Precise Characterization and 3-Dimensional Reconstruction of the Autonomic Nerve Distribution of the Human Ureter.

PURPOSE: We sought to characterize and 3-dimensionally reconstruct the distribution of the autonomic innervation of the human ureter. MATERIALS AND METHODS: Three male and 3 female pairs of ureters were evaluated at 2 mm serial transverse sections along the entire length of the ureter. The location of nerve tissue was immunohistochemically identified using the neuronal marker, S100 protein. ImageJ software was used to calculate nerve count and density in the adventitia and smooth muscle. Blender® graphics software was used to create a 3-dimensional reconstruction of autonomic nerve distribution. RESULTS: Within the adventitia nerve density was highest in the mid and distal ureter (females 2.87 and 2.71 nerves per mm2, and males 1.68 and 1.69 nerves per mm2) relative to the proximal ureter (females and males 1.94 and 1.22 nerves per mm2, respectively, p >0.0005). Females had significantly higher nerve density throughout the adventitia, especially in the distal ureter (2.87 vs 1.68 nerves per mm2, p <0.0005). In smooth muscle the nerve density progressively increased from the proximal to the distal ureter (p >0.0005). Smooth muscle nerve density was similar in the 2 genders (p = 0.928). However, in females nerve density was significantly higher in the first 2 cm of the distal ureter relative to the second 2 cm (3.6 vs 1.5 nerves per mm2, p <0.001) but not in males (3.0 vs 2.1 nerves per mm2, p = 0.126). CONCLUSIONS: Nerve density was highly concentrated at the distal ureter in the adventitia and smooth muscle of the male and female human ureters. The female ureter had greater nerve density in the adventitia, and in smooth muscle nerves were significantly concentrated at the ureteral orifice and the ureteral tunnel.

Conditional Creation and Rescue of Nipbl-Deficiency in Mice Reveals Multiple Determinants of Risk for Congenital Heart Defects.

Elucidating the causes of congenital heart defects is made difficult by the complex morphogenesis of the mammalian heart, which takes place early in development, involves contributions from multiple germ layers, and is controlled by many genes. Here, we use a conditional/invertible genetic strategy to identify the cell lineage(s) responsible for the development of heart defects in a Nipbl-deficient mouse model of Cornelia de Lange Syndrome, in which global yet subtle transcriptional dysregulation leads to development of atrial septal defects (ASDs) at high frequency. Using an approach that allows for recombinase-mediated creation or rescue of Nipbl deficiency in different lineages, we uncover complex interactions between the cardiac mesoderm, endoderm, and the rest of the embryo, whereby the risk conferred by genetic abnormality in any one lineage is modified, in a surprisingly non-additive way, by the status of others. We argue that these results are best understood in the context of a model in which the risk of heart defects is associated with the adequacy of early progenitor cell populations relative to the sizes of the structures they must eventually form.

Kelly procedure for exstrophy or epispadias patients: Anatomical description of the pudendal neurovasculature.

INTRODUCTION: Adequate penile length in males with bladder exstrophy or epispadias is a major challenge. Kelly previously described a surgical technique of a single stage reconstruction for patients with exstrophy or epispadias that potentially achieves significant penile lengthening by completely detaching the insertion of the corpora cavernosa from the ischiopubic rami. However, because of the possibility of damage to the pudendal neurovascular supply that may lead to partial or complete penile loss, this technique has not gained popularity. The aim of this study is to describe the surgical anatomic relationship of the pudendal neurovascular bundle (NVB) to the ischiopubic rami and to determine a safer approach to dissection during the Kelly procedure. METHODS: We performed meticulous dissection in three formalin-fixed and one fresh adult male cadavers to demonstrate the anatomical relationships between the pudendal neurovascular supply of the penis and the cavernosal insertion to the ischiopubic ramus. RESULTS AND DISCUSSION: We demonstrated the relationships and distance between the NVB and the area of separation between the crus and the ischiopubic ramus at the level of the periosteum. The insertion of the crus to the ischiopubic ramus is inferior lateral, whereas the NVB lies at a superior medial position. This anatomical relationship is best visualized when the dissection is carried out starting from the distal portion of the NVB and proceeding proximally. This area of the periosteum is avascular and the NVB can be preserved safely as long as the dissection is conducted at that subperiosteal level. Based on this cadaver dissection study, we suppose that detaching the corporal cavernosa from the pubic bones at the subperiosteal level allows for a safe distance to be maintained from the pudendal nerve at all times. We believe that if a surgeon performs the dissection inferiorly and laterally, the corpora cavernosa can be safely detached from the ischiopubic ramus and injury to the pudendal vessels and nerve can be avoided. However, it must be noted that there are limitations to applying the results from this study of normal, adult cadavers to the anatomy of children and adolescents with exstrophy or epispadias, who form the largest proportion of patients who are candidates for this procedure. CONCLUSION: This anatomical study demonstrates the relationship between the pudendal NVB, the crus, and the ischiopubic ramus. We demonstrated how the separation of the crus from the ischiopubic periosteum might be performed more safely.

