posttranscriptional level. Because the ETB-mediated dilation in thoracic aorta was also reduced, downregulation of the ETB receptor apparently is not restricted to cerebral arteries. The nitric oxide-cyclic guanosine monophosphate system in BA is, however, intact.

Mesenteric vascular responses to i.v. administration of lipoxin A4 and lipoxin B4 in the conscious rat.

Lipoxin A4 and lipoxin B4 are newly discovered lipoxygenase-interacting products of leukocytes which might have a role in cardiovascular events associated with anaphylaxis. We have tested this possibility by systemic administration of both LXA4 and LXB4 to the conscious rat while monitoring systemic and regional hemodynamic changes. LXA4 and LXB4 (1-100 micrograms/kg) produced dose-dependent constriction of the mesenteric vessels, up to +123 +/- 23% and +50 +/- 9% for LXA4/B4, respectively. Dose-related changes were not observed in arterial blood pressure, heart rate, renal (LXB4) and hindquarter blood flow. We suggest that LXA4 and LXB4 might affect selective vascular beds, such as the mesenteric vessels, and contribute to variations in blood flow in specific pathophysiological states.

Hypothalamic opioid mu-receptors regulate discrete hemodynamic functions in the conscious rat.

The effect of the selective mu-opioid agonist D-Ala2-Me-Phe4-Gly-ol5-enkephalin (DAGO), injected into the medial preoptic nucleus of hypothalamus, on cardiac output and regional blood flow was studied in the conscious rat and the effect of DAGO on renal sympathetic nerve activity and renal blood flow was studied in anesthetized rats. In conscious rats, injections of DAGO (1 or 10 nmol) into the preoptic nucleus increased the blood pressure in a dose-related manner. The maximum rises of mean arterial pressure and pulse pressure after the larger dose were +23 +/- 5 mmHg (mean +/- SEM, P less than 0.01) and +17 +/- 3 mmHg (P less than 0.01), respectively. A small dose (0.1 nmol) increased heart rate (+47 +/- 13 bpm, P less than 0.05); the 1 nmol dose produced bradycardia (-39 +/- 11 bpm, P less than 0.05), while the 10 nmol dose initially decreased heart rate (-68 +/- 15 bpm (P less than 0.01) and then gradually increased heart rate to a maximum of +74 +/- 13 bpm, (P less than 0.01). A long-lasting increase in cardiac output was also elicited by DAGO, with maximum changes after 1 and 10 nmol of +14 +/- 6% and +22 +/- 7% (P less than 0.01), respectively. Blood flow in the hindquarters increased after DAGO but the mesenteric and renal blood flow decreased in a dose-related manner. Significant responses in hindquarter and mesenteric blood flow after DAGO were independent of systemic hemodynamic responses at the dose of 0.1 nmol. The vascular resistance in the hindquarters significantly decreased after a small dose of DAGO while the larger doses dose-dependently increased mesenteric and renal vascular resistance. A crucial role of the sympathetic nervous system in the hemodynamic effects of DAGO was demonstrated: (1) by the profound activation of renal sympathetic nerve activity after injections of DAGO (1 nmol/100 nl) into the preoptic nucleus, (2) by blockade of the pressor, tachycardic and regional hemodynamic effects of DAGO (1 nmol) by the ganglion blocker chlorisondamine (5 mg/kg i.v.). The results suggest that the pressor effect of DAGO in preoptic nucleus is due primarily to an increase in cardiac output. The differential changes in blood flow in organs further suggest that the opioid mu-receptors in the preoptic nucleus might be involved in the integration of peripheral blood flow in the hypothalamus during affective behavior.

Respiratory mu-opioid and benzodiazepine interactions in the unrestrained rat.

Interactions of mu-opioid receptors with the benzodiazepine system were studied by examining the modulatory effects of flumazenil (a benzodiazepine antagonist) and alprazolam (a benzodiazepine agonist) on the respiratory effects of the opioid peptide dermorphin. Dermorphin, 1-30 nmol administered i.c.v., to conscious, unrestrained rats decreased ventilation rate (VR) and minute volume (MV) dose-dependently. The ventilatory depression was antagonized by naloxone and by the benzodiazepine antagonist flumazenil. The benzodiazepine alprazolam potentiated the respiratory inhibition of a small (1 nmol) dose of dermorphin but antagonized that of a higher dose (3 nmol). The results suggest that the benzodiazepine/GABA receptor complex modulates respiratory depression induced by central mu-receptor stimulation in the rat.

