Human Cardiac Development.

Many aspects of heart development are topographically complex and require three-dimensional (3D) reconstruction to understand the pertinent morphology. We have recently completed a comprehensive primer of human cardiac development that is based on firsthand segmentation of structures of interest in histological sections. We visualized the hearts of 12 human embryos between their first appearance at 3.5 weeks and the end of the embryonic period at 8 weeks. The models were presented as calibrated, interactive, 3D portable document format (PDF) files. We used them to describe the appearance and the subsequent remodeling of around 70 different structures incrementally for each of the reconstructed stages. In this chapter, we begin our account by describing the formation of the single heart tube, which occurs at the end of the fourth week subsequent to conception. We describe its looping in the fifth week, the formation of the cardiac compartments in the sixth week, and, finally, the septation of these compartments into the physically separated left- and right-sided circulations in the seventh and eighth weeks. The phases are successive, albeit partially overlapping. Thus, the basic cardiac layout is established between 26 and 32 days after fertilization and is described as Carnegie stages (CSs) 9 through 14, with development in the outlet component trailing that in the inlet parts. Septation at the venous pole is completed at CS17, equivalent to almost 6 weeks of development. During Carnegie stages 17 and 18, in the seventh week, the outflow tract and arterial pole undergo major remodeling, including incorporation of the proximal portion of the outflow tract into the ventricles and transfer of the spiraling course of the subaortic and subpulmonary channels to the intrapericardial arterial trunks. Remodeling of the interventricular foramen, with its eventual closure, is complete at CS20, which occurs at the end of the seventh week. We provide quantitative correlations between the age of human and mouse embryos as well as the Carnegie stages of development. We have also set our descriptions in the context of variations in the timing of developmental features.

Cardiac development demystified by use of the HDBR atlas.

Much has been learned over the last half century regarding the molecular and genetic changes that take place during cardiac development. As yet, however, these advances have not been translated into knowledge regarding the marked changes that take place in the anatomical arrangements of the different cardiac components. As such, therefore, many aspects of cardiac development are still described on the basis of speculation rather than evidence. In this review, we show how controversial aspects of development can readily be arbitrated by the interested spectator by taking advantage of the material now gathered together in the Human Developmental Biology Resource; HDBR. We use the material to demonstrate the changes taking place during the formation of the ventricular loop, the expansion of the atrioventricular canal, the incorporation of the systemic venous sinus, the formation of the pulmonary vein, the process of atrial septation, the remodelling of the pharyngeal arches, the major changes occurring during formation of the outflow tract, the closure of the embryonic interventricular communication, and the formation of the ventricular walls. We suggest that access to the resource makes it possible for the interested observer to arbitrate, for themselves, the ongoing controversies that continue to plague the understanding of cardiac development.

Revisiting the anatomy of the left ventricle in the light of knowledge of its development.

Despite centuries of investigation, certain aspects of left ventricular anatomy remain either controversial or uncertain. We make no claims to have resolved these issues, but our review, based on our current knowledge of development, hopefully identifies the issues requiring further investigation. When first formed, the left ventricle had only inlet and apical components. With the expansion of the atrioventricular canal, the developing ventricle cedes part of its inlet to the right ventricle whilst retaining the larger parts of the cushions dividing the atrioventricular canal. Further remodelling of the interventricular communication provides the ventricle with its outlet, with the aortic root being transferred to the left ventricle along with the newly formed myocardium supporting its leaflets. The definitive ventricle possesses inlet, apical and outlet parts. The inlet component is guarded by the mitral valve, with its leaflets, in the normal heart, supported by papillary muscles located infero-septally and supero-laterally. There is but a solitary zone of apposition between the leaflets, which we suggest are best described as being aortic and mural. The trabeculated component extends beyond the inlet to the apex and is confluent with the outlet part, which supports the aortic root. The leaflets of the aortic valve are supported in semilunar fashion within the root, with the ventricular cavity extending to the sinutubular junction. The myocardial-arterial junction, however, stops well short of the sinutubular junction, with myocardium found only at the bases of the sinuses, giving rise to the coronary arteries. We argue that the relationships between the various components should now be described using attitudinally appropriate terms rather than describing them as if the heart is removed from the body and positioned on its apex.

