Development of the sympathetic trunks in human embryos.

Although the development of the sympathetic trunks was first described >100 years ago, the topographic aspect of their development has received relatively little attention. We visualised the sympathetic trunks in human embryos of 4.5-10 weeks post-fertilisation, using Amira 3D-reconstruction and Cinema 4D-remodelling software. Scattered, intensely staining neural crest-derived ganglionic cells that soon formed longitudinal columns were first seen laterally to the dorsal aorta in the cervical and upper thoracic regions of Carnegie stage (CS)14 embryos. Nerve fibres extending from the communicating branches with the spinal cord reached the trunks at CS15-16 and became incorporated randomly between ganglionic cells. After CS18, ganglionic cells became organised as irregular agglomerates (ganglia) on a craniocaudally continuous cord of nerve fibres, with dorsally more ganglionic cells and ventrally more fibres. Accordingly, the trunks assumed a "pearls-on-a-string" appearance, but size and distribution of the pearls were markedly heterogeneous. The change in position of the sympathetic trunks from lateral (para-aortic) to dorsolateral (prevertebral or paravertebral) is a criterion to distinguish the "primary" and "secondary" sympathetic trunks. We investigated the position of the trunks at vertebral levels T2, T7, L1 and S1. During CS14, the trunks occupied a para-aortic position, which changed into a prevertebral position in the cervical and upper thoracic regions during CS15, and in the lower thoracic and lumbar regions during CS18 and CS20, respectively. The thoracic sympathetic trunks continued to move further dorsally and attained a paravertebral position at CS23. The sacral trunks retained their para-aortic and prevertebral position, and converged into a single column in front of the coccyx. Based on our present and earlier morphometric measurements and literature data, we argue that differential growth accounts for the regional differences in position of the sympathetic trunks.

Extrinsic innervation of the pelvic organs in the lesser pelvis of human embryos.

Realistic models to understand the developmental appearance of the pelvic nervous system in mammals are scarce. We visualized the development of the inferior hypogastric plexus and its preganglionic connections in human embryos at 4-8 weeks post-fertilization, using Amira 3D reconstruction and Cinema 4D-remodelling software. We defined the embryonic lesser pelvis as the pelvic area caudal to both umbilical arteries and containing the hindgut. Neural crest cells (NCCs) appeared dorsolateral to the median sacral artery near vertebra S1 at ~5 weeks and had extended to vertebra S5 1 day later. Once para-arterial, NCCs either formed sympathetic ganglia or continued to migrate ventrally to the pre-arterial region, where they formed large bilateral inferior hypogastric ganglionic cell clusters (IHGCs). Unlike more cranial pre-aortic plexuses, both IHGCs did not merge because the 'pelvic pouch', a temporary caudal extension of the peritoneal cavity, interposed. Although NCCs in the sacral area started to migrate later, they reached their pre-arterial position simultaneously with the NCCs in the thoracolumbar regions. Accordingly, the superior hypogastric nerve, a caudal extension of the lumbar splanchnic nerves along the superior rectal artery, contacted the IHGCs only 1 day later than the lumbar splanchnic nerves contacted the inferior mesenteric ganglion. The superior hypogastric nerve subsequently splits to become the superior hypogastric plexus. The IHGCs had two additional sources of preganglionic innervation, of which the pelvic splanchnic nerves arrived at ~6.5 weeks and the sacral splanchnic nerves only at ~8 weeks. After all preganglionic connections had formed, separate parts of the inferior hypogastric plexus formed at the bladder neck and distal hindgut.

Development of extrinsic innervation in the abdominal intestines of human embryos.

Compared to the intrinsic enteric nervous system (ENS), development of the extrinsic ENS is poorly documented, even though its presence is easily detectable with histological techniques. We visualised its development in human embryos and foetuses of 4-9.5 weeks post-fertilisation using Amira 3D-reconstruction and Cinema 4D-remodelling software. The extrinsic ENS originated from small, basophilic neural crest cells (NCCs) that migrated to the para-aortic region and then continued ventrally to the pre-aortic region, where they formed autonomic pre-aortic plexuses. From here, nerve fibres extended along the ventral abdominal arteries and finally connected to the intrinsic system. Schwann cell precursors (SCPs), a subgroup of NCCs that migrate on nerve fibres, showed region-specific differences in differentiation. SCPs developed into scattered chromaffin cells of the adrenal medulla dorsolateral to the coeliac artery (CA) and into more tightly packed chromaffin cells of the para-aortic bodies ventrolateral to the inferior mesenteric artery (IMA), with reciprocal topographic gradients between both fates. The extrinsic ENS first extended along the CA and then along the superior mesenteric artery (SMA) and IMA 5 days later. Apart from the branch to the caecum, extrinsic nerves did not extend along SMA branches in the herniated parts of the midgut until the gut loops had returned in the abdominal cavity, suggesting a permissive role of the intraperitoneal environment. Accordingly, extrinsic innervation had not yet reached the distal (colonic) loop of the midgut at 9.5 weeks development. Based on intrinsic ENS-dependent architectural remodelling of the gut layers, extrinsic innervation followed intrinsic innervation 3-4 Carnegie stages later.

The development of the dorsal mesentery in human embryos and fetuses.

The vertebrate intestine has a continuous dorsal mesentery between pharynx and anus that facilitates intestinal mobility. Based on width and fate the dorsal mesentery can be subdivided into that of the caudal foregut, midgut, and hindgut. The dorsal mesentery of stomach and duodenum is wide and topographically complex due to strong and asymmetric growth of the stomach. The associated formation of the lesser sac partitions the dorsal mesentery into the right-sided "caval fold" that serves as conduit for the inferior caval vein and the left-sided mesogastrium. The thin dorsal mesentery of the midgut originates between the base of the superior and inferior mesenteric arteries, and follows the transient increase in intestinal growth that results in small-intestinal looping, intestinal herniation and, subsequently, return. The following fixation of a large portion of the abdominal dorsal mesentery to the dorsal peritoneal wall by adhesion and fusion is only seen in primates and is often incomplete. Adhesion and fusion of mesothelial surfaces in the lesser pelvis results in the formation of the "mesorectum". Whether Toldt's and Denonvilliers' "fasciae of fusion" identify the location of the original mesothelial surfaces or, alternatively, represent the effects of postnatal wear and tear due to intestinal motility and intra-abdominal pressure changes, remains to be shown. "Malrotations" are characterized by growth defects of the intestinal loops with an ischemic origin and a narrow mesenteric root due to insufficient adhesion and fusion.

Effect of L-arginine on energy metabolism, skeletal muscle and brown adipose tissue in South Asian and Europid prediabetic men: a randomised double-blinded crossover study.

AIMS/HYPOTHESIS: Individuals of South Asian origin are at increased risk of developing type 2 diabetes mellitus and associated comorbidities compared with Europids. Disturbances in energy metabolism may contribute to this increased risk. Skeletal muscle and possibly also brown adipose tissue (BAT) are involved in human energy metabolism and nitric oxide (NO) is suggested to play a pivotal role in regulating mitochondrial biogenesis in both tissues. We aimed to investigate the effects of 6 weeks of supplementation with L-arginine, a precursor of NO, on energy metabolism by BAT and skeletal muscle, as well as glucose metabolism in South Asian men compared with men of European descent. METHODS: We included ten Dutch South Asian men (age 46.5 ± 2.8 years, BMI 30.1 ± 1.1 kg/m2) and ten Dutch men of European descent, that were similar with respect to age and BMI, with prediabetes (fasting plasma glucose level 5.6-6.9 mmol/l or plasma glucose levels 2 h after an OGTT 7.8-11.1 mmol/l). Participants took either L-arginine (9 g/day) or placebo orally for 6 weeks in a randomised double-blind crossover study. Participants were eligible to participate in the study when they were aged between 40 and 55 years, had a BMI between 25 and 35 kg/m2 and did not have type 2 diabetes. Furthermore, ethnicity was defined as having four grandparents of South Asian or white European origin, respectively. Blinding of treatment was done by the pharmacy (Hankintatukku) and an independent researcher from Leiden University Medical Center randomly assigned treatments by providing a coded list. All people involved in the study as well as participants were blinded to group assignment. After each intervention, glucose tolerance was determined by OGTT and basal metabolic rate (BMR) was determined by indirect calorimetry; BAT activity was assessed by cold-induced [18F]fluorodeoxyglucose ([18F]FDG) positron emission tomography-computed tomography scanning. In addition, a fasting skeletal muscle biopsy was taken and analysed ex vivo for respiratory capacity using a multisubstrate protocol. The primary study endpoint was the effect of L-arginine on BAT volume and activity. RESULTS: L-Arginine did not affect BMR, [18F]FDG uptake by BAT or skeletal muscle respiration in either ethnicity. During OGTT, L-arginine lowered plasma glucose concentrations (AUC0-2 h - 9%, p < 0.01), insulin excursion (AUC0-2 h - 26%, p < 0.05) and peak insulin concentrations (-26%, p < 0.05) in Europid but not South Asian men. This coincided with enhanced cold-induced glucose oxidation (+44%, p < 0.05) in Europids only. Of note, in skeletal muscle biopsies several respiration states were consistently lower in South Asian men compared with Europid men. CONCLUSIONS/INTERPRETATION: L-Arginine supplementation does not affect BMR, [18F]FDG uptake by BAT, or skeletal muscle mitochondrial respiration in Europid and South Asian overweight and prediabetic men. However, L-arginine improves glucose tolerance in Europids but not in South Asians. Furthermore, South Asian men have lower skeletal muscle oxidative capacity than men of European descent. FUNDING: This study was funded by the EU FP7 project DIABAT, the Netherlands Organization for Scientific Research, the Dutch Diabetes Research Foundation and the Dutch Heart Foundation. TRIAL REGISTRATION: ClinicalTrials.gov NCT02291458.