[Show abstract][Hide abstract] ABSTRACT: A homozygous mutation of the endothelin B receptor (EDNRB) gene in spotting lethal (sl/sl) rats leads to Hirschsprung's disease (HSCR) with long segmented aganglionosis. However, the effects on the development of the enteric nervous system (ENS) promoted by a heterozygous mutation of the EDNRB gene are not known. The present study aimed to describe and morphometrically assess the phenotypic abnormalities of the ENS in heterozygous (+/sl) EDNRB deficient rats in comparison with homozygous (sl/sl) EDNRB deficient and wild-type (+/+) rats.
The distal small intestine, caecum, and colon were obtained from sl/sl, +/sl, and +/+ rats. To demonstrate the three dimensional organisation of the ENS, the intestinal wall was microdissected into wholemounts and incubated against the pan-neuronal marker protein gene product 9.5. Assessment of the ENS included morphometric quantification of ganglionic size and density, the number of nerve cells per ganglia, and the diameter of nerve fibre strands within both the myenteric and submucous plexus.
Sl/sl rats were characterised by complete aganglionosis resembling the same histopathological features observed in patients with HSCR. +/sl rats revealed more subtle abnormalities of the ENS: the submucous plexus was characterised by a significantly increased ganglionic size and density, and the presence of hypertrophied nerve fibre strands. Morphometric evaluation of the myenteric plexus did not show statistically significant differences between +/sl and +/+ rats.
In contrast with sl/sl rats, +/sl rats display non-aganglionated malformations of the ENS. Interestingly, these innervational abnormalities resemble the histopathological criteria for intestinal neuronal dysplasia (IND). Although IND has been described in several intestinal motility disorders, the concept of a clearly defined clinical-histopathological entity is still controversially discussed. The present findings support the concept of IND based on clearly defined morphological criteria suggesting a genetic link, and thus may provide a model for human IND. Furthermore, the data underline the critical role of the "gene dose" for the phenotypic effects promoted by the EDNRB/EDN3 system and confirm that the development of the ENS is not an "all or none" phenomenon.
[Show abstract][Hide abstract] ABSTRACT: Erythropoietin (EPO) and its receptor play a major role in embryonic brain, are weakly expressed in normal postnatal/adult brain and up-regulated upon metabolic stress. EPO protects neurons from hypoxic/ ischemic injury. The objective of this trial is to study the safety and efficacy of recombinant human EPO (rhEPO) for treatment of ischemic stroke in man.
The trial consisted of a safety part and an efficacy part. In the safety study, 13 patients received rhEPO intravenously (3.3 X 10(4) IU/50 ml/30 min) once daily for the first 3 days after stroke. In the double-blind randomized proof-of-concept trial, 40 patients received either rhEPO or saline. Inclusion criteria were age <80 years, ischemic stroke within the middle cerebral artery territory confirmed by diffusion-weighted MRI, symptom onset <8 hr before drug administration, and deficits on stroke scales. The study endpoints were functional outcome at day 30 (Barthel Index, modified Rankin scale), NIH and Scandinavian stroke scales, evolution of infarct size (sequential MRI evaluation using diffusion-weighted [DWI] and fluid-attenuated inversion recovery sequences [FLAIR]) and the damage marker S100ss.
No safety concerns were identified. Cerebrospinal fluid EPO increased to 60-100 times that of nontreated patients, proving that intravenously administered rhEPO reaches the brain. In the efficacy trial, patients received rhEPO within 5 hr of onset of symptoms (median, range 2:40-7:55). Admission neurologic scores and serum S100beta concentrations were strong predictors ofoutcome. Analysis of covariance controlled for these two variables indicated that rhEPO treatment was associated with an improvement in follow-up and outcome scales. A strong trend for reduction in infarct size in rhEPO patients as compared to controls was observed by MRI.
Intravenous high-dose rhEPO is well tolerated in acute ischemic stroke and associated with an improvement in clinical outcome at 1 month. A larger scale clinical trial is warranted.