Development of the arterial roots and ventricular outflow tracts.

The separation of the outflow tract of the developing heart into the systemic and pulmonary arterial channels remains controversial and poorly understood. The definitive outflow tracts have three components. The developing outflow tract, in contrast, has usually been described in two parts. When the tract has exclusively myocardial walls, such bipartite description is justified, with an obvious dogleg bend separating proximal and distal components. With the addition of non-myocardial walls distally, it becomes possible to recognise three parts. The middle part, which initially still has myocardial walls, contains within its lumen a pair of intercalated valvar swellings. The swellings interdigitate with the distal ends of major outflow cushions, formed by the remodelling of cardiac jelly, to form the primordiums of the arterial roots. The proximal parts of the major cushions, occupying the proximal part of the outflow tract, which also has myocardial walls, themselves fuse and muscularise. The myocardial shelf thus formed remodels to become the free-standing subpulmonary infundibulum. Details of all these processes are currently lacking. In this account, we describe the anatomical changes seen during the overall remodelling. Our interpretations are based on the interrogation of serially sectioned histological and high-resolution episcopic microscopy datasets prepared from developing human and mouse embryos, with some of the datasets processed and reconstructed to reveal the specific nature of the tissues contributing to the separation of the outflow channels. Our findings confirm that the tripartite postnatal arrangement can be correlated with the changes occurring during development.

Morphogenetic processes in the development and evolution of the arteries of the pharyngeal arches: their relations to congenital cardiovascular malformations.

The heart and aortic arch arteries in amniotes form a double circulation, taking oxygenated blood from the heart to the body and deoxygenated blood to the lungs. These major vessels are formed in embryonic development from a series of paired and symmetrical arteries that undergo a complex remodelling process to form the asymmetric arch arteries in the adult. These embryonic arteries form in the pharyngeal arches, which are symmetrical bulges on the lateral surface of the head. The pharyngeal arches, and their associated arteries, are found in all classes of vertebrates, but the number varies, typically with the number of arches reducing through evolution. For example, jawed vertebrates have six pairs of pharyngeal arch arteries but amniotes, a clade of tetrapod vertebrates, have five pairs. This had led to the unusual numbering system attributed to each of the pharyngeal arch arteries in amniotes (1, 2, 3, 4, and 6). We, therefore, propose that these instead be given names to reflect the vessel: mandibular (1st), hyoid (2nd), carotid (3rd), aortic (4th) and pulmonary (most caudal). Aberrant arch artery formation or remodelling leads to life-threatening congenital cardiovascular malformations, such as interruption of the aortic arch, cervical origin of arteries, and vascular rings. We discuss why an alleged fifth arch artery has erroneously been used to interpret congenital cardiac lesions, which are better explained as abnormal collateral channels, or remodelling of the aortic sac.

The advantages of naming rather than numbering the arteries of the pharyngeal arches.

Controversies continue as to how many pharyngeal arches, with their contained arteries, are to be found in the developing human. Resolving these controversies is of significance to paediatric cardiologists since many investigating abnormalities of the extrapericardial arterial pathways interpret their findings on the basis of persistence of a fifth set of such arteries within an overall complement of six sets. The evidence supporting such an interpretation is open to question. In this review, we present the history of the existence of six such arteries, emphasising that the initial accounts of human development had provided evidence for the existence of only five sets. We summarise the current evidence that substantiates these initial findings. We then show that the lesions interpreted on the basis of persistence of the non-existing fifth arch arteries are well described on the basis of the persistence of collateral channels, known to exist during normal development, or alternatively due to remodelling of the aortic sac.

A revised terminology for the pharyngeal arches and the arch arteries.