Evidence for differential opioid mu 1- and mu 2-receptor-mediated regulation of heart rate in the conscious rat.

The possibility that mu-opioid-induced tachycardia and bradycardia could be mediated by different subtypes of the mu-receptor was studied in conscious Sprague-Dawley rats. The selective mu-receptor agonist dermorphin and its analog, TAPS (Tyr-D-Arg-Phe-sarcosine), a putative mu 1-receptor agonist, were given centrally. Tyr-D-Arg-Phe-sarcosine increased the heart rate, the response being inversely correlated to the dose (an increase of 71 +/- 22, 49 +/- 14 and 30 +/- 17 beats/min at doses of 0.3, 3 and 30 pmol, respectively). Dermorphin induced less clear changes in heart rate (maximum increase of 39 +/- 14 beats/min at the dose of 1 pmol). After treatment with the mu 1-selective antagonist naloxonazine (NAZ), TAPS 30 pmol and dermorphin 1 pmol decreased heart rate by -22 +/- 10 and -24 +/- 7 bpm, respectively. The bradycardiac effect of larger doses of dermorphin was potentiated by NAZ (from -25 +/- 8 to -97 +/- 22 bpm) but abolished by the non-selective antagonist naloxone. These data suggest that the high affinity mu 1-opioid receptors mediate tachycardic responses and mu 2-receptors mediate bradycardic responses.

Endothelin B receptor deficient transgenic rescue rats: a rescue phenomenon in the brain.

Homozygous endothelin B receptor deficiency leads to congenital aganglionosis of the gut in rats and mice, equivalent to human Hirschsprung disease. Homozygous endothelin B receptor deficient rats (spotting lethal rats, sl/sl) are characterized not only by this developmental disorder of the enteric nervous system, which limits their life span to 3-4 weeks, but exhibit an increased rate of apoptosis in the dentate gyrus compared to wildtype (+/+) rats. Recently, endothelin B receptor deficient transgenic rescue rats (sl/sl, tg/tg) were created to further investigate the role of the endothelin B receptor in mature animals. Linkage of the human dopamine-beta-hydroxylase promoter to the rat endothelin B receptor gene and expression of this transgenic construct results in normal development of the enteric nervous system. We investigated the expression pattern of this transgenic construct in the brain by using reverse transcriptase polymerase chain reaction. Unexpectedly, transgene mRNA expression was not restricted to the brain stem where adrenergic and noradrenergic nuclei are known to be present but, in addition, was also detectable in hippocampus and cortex. Using in situ tailing technique, cleaved caspase-3 immunohistochemistry and analysis of hematoxylin-eosin-stained serial sections, we found that all studied transgenic animals were rescued from the increased rate of apoptosis in the dentate gyrus characteristic for non-transgenic sl/sl rats. This finding supports our previous observation that the endothelin B receptor might be an important regulatory element supporting cellular survival in the hippocampus during postnatal development. The endothelin B receptor deficient transgenic rescue rats used here are rescued from developmental disorders both in the gut and in the brain.

Endothelin B receptor-deficient rats as a subtraction model to study the cerebral endothelin system.

Endothelins, due to their potent vasoactivity and mitogenicity, appear to play an important role in the brain, where all components of the endothelin system, peptides, receptors and converting enzyme, are expressed. To further elucidate the role of the cerebral endothelin system, astrocytes and cerebral vessels from sl/sl rats, devoid of functional endothelin B receptors, have been employed. Astrocytes from sl/sl rats display the following abnormalities as compared to wild-type (+/+) cells: (i) elevated basal extracellular endothelin-1 levels; (ii) exclusive presence of functional endothelin A receptors; (iii) increased extracellular endothelin-1 levels upon endothelin A receptor blockade; (iv) augmented basal endothelin-converting enzyme activity; (v) altered calcium response to endothelin-1. The basilar artery of sl/sl rats shows an enhanced constricting response to endothelin-1 and fails to dilate in response to endothelin-3, shifting the endothelin vasomotor balance to constriction. In conclusion, endothelin B receptors may be essential for restricting extracellular endothelin-1 levels in the brain, as well as for a balanced cerebral vasomotor action of endothelins.

