Colostomy Delays Cell Loss in the Brain and Improves Juvenile Survival in a Neonatal Rat Model of Hirschsprung's Disease.

Hirschsprung's disease is a congenital malformation characterized by the absence of enteric ganglia in the distal intestine and gut obstruction. Our previous study indicates the brain pathology during the disease progression. A subpopulation of Hirschsprung's disease patients is also associated with anomalies of the central nervous system. In the investigation, we studied a rat model of Hirschsprung's disease, known as spotting lethal (sl/sl) ETB-/- rats, which carries a spontaneous deletion in endothelin receptor B (human gene name: EDNRB) and manifests a similar phenotype as humans with Hirschsprung's disease. Homozygous mutant sl/sl rats were successfully rescued from premature death by performing colostomy and dramatically survived to their juvenile age. By the body weight measured, their body growth was not revealed to be significantly different between ETB-/- and wildtype ETB+/+ or heterozygous (+/sl) ETB+/- groups while all underwent the same colostomy. Cell loss was investigated in several brain regions by using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay (TUNEL) in ETB+/+, ETB-/-, and ETB+/- rats. Number of TUNEL-positive cells in the cerebellum and the hippocampus of ETB-/- rats was significantly increased compared with that of the ETB+/+ and ETB+/- rats. TUNEL-positive cells were observed in the molecular layer and granular cell layers of the cerebellum. In contrast, no significant difference in the density of TUNEL-positive cells was revealed in the cerebral cortex. These results suggest that either endothelin receptor B sl mutation or colostomy has predominant lasting effects on the cell survival/loss in the cerebellum and hippocampus of adult ETB-/- rats. Our findings provide the information on cellular changes in the brains of patients with Hirschsprung's disease due to congenital EDNRB mutation as well as clinically relevant interventions.

Structural heart defects associated with ETB mutation, a cause of Hirschsprung disease.

BACKGROUND: HSCR, a colonic neurocristopathy affecting 1/5000 births, is suggested to associate with cardiac septal defects and conotruncal malformations. However, we question subtle cardiac changes maybe more commonly present due to multi-regulations by HSCR candidate genes, in this instance, ETB. To investigate, we compared the cardiac morphology and quantitative measurements of sl/sl rat to those of the control group. METHODS: Eleven neonatal rats were generated from heterozygote (ETB+/-) crossbreeding. Age and bodyweight were recorded at time of sacrifice. Diffusion-staining protocols with 1.5% iodine solution was completed prior to micro-CT scanning. All rats were scanned using an in vivo micro-CT scanner, Caliper Quantum FX, followed by two quality-control scans using a custom-built ex vivo micro-CT system. All scans were reviewed for gross cardiac dysmorphology. Micro-CT data were segmented semi-automatically post-NLM filtering for: whole-heart, LV, RV, LA, RA, and aortic arch. Measurements were taken with Drishti. Following image analysis, PCR genotyping of rats was performed: five sl/sl rats, three wildtype, and three heterozygotes. Statistical comparisons on organ volume, growth rate, and organ volume/bodyweight ratios were made between sl/sl and the control group. RESULTS: Cardiac morphology and constituents were preserved. However, significant volumetric reductions were recorded in sl/sl rats with respect to the control: whole heart (38.70%, p value = 0.02); LV (41.22%, p value = 0.01), RV (46.15%, p value = 0.02), LA (44.93%, p value = 0.06), and RA (39.49%, p value = 0.02). Consistent trend was observed in growth rate (~ 20%) and organ-volume/bodyweight ratios (~ 25%). On the contrary, measurements on aortic arch demonstrated no significant difference among the two groups. CONCLUSION: Despite the presence of normal morphology, significant cardiac growth retardation was detected in sl/sl rat, supporting the likely association of cardiac anomalies with HSCR, at least in ETB-/- subtype. Structural reduction was likely due to a combination of failure to thrive from enteric dysfunction, alterations to CaNCC colonization, and importantly coronary hypoperfusion from elevated ET-1/ETA-mediated hypervasoconstriction. Little correlation was detected between aortic arch development and sl/sl rat, supporting minor ETB role in large vessels. Although further clinical study is warranted, HSCR patients may likely require cardiac assessment in view of potential congenital cardiac defects.

Brain size reductions associated with endothelin B receptor mutation, a cause of Hirschsprung's disease.