Redefining the Autonomic Nerve Distribution of the Bladder Using 3-Dimensional Image Reconstruction.

PURPOSE: We sought to create a 3-dimensional reconstruction of the autonomic nervous tissue innervating the bladder using male and female cadaver histopathology. MATERIALS AND METHODS: We obtained bladder tissue from a male and a female cadaver. Axial cross sections of the bladder were generated at 3 to 5 mm intervals and stained with S100 protein. We recorded the distance between autonomic nerves and bladder mucosa. We manually demarcated nerve tracings using ImageScope software (Aperio, Vista, California), which we imported into Blender™ graphics software to generate 3-dimensional reconstructions of autonomic nerve anatomy. RESULTS: Mean nerve density ranged from 0.099 to 0.602 and 0.012 to 0.383 nerves per mm2 in female and male slides, respectively. The highest concentrations of autonomic innervation were located in the posterior aspect of the bladder neck in the female specimen and in the posterior region of the prostatic urethra in the male specimen. Nerve density at all levels of the proximal urethra and bladder neck was significantly higher in posterior vs anterior regions in female specimens (0.957 vs 0.169 nerves per mm2, p<0.001) and male specimens (0.509 vs 0.206 nerves per mm2, p=0.04). CONCLUSIONS: Novel 3-dimensional reconstruction of the bladder is feasible and may help redefine our understanding of human bladder innervation. Autonomic innervation of the bladder is highly focused in the posterior aspect of the proximal urethra and bladder neck in male and female bladders.

Intrarenal and extrarenal autonomic nervous system redefined.

PURPOSE: The autonomic nervous supply to the kidneys is involved in the development of several diseases including hypertension. The neural distribution at the segmental vessels and intrarenal vasculature has not been well characterized. Thus, we evaluated the autonomic nerve distribution from the great vessels to the renal cortex in a cadaveric model. MATERIALS AND METHODS: We performed a detailed anatomical nerve dissection from the inferior mesenteric artery to the renal operculum in 2 human cadaveric torsos. Autonomic nerve fibers were verified by dissecting the greater splanchnic, sympathetic trunk and ganglia. We then systematically cross-sectioned the kidneys in 12, 1 mm slices across 3.6 cm, and stained the slices for histopathological analysis of neural tissue in relation to segmental arteries and other anatomical landmarks. Advanced reconstructive software was used to create a 3-dimensional computer image. RESULTS: Autonomic nerve fibers are located almost exclusively anteriorly on the main renal arteries and segmental arteries, and are absent from veins. Histopathology revealed that the intrarenal nerves continued to track exclusively with the arteries but were more circumferentially distributed. There is minimal nerve tissue around the veins. Many nerves were within a few millimeters of the renal collecting system. CONCLUSIONS: The autonomic nerves supplying the kidney maintain their distribution almost exclusively along the anterior surface of arteries as they pass from the aorta to the segmental arteries. Once inside the renal parenchyma, the nerves are circumferentially distributed around the renal arteries and are in close proximity to the renal collecting system.

Altering HIF-1α through 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure affects coronary vessel development.