Upregulation of intercellular adhesion molecule 1 (ICAM-1) on brain microvascular endothelial cells in rat ischemic cortex.

The expression of intercellular adhesion molecule 1 (ICAM-1) was studied in rat focal ischemic cortex. A significant increase in ICAM-1 mRNA expression in the ischemic cortex over levels in contralateral (nonischemic) site was observed by means of Northern blot analysis following either permanent or temporary occlusion with reperfusion of the middle cerebral artery (PMCAO or MCAO with reperfusion) in spontaneously hypertensive rats. In the ischemic cortex, levels of ICAM-1 mRNA increased significantly at 3 h (2.6-fold, n = 3, P < 0.05), peaked at 6 to 12 h (6.0-fold, P < 0.01) and remained elevated up to 5 days (2.5-fold, P < 0.05) after PMCAO. The profile of ICAM-1 mRNA expression in the ischemic cortex following MCAO with reperfusion was similar to that following PMCAO, except that ICAM-1 mRNA was significantly increased as early as 1 h (6.3-fold, n = 3, P < 0.05) and then gradually reached a peak at 12 h (12-fold, P < 0.01) after reperfusion. ICAM-1 mRNA expression in ischemic cortex following PMCAO was significantly greater in hypertensive rats than in two normotensive rat strains. Immunostaining using anti-ICAM-1 antibodies indicated that upregulated ICAM-1 expression was localized to endothelial cells of intraparenchymal blood vessels in the ischemic but not contralateral cortex. The data suggest that an upregulation of ICAM-1 mRNA and protein on brain capillary endothelium may play an important role in leukocyte migration into ischemic brain tissue.

Temporal profile of expression and cellular localization of inducible nitric oxide synthase, interleukin-1beta and interleukin converting enzyme after cryogenic lesion of the rat parietal cortex.

We used in situ hybridization, RT PCR and immunohistochemistry to study the time course of expression and the cellular localization of inducible nitric oxide synthase (iNOS) and interleukin-1beta (IL-1beta) during the first 7 days after induction of a standardized cryogenic lesion on the right parietal cortex in male rats. Cryogenic lesion induced iNOS mRNA in the lesioned hemisphere after 6 to 72 h with a maximum (15+/-2 cells/mm2, n=4, p<0.01 vs. sham) at 24 h. Microglia, invading monocytes and granulocytes in and around the lesion expressed iNOS immunoreactivity starting at 12 h and peaking (29+/-10 cells/mm2, n=4, p<0.05 vs. sham) at 24 h after lesion. Induction of IL-1beta mRNA expression was immediate with a peak (9+/-1 cells/mm2, n=4, p<0.01 vs. sham) at 24 h after cryogenic lesion. The number of round cells with IL-1beta immunoreactivity around the lesion was maximal (8+/-2 cells/0.1 mm2, n=3, p<0.01 vs. sham) at 24 h. A weak astrocytic expression of IL-1beta-immunoreactivity was seen in sham animal brains. Astrocytic IL-1beta-expression was significantly increased in the lesion hemisphere and both hippocampi. Interleukin converting enzyme (ICE) was expressed in astrocytes and microglia around the lesion 6 h after injury. The number of ICE immunoreactive cells (8+/-2 cells/0. 1 mm2, n=3, p<0.05 vs. sham) peaked at 72 h after lesion. Neuronal expression of ICE and IL-1beta was seen in the lesion periphery 72 h and 7 days after injury. At this time, morphological features of apoptosis were evident in cells in the lesion periphery. The data indicate an early activation of microglia and monocyte invasion into the lesion hemisphere leading to multicellular expression of iNOS, ICE, and IL-1beta. These events may contribute to the expansion of neuronal damage after brain injury.

The supraoptic nuclei in vasopressin and hemodynamic responses to hemorrhage in rats.

Cardiovascular and vasopressin (AVP) responses to hemorrhage were studied in rats with lesions of the hypothalamic supraoptic nuclei (SONL). Bleeding caused hypotension and increase in heart rate (HR) and AVP. SONL rats failed to fully recover from bleeding as compared to normal rats. Plasma AVP in SONL rats was in the normal in basal conditions, but failed to increase to levels attained in normal rats throughout the post-hemorrhage period. These data suggest that the supraoptic nuclei are the primary regulatory sites for AVP release in response to hemorrhage and that lack of adequate AVP release significantly retards blood pressure recovery after bleeding.