BACKGROUND: ETB has been reported to regulate neurogenesis and vasoregulation in foetal development. Its dysfunction was known to cause HSCR, an aganglionic colonic disorder with syndromic forms reported to associate with both small heads and developmental delay. We therefore asked, "is CNS maldevelopment a more general feature of ETB mutation?" To investigate, we reviewed the micro-CT scans of an ETB-/- model animal, sl/sl rat, and quantitatively evaluated the structural changes of its brain constituents. METHODS: Eleven neonatal rats generated from ETB+/- cross breeding were sacrificed. Micro-CT scans were completed following 1.5% iodine-staining protocols. All scans were reviewed for morphological changes. Selected organs were segmented semi-automatically post-NLM filtering: TBr, T-CC, T-CP, OB, Med, Cer, Pit, and S&I Col. Volumetric measurements were made using Drishti rendering software. Rat genotyping was completed following analysis. Statistical comparisons on organ volume, organ growth rate, and organ volume/bodyweight ratios were made between sl/sl and the control groups based on autosomal recessive inheritance. One-way ANOVA was also performed to evaluate potential dose-dependent effect. RESULTS: sl/sl rat has 16.32% lower body weight with 3.53% lower growth rate than the control group. Gross intracranial morphology was preserved in sl/sl rats. However, significant volumetric reduction of 20.33% was detected in TBr; similar reductions were extended to the measurements of T-CC, T-CP, OB, Med, and Pit. Consistently, lower brain and selected constituent growth rates were detected in sl/sl rat, ranging from 6.21% to 11.51% reduction. Lower organ volume/bodyweight ratio was detected in sl/sl rats, reflecting disproportional neural changes with respect to body size. No consistent linear relationships exist between ETB copies and intracranial organ size or growth rates. CONCLUSION: Although ETB-/- mutant has a normal CNS morphology, significant size reductions in brain and constituents were detected. These structural changes likely arise from a combination of factors secondary to dysfunctional ET-1/ET-3/ETB signalling, including global growth impairment from HSCR-induced malnutrition and dysregulations in the neurogenesis, angiogenesis, and cerebral vascular control. These changes have important clinical implications, such as autonomic dysfunction or intellectual delay. Although further human study is warranted, our study suggested comprehensive managements are required for HSCR patients, at least in ETB-/- subtype.

Reduced cell proliferation and increased apoptosis in the hippocampal formation in a rat model of Hirschsprung's disease.

Hirschsprung's disease (HSCR) is a congenital malformation characterized by the absence of enteric ganglia in the distal intestine and gut obstruction. Some HSCR patients also have associated neurological symptoms. We studied a rat model of HSCR, also known as spotting lethal (sl/sl) rat, which carries a spontaneous deletion in the gene of endothelin receptor B (EDNRB) and a similar phenotype as humans with HSCR. We focused on the changes in cell proliferation and apoptosis in the hippocampal formation of the sl/sl rat. Proliferating cells in wildtype (+/+), heterozygous (+/sl) and homozygous (sl/sl) rats were labelled by intraperitoneal injection of 5-bromo-2'-deoxyuridine (BrdU) at postnatal day 2. The density of proliferating cells in the CA1 and CA3 regions of the hippocampus and dentate gyrus of sl/sl rats was significantly reduced compared to +/+ rats. The effect of EDNRB mutation on cell apoptosis was examined by using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling assay. This showed that the density of apoptotic cells in the hippocampal formation, particularly in the CA1 region of sl/sl rats, was significantly increased compared to +/+ rats. The expression of brain derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) was measured with ELISA in the hippocampal formation, but no difference was revealed between genotypes. These results suggest that EDNRB mutation reduces cell proliferation and increases apoptosis in the hippocampal formation of the sl/sl rat, but does not alter the levels of BDNF and GDNF. Our findings provide an insight into the cellular changes in the brains of HSCR patients caused by EDNRB mutation.

Surgical Intervention to Rescue Hirschsprung Disease in a Rat Model.

BACKGROUND/AIMS: Rats with a spontaneous null mutation in endothelin receptor type B or Ednrb (sl/sl; spotting lethal) lack enteric neurons in the distal bowel and usually die within the first week after birth. This early postnatal lethality limits their use for examining the potential of cell therapy to treat Hirschsprung disease, and for studies of the influence of EDNRB on the mature CNS and vascular systems. METHODS: We have developed a surgical intervention to prolong the life of the spotting lethal sl/sl rat, in which we perform a colostomy on postnatal (P) day 4-6 rats to avoid the fatal obstruction caused by the lack of colonic enteric neurons. RESULTS: The stomas remained patent and functional and the rats matured normally following surgery. Weight gains were comparable between control and Hirschsprung phenotype (sl/sl) rats, which were followed until 4 weeks after surgery (5 weeks old). We confirmed the absence of enteric neurons in the distal colon of rats whose lives were saved by the surgical intervention. CONCLUSIONS: This study provides a novel approach for studying EDNRB signalling in multiple organ systems in mature rats, including an animal model to study the efficacy of cell therapy to treat Hirschsprung disease.