Molecular Medicine 08/2002; 8(8):495-505. · 4.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The role of functional endothelin-B (ETB)-receptors on neuronal survival upon hypoxia-ischemia (HI) has been investigated in 14-day-old ETB-receptor-deficient spotting lethal (sl/sl) and wildtype (+/+) rats. Carotid ligation followed by exposure to 8% oxygen for 2 h produced distinct cortical and hippocampal neuronal damage. Damage severity 24 h after HI was mild to intermediate in +/+ rats whereas large cortical infarcts and profound apoptosis of the hippocampus evolved in sl/sl rats. The number of apoptotic cells in the dentate 24 h after HI amounted to 30 +/- 7 cells/0.1 mm(2) in sl/sl compared to 9 +/- 3 cells/0.1 mm(2) in wildtype rats (mean +/- S.E.M., n=10-11, P=0.0093). In-vitro hypoxia (15 h) resulted in a comparable increase in cell death in primary pure neuronal hippocampal cultures from both groups (49.8 +/- 1.6% in sl/sl, 51.4 +/- 0.9% in +/+, mean +/- S.E.M., n=5, P=0.0560). To conclude, absence of functional ETB receptors is associated with an increased susceptibility to HI in-vivo, which is not intrinsic to neurons. Antagonism of ETB receptors seems not to be desirable in ischemic stroke.
Brain Research 08/2002; 945(1):144-9. DOI:10.1016/S0006-8993(02)02911-6 · 2.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Molecular Medicine 02/2001; 78(11):633-41. DOI:10.1007/s001090000158 · 5.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Spotting lethal (sl) rats, a model for Hirschsprung's disease, recently have been found to carry a deletion in the endothelin B (ET(B)) gene, causing functional lack of ET(B) receptors. The ET(B) receptor mediates, together with and in counterbalance to the ET(A) receptor, endothelin actions on vessels, cell proliferation, and migration. The authors investigated the effect of homozygosity (sI/sI) or heterozygosity (+/sl) on phenotype, intestinal morphology, and survival.
Weight, circumference, and serum albumin were measured. Histological tests of major organs and immunoperoxidase reaction for Peripherin, glial fibrillary acid protein (GFAP), and S-100 in small and large intestine were performed. Peripherin-immunostained sections of colon and jejunum were analyzed morphometrically. Screening for sepsis included search for enterocolitis, bacterial infection, endotoxin, and iNOS mRNA.
Sl/sl rats died within 4 weeks of life, showing an early and a later death group. Serum albumin levels were decreased in sl/sl rats, whereas signs of sepsis were rare. Immunostaining uncovered alterations in nerve and glial cells in the whole gut of sl/sl rats, and to a subtle degree also in +/sl rats, which appear clinically normal. Morphometric quantification yielded statistically significant alterations in sl/sl rats only. No obvious abnormalities were found in other organs.
Sl/sl rats die from malnutrition rather than sepsis, too early for ischemic complications to occur. Rats of the later death group are a suitable model for studying the ET8 receptor in vivo. Subtle abnormalities in the enteric nervous system of heterozygous rats underline the critical role of the "gene dose" for functional compensation.
Journal of Pediatric Surgery 04/2000; 35(3):480-8. DOI:10.1016/S0022-3468(00)90218-5 · 1.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The dentate gyrus retains neuronal proliferative potential throughout life. Using immature endothelin B receptor-deficient (sl/sl) rats, a rabbit model of pneumococcal meningitis and autopsy brains from humans who died from pneumococcal meningitis, we explored the role of endothelin B receptors in physiological and pathological neuronal apoptosis in the dentate gyrus. At postnatal days 3-4, the rate of apoptosis in the dentate gyrus was high in all rats, declining to low levels in wild-type rats (+/+) on days 14 and 22, but remaining high in both homozygous (sl/sl) and heterozygous (sl/+) endothelin B receptor-deficient rats. Increased apoptosis was not significantly compensated for by neuronal proliferation. Hippocampal neuronal cultures also exhibited genotype-dependent apoptosis with the highest rate in neurons from homozygous endothelin B receptor-deficient (sl/sl) rats. In rabbit and human pneumococcal meningitis, increased apoptosis in the dentate gyrus was associated with loss of neuronal endothelin B receptor immunoreactivity. In conclusion, endothelin B receptors appear to act as neuronal survival factors in the dentate gyrus in rodents and man, both during postnatal development and under pathological conditions.
[Show abstract][Hide abstract] ABSTRACT: 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.