Differential tissue hypoxia drives normal cardiogenic events including coronary vessel development. This requirement renders cardiogenic processes potentially susceptible to teratogens that activate a transcriptional pathway that intersects with the hypoxia-inducible factor (HIF-1) pathway. The potent toxin 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to cause cardiovascular defects by way of reduced myocardial hypoxia, inhibition of angiogenic stimuli, and alterations in responsiveness of endothelial cells to those stimuli. Our working hypothesis is that HIF-1 levels and thus HIF-1 signaling in the developing myocardium will be reduced by TCDD treatment in vivo during a critical stage and in particularly sensitive sites during heart morphogenesis. This inadequate HIF-1 signaling will subsequently result in outflow tract (OFT) and coronary vasculature defects. Our current data using the chicken embryo model showed a marked decrease in the intensity of immunostaining for HIF-1α nuclear expression in the OFT myocardium of TCDD-treated embryos. This area at the base of the OFT is particularly hypoxic during normal development; where endothelial cells initially form a concentrated anastomosing network known as the peritruncal ring; and where the left and right coronary arteries eventually connect to the aortic lumen. Consistent with this finding, anomalies of the proximal coronaries were detected after TCDD treatment and HIF-1α protein levels decreased in a TCDD dose-dependent manner.

Expression of lymphatic markers during avian and mouse cardiogenesis.

The adult heart has been reported to have an extensive lymphatic system, yet the development of this important system during cardiogenesis is still largely unexplored. The nuclear-localized transcription factor Prox-1 identified a sheet of Prox-1-positive cells on the developing aorta and pulmonary trunk in avian and murine embryos just before septation of the four heart chambers. The cells coalesced into a branching lymphatic network that spread within the epicardium to cover the heart. These vessels eventually expressed the lymphatic markers LYVE-1, VEGFR-3, and podoplanin. Before the Prox-1-positive cells were detected in the mouse epicardium, LYVE-1, a homologue of the CD44 glycoprotein, was primarily expressed in individual epicardial cells. Similar staining patterns were observed for CD44 in avian embryos. The proximity of these LYVE-1/CD44-positive mesenchymal cells to Prox-1-positive vessels suggests that they may become incorporated into the lymphatics. Unexpectedly, we detected LYVE-1/PECAM/VEGFR-3-positive vessels within the embryonic and adult myocardium, which remained Prox-1/podoplanin-negative. Lymphatic markers were surprisingly found in adult rat and embryonic mouse epicardial cell lines, with Prox-1 also exhibiting nuclear-localized expression in primary cultures of embryonic avian epicardial cells. Our data identified three types of cells in the embryonic heart expressing lymphatic markers: (1) Prox-1-positive cells from an extracardiac source that migrate within the serosa of the outflow tract into the epicardium of the developing heart, (2) individual LYVE-1-positive cells in the epicardium that may be incorporated into the Prox-1-positive lymphatic vasculature, and (3) LYVE-1-positive cells/vessels in the myocardium that do not become Prox-1-positive even in the adult heart.

Altered hypoxia-inducible factor-1 alpha expression levels correlate with coronary vessel anomalies.

The outflow tract myocardium and other regions corresponding to the location of the major coronary vessels of the developing chicken heart, display a high level of hypoxia as assessed by the hypoxia indicator EF5. The EF5-positive tissues were also specifically positive for nuclear-localized hypoxia inducible factor-1 alpha (HIF-1alpha), the oxygen-sensitive component of the hypoxia inducible factor-1 (HIF-1) heterodimer. This led to our hypothesis that there is a "template" of hypoxic tissue that determines the stereotyped pattern of the major coronary vessels. In this study, we disturbed this template by altering ambient oxygen levels (hypoxia 15%; hyperoxia 75-40%) during the early phases of avian coronary vessel development, in order to alter tissue hypoxia, HIF-1alpha protein expression, and its downstream target genes without high mortality. We also altered HIF-1alpha gene expression in the embryonic outflow tract cardiomyocytes by injecting an adenovirus containing a constitutively active form of HIF-1alpha (AdCA5). We assayed for coronary anomalies using anti-alpha-smooth muscle actin immunohistology. When incubated under abnormal oxygen levels or injected with a low titer of the AdCA5, coronary arteries displayed deviations from their normal proximal connections to the aorta. These deviations were similar to known clinical anomalies of coronary arteries. These findings indicated that developing coronary vessels may be subject to a level of regulation that is dependent on differential oxygen levels within cardiac tissues and subsequent HIF-1 regulation of gene expression.