Deficiency in endothelin receptor B reduces proliferation of neuronal progenitors and increases apoptosis in postnatal rat cerebellum.

Endothelins regulate cellular functions in the mammalian brain through the endothelin receptors A and B (EDNRA and EDNRB). In this study, we investigated the role of EDNRB on cell proliferation in the cerebellum by using the spotting lethal (sl) rat, which carries a naturally occurring deletion in the EDNRB gene. Proliferating cells in the three genotypes, wild-type (+/+), heterozygous (+/sl) and homozygous mutant (sl/sl) rats were labelled by intraperitoneal injection of 5-bromo-2'-deoxyuridine (BrdU) at postnatal day 2. The density of BrdU-positive cells (per mm(2)) in the external germinal layer of sl/sl rats (Mean +/- SEM, 977 +/- 388) was significantly reduced compared to +/+ (4915 +/- 631) and +/sl (2304 +/- 557) rats. Subsequently, we examined the effects of EDNRB mutation on neural apoptosis by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labelling assay. This showed that the density of apoptotic cells in the cerebella of sl/sl rats (9.3 +/- 0.5/mm(2)) was significantly more increased than +/+ rats (4 +/- 0.7). The expression of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) were measured with standard ELISA, but were unchanged in all genotypes. These results suggest that ENDRB mediates neural proliferation and have anti-apoptotic effects in the cerebellum of the postnatal rat, and that these effects are independent of changes in the expression of BDNF and GDNF. Our findings will lead to better understanding of the morphological changes in the cerebellum of Hirschsprung's disease patients with congenital EDNRB mutation.

Activation of alpha 2A adrenoceptors alters dendritic spine development and the expression of spinophilin in cultured cortical neurones.

alpha2 adrenoceptors have been shown to regulate the development of dendrites in mammalian cortical neurones. In this study we have investigated how agonists of alpha2 adrenoceptors affect length and density of dendritic spines in cultured cortical neurones from C57/B6 mice. A twenty-four hour incubation of 14 day old cultured neurones with UK 14304, an alpha2-adrenoceptor agonist, resulted in a significant increase in the average length and density of dendritic spines. Furthermore, incubation of neurones with the selective alpha 2A agonist guanfacine resulted in 1.2-fold increase in spine length and 1.8-fold increase in spine density. These effects were blocked by RX 821002 and BRL 44408, alpha2- and alpha 2A-adrenoceptor antagonists, respectively. The observed changes in the density and length of dendritic spines were correlated with increased expression of spinophilin, a key cytoskeletal protein in the formation and maintenance of dendritic spines, and a decrease in the phosphorylation of spinophilin on serine residues. The latter finding points to a possible mechanism by which adrenoceptors may regulate spinophilin function in dendritic spine development and structure in cortical neurones in vitro.

Defective NF-kappaB activation in virus-infected neuronal cells is restored by genetic complementation.

The interferon-beta (IFNbeta) gene is not inducible in neuronal cells in response to measles virus (MV) due to lack of nuclear factor kappa B (NF-kappaB) activation. NF-kappaB is normally sequestered in the cytoplasm by an inhibitor (IkappaBalpha). Previously, the authors demonstrated that the failure to activate neuronal NF-kappaB by MV was due to the inability to phosphorylate and degrade its inhibitor, IkappaBalpha. Here the authors demonstrate that transient transfection of a brain cDNA library into neuronal cells restores the ability of MV to activate NF-kappaB. In addition, tumor necrosis factor-alpha (TNFalpha), but not interleukin-1 (IL-1) or lipopolysaccharide (LPS), stimulation resulted in IkappaBalpha phosphorylation and degradation in two neuronal cell lines. These results indicate that failure of MV to activate neuronal NF-kappaB is due to a signaling defect and that MV utilizes an NF-kappaB signaling pathway distinct from that of TNFalpha, but may overlap with that for IL-1 and LPS.