The pharyngeal arches are a series of bulges found on the lateral surface of the head of vertebrate embryos. In humans, and other amniotes, there are five pharyngeal arches and traditionally these have been labelled from cranial to caudal-1, 2, 3, 4 and 6. This numbering is odd-there is no '5'. Two reasons have been given for this. One is that during development, a 'fifth' arch forms transiently but is not fully realised. The second is that this numbering fits with the evolutionary history of the pharyngeal arches. Recent studies, however, have shown that neither of these justifications have basis. The traditional labelling is problematic as it causes confusion to those trying to understand the development of the pharyngeal arches. In particular, it creates difficulties in the field of congenital cardiac malformations, where it is common to find congenital cardiac lesions interpreted on the basis of persistence of the postulated arteries of the fifth arch. To resolve these problems and to take account of the recent studies that have clarified pharyngeal arch development, we propose a new terminology for the pharyngeal arches. In this revised scheme, the pharyngeal arches are to be labelled as follows-the first, most cranial, the mandibular (M), the second, the hyoid (H), the third, the carotid (C), the fourth, the aortic (A) and the last, most caudal, the pulmonary (P).

A pictorial account of the human embryonic heart between 3.5 and 8 weeks of development.

Heart development is topographically complex and requires visualization to understand its progression. No comprehensive 3-dimensional primer of human cardiac development is currently available. We prepared detailed reconstructions of 12 hearts between 3.5 and 8 weeks post fertilization, using Amira® 3D-reconstruction and Cinema4D®-remodeling software. The models were visualized as calibrated interactive 3D-PDFs. We describe the developmental appearance and subsequent remodeling of 70 different structures incrementally, using sequential segmental analysis. Pictorial timelines of structures highlight age-dependent events, while graphs visualize growth and spiraling of the wall of the heart tube. The basic cardiac layout is established between 3.5 and 4.5 weeks. Septation at the venous pole is completed at 6 weeks. Between 5.5 and 6.5 weeks, as the outflow tract becomes incorporated in the ventricles, the spiraling course of its subaortic and subpulmonary channels is transferred to the intrapericardial arterial trunks. The remodeling of the interventricular foramen is complete at 7 weeks.

Miniseries 1-Part I: the Development of the atrioventricular conduction axis.

Despite years of research, many details of the formation of the atrioventricular conduction axis remain uncertain. In this study, we aimed to clarify the situation. We studied three-dimensional reconstructions of serial histological sections and episcopic datasets of human embryos, supplementing these findings with assessment of material housed at the Human Developmental Biological Resource. We also examined serially sectioned human foetal hearts between 10 and 30 weeks of gestation. The conduction axis originates from the primary interventricular ring, which is initially at right angles to the plane of the atrioventricular canal, with which it co-localizes in the lesser curvature of the heart loop. With rightward expansion of the atrioventricular canal, the primary ring bends rightward, encircling the newly forming right atrioventricular junction. Subsequent to remodelling of the outflow tract, part of the primary ring remains localized on the crest of the muscular ventricular septum. By 7 weeks, its atrioventricular part has extended perpendicular to the septal parts. The atrioventricular node is formed at the inferior transition between the ventricular and atrial parts, with the transition itself marking the site of the penetrating atrioventricular bundle. Only subsequent to muscularization of the true second atrial septum does it become possible to recognize the definitive node. The conversion of the developmental arrangement into the definitive situation as seen postnatally requires additional remodelling in the first month of foetal development, concomitant with formation of the inferior pyramidal space and the infero-septal recess of the subaortic outflow tract.

The development and structure of the mesentery.

The position of abdominal organs, and mechanisms by which these are centrally connected, are currently described in peritoneal terms. As part of the peritoneal model of abdominal anatomy, there are multiple mesenteries. Recent findings point to an alternative model in which digestive organs are connected to a single mesentery. Given that direct evidence of this is currently lacking, we investigated the development and shape of the entire mesentery. Here we confirm that, within the abdomen, there is one mesentery in which all abdominal digestive organs develop and remain connected to. We show that all abdominopelvic organs are organised into two, discrete anatomical domains, the mesenteric and non-mesenteric domain. A similar organisation occurs across a range of animal species. The findings clarify the anatomical foundation of the abdomen; at the foundation level, the abdomen comprises a visceral (i.e. mesenteric) and somatic (i.e. musculoskeletal) frame. The organisation at that level is a fundamental order that explains the positional anatomy of all abdominopelvic organs, vasculature and peritoneum. Collectively, the findings provide a novel start point from which to systemically characterise the abdomen and its contents.