Mouse and human phenotypes indicate a critical conserved role for ERK2 signaling in neural crest development.

Disrupted ERK1/2 (MAPK3/MAPK1) MAPK signaling has been associated with several developmental syndromes in humans; however, mutations in ERK1 or ERK2 have not been described. We demonstrate haplo-insufficient ERK2 expression in patients with a novel approximately 1 Mb micro-deletion in distal 22q11.2, a region that includes ERK2. These patients exhibit conotruncal and craniofacial anomalies that arise from perturbation of neural crest development and exhibit defects comparable to the DiGeorge syndrome spectrum. Remarkably, these defects are replicated in mice by conditional inactivation of ERK2 in the developing neural crest. Inactivation of upstream elements of the ERK cascade (B-Raf and C-Raf, MEK1 and MEK2) or a downstream effector, the transcription factor serum response factor resulted in analogous developmental defects. Our findings demonstrate that mammalian neural crest development is critically dependent on a RAF/MEK/ERK/serum response factor signaling pathway and suggest that the craniofacial and cardiac outflow tract defects observed in patients with a distal 22q11.2 micro-deletion are explained by deficiencies in neural crest autonomous ERK2 signaling.

Apoptosis in the developing mouse heart.

Apoptosis occurs at high frequency in the myocardium of the developing avian cardiac outflow tract (OFT). Up- or down-regulating apoptosis results in defects resembling human conotruncal heart anomalies. This finding suggested that regulated levels of apoptosis are critical for normal morphogenesis of the four-chambered heart. Recent evidence supports an important role for hypoxia of the OFT myocardium in regulating cell death and vasculogenesis. The purpose of this study was to determine whether apoptosis in the outflow tract myocardium occurs in the mouse heart during developmental stages comparable to the avian heart and to determine whether differential hypoxia is also present at this site in the murine heart. Apoptosis was detected using a fluorescent vital dye, Lysotracker Red (LTR), in the OFT myocardium of the mouse starting at embryonic day (E) 12.5, peaking at E13.5-14.5, and declining thereafter to low or background levels by E18.5. In addition, high levels of apoptosis were detected in other cardiac regions, including the apices of the ventricles and along the interventricular sulcus. Apoptosis in the myocardium was detected by double-labeling with LTR and cardiomyocyte markers. Terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) and immunostaining for cleaved Caspase-3 were used to confirm the LTR results. At the peak of OFT apoptosis in the mouse, the OFT myocardium was relatively hypoxic, as indicated by specific and intense EF5 staining and HIF1alpha nuclear localization, and was surrounded by the developing vasculature as in the chicken embryo. These findings suggest that cardiomyocyte apoptosis is an evolutionarily conserved mechanism for normal morphogenesis of the outflow tract myocardium in avian and mammalian species.

Differential levels of tissue hypoxia in the developing chicken heart.

Tissue hypoxia plays a critical role in normal development, including cardiogenesis. Previously, we showed that oxygen concentration, as assessed by the hypoxia indicator EF5, is lowest in the outflow tract (OFT) myocardium of the developing chicken heart and may be regulating events in OFT morphogenesis. In this study, we identified additional areas of the embryonic chicken heart that were intensely positive for EF5 within the myocardium in discrete regions of the atrial wall and the interventricular septum (IVS). The region of the IVS that is EF5-positive includes a portion of the developing central conduction system identified by HNK-1 co-immunostaining. The EF5 positive tissues were also specifically positive for nuclear-localized hypoxia inducible factor 1alpha (HIF-1alpha), the oxygen-sensitive component of the hypoxia inducible factor 1 (HIF-1) heterodimer. The pattern of the most intensely EF5-stained myocardial regions of the atria and IVS resemble the pattern of the major coronary vessels that form in later stages within or immediately adjacent to these particular regions. These vessels include the sinoatrial nodal artery that is a branch of the right coronary artery within the atrial wall and the anterior/posterior interventricular vessels of the IVS. These findings indicate that a portion of the developing central conduction system and the patterning of coronary vessels may be subject to a level of regulation that is dependent on differential oxygen concentration within cardiac tissues and subsequent HIF-1 regulation of gene expression.