M.C. DeRuiter

Decellularization of rat aortic valve allografts reduces leaflet destruction and extracellular matrix remodeling.

OBJECTIVES Decellularization of aortic valve allografts in advance of transplantation is a promising approach to overcome immune-induced early graft failure. In this study the effects of in vitro cell extraction on extracellular matrix molecules and in vivo remodeling of decellularized aortic valves were investigated in a heterotopic aortic valve rat implantation model. METHODS Rat aortic valve conduits were decellularized by a 2