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.

Inferior Extensions of the Atrioventricular Node.

The pathways for excitation of the atrioventricular node enter either superiorly, as the so-called 'fast' pathway, or inferiorly as the 'slow' pathway. However, knowledge of the specific anatomical details of these pathways is limited. Most of the experimental studies that established the existence of these pathways were conducted in mammalian hearts, which have subtle differences to human hearts. In this review, the authors summarise their recent experiences investigating human cardiac development, correlating these results with the arrangement of the connections between the atrial myocardium and the compact atrioventricular node as revealed by serial sectioning of adult human hearts. They discuss the contributions made from the atrioventricular canal myocardium, as opposed to the primary ring. Both these rings are incorporated into the atrial vestibules, albeit with the primary ring contributing only to the tricuspid vestibule. The atrial septal cardiomyocytes are relatively late contributors to the nodal inputs. Finally, they relate our findings of human cardiac development to the postnatal arrangement.

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.

Pseudolesion in the right parafissural liver parenchyma on CT: The base is found in embryology and collagen content.

BACKGROUND: Computed tomography (CT) images of livers may show a hypo-attenuated structure alongside the falciform ligament, which can be a focal fatty pseudolesion and can mimic a malignancy. The preferred location is on the right parafissural site, ventral in segment IVa/b. The etiology is not clear, nor is it known how the histology of this location develops. These are evaluated in this study. METHODS: 40 adult cadavers with autopsy and / or postmortem CT in a university hospital and a forensic center were included. Liver biopsies were taken at the left side of the falciform ligament as control, and at the right side as the possible precursor of a pseudolesion; these were examined for collagen and fat content. Cadavers with steatotic (>5% fat) or fibrotic (>2% collagen) control samples were excluded. RESULTS: Significantly more collagen was present in the right parafissural liver parenchyma: median 0.68% (IQR: 0.32-1.17%), compared to the left side 0.48% (IQR: 0.21-0.75%) (p 0.008), with equal fat content and CT attenuation values. The etiophysiology goes back to the demise of the umbilical venes in the early embryonic and neonatal period. CONCLUSIONS: The right parafissural area contains more collagen and an equal amount of fat compared to the control left side. This supports the hypothesis of delayed, 'third' inflow: the postnatal change in blood supply from umbilical to portal leaves the downstream parafissural area hypoperfused leading to hypoxia which in turn results in collagen accumulation and the persistence of paraumbilical veins of Sappey.

Interactive three-dimensional teaching models of the female and male pelvic floor.

Controversies regarding structure and function of the pelvic floor persist because of its poor accessibility and complex anatomical architecture. Most data are based on dissection. This "surgical" approach requires profound prior knowledge, because applying the scalpel precludes a "second look." The "sectional" approach does not entail these limitations, but requires segmentation of structures and three-dimensional reconstruction. This approach has produced several "Visible Human Projects." We dealt with limited spatial resolution and difficult-to-segment structures by proceeding from clear-cut to more fuzzy boundaries and comparing segmentation between investigators. We observed that the bicipital levator ani muscle consisted of pubovisceral and puborectal portions; that the pubovisceral muscle formed, together with rectococcygeal and rectoperineal muscles, a rectal diaphragm; that the external anal sphincter consisted of its subcutaneous portion and the puborectal muscle only; that the striated urethral sphincter had three parts, of which the middle (urethral compressor) was best developed in females and the circular lower ("membranous") best in males; that the rectourethral muscle, an anterior extension of the rectal longitudinal smooth muscle, developed a fibrous node in its center (perineal body); that the perineal body was much better developed in females than males, so that the rectourethral subdivision into posterior rectoperineal and anterior deep perineal muscles was more obvious in females; that the superficial transverse perineal muscle attached to the fibrous septa of the ischioanal fat; and that the uterosacral ligaments and mesorectal fascia colocalized. To facilitate comprehension of the modified topography we provide interactive 3D-PDFs that are freely available for teaching purposes. Clin. Anat. 33:275-285, 2020. © 2019 Wiley Periodicals, Inc.

Anatomy of rodent and human livers: What are the differences?

The size of the liver of terrestrial mammals obeys the allometric scaling law over a weight range of >3 ∗ 106. Since scaling reflects adaptive changes in size or scale among otherwise similar animals, we can expect to observe more similarities than differences between rodent and human livers. Obvious differences, such as the presence (rodents) or absence (humans) of lobation and the presence (mice, humans) or absence (rats) of a gallbladder, suggest qualitative differences between the livers of these species. After review, however, we conclude that these dissimilarities represent relatively small quantitative differences. The microarchitecture of the liver is very similar among mammalian species and best represented by the lobular concept, with the biggest difference present in the degree of connective tissue development in the portal tracts. Although larger mammals have larger lobules, increasing size of the liver is mainly accomplished by increasing the number of lobules. The increasing role of the hepatic artery in lobular perfusion of larger species is, perhaps, the most important and least known difference between small and large livers, because it profoundly affects not only interventions like liver transplantations, but also calculations of liver function.

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.

Remodeling of the Embryonic Interventricular Communication in Regard to the Description and Classification of Ventricular Septal Defects.

Ventricular septal defects are the commonest congenital cardiac malformations. Appropriate knowledge of the steps involved in completion of ventricular septation should provide clues as to the morphology of the different phenotypes. Currently, however, consensus is lacking regarding the components of the developing ventricular septum, and how best to describe the different phenotypes seen in postnatal life. We have reassessed the previous investigations devoted to closure of the embryonic interventricular communication. On this basis, we discuss how studies in the early part of the 20th century correctly identified the steps involved in the remodeling of the embryonic interventricular foramen subsequent to the stage at which the outflow tract arises entirely above the cavity of the developing right ventricle. There has, however, already been remodeling of the foramen from the stage at which the atrioventricular canal is supported exclusively by the developing left ventricle. We show how these temporal changes in morphology can provide explanations for the different ventricular septal defects seen in the clinical setting. Thus, muscular defects represent inappropriate coalescence of muscular ventricular septum. The channels that are perimembranous are due to failure of closure of the persisting embryonic interventricular foramen. Those that are doubly committed and juxta-arterial reflect failure of formation of the free-standing subpulmonary muscular infundibular sleeve. The findings also point to the importance of appropriate alignment, during development, between the developing atrial and ventricular septums, and between the apical component of the ventricular septum and the ventricular outlet components. Anat Rec, 302:19-31, 2019. © 2018 Wiley Periodicals, Inc.

Innervation of the human spleen: A complete hilum-embedding approach.

INTRODUCTION: The spleen is hypothesized to play a role in the autonomic nervous system (ANS)-mediated control of host defence, but the neuroanatomical evidence for this assumption rests on a sparse number of studies, which mutually disagree with respect to the existence of cholinergic or vagal innervation. METHODS: We conducted an immuno- and enzyme-histochemical study of the innervation of the human spleen using a complete hilum-embedding approach to ensure that only nerves that entered or left the spleen were studied, and that all splenic nerves were included in the sampled area. Furthermore, a complete embedded spleen was serially sectioned to prepare a 3D reconstruction of the hilar nerve plexus. RESULTS: All detected nerves entering the spleen arise from the nerve plexus that surrounds branches of the splenic artery and are catecholaminergic. Inside the spleen these nerves continue within the adventitia of the white pulpal central arteries and red pulpal arterioles. Staining for either choline acetyltransferase or acetylcholinesterase did not reveal any evidence for cholinergic innervation of the human spleen, irrespective of the type of fixation (regularly fixed, fresh-frozen post-fixed or fresh-frozen cryoslides). Furthermore, no positive VIP staining was observed (VIP is often co-expressed in postganglionic parasympathetic nerves). CONCLUSION: Our comprehensive approach did not produce any evidence for a direct cholinergic (or VIP-ergic) innervation of the spleen. This finding does not rule out (indirect) vagal innervation via postganglionic non-cholinergic periarterial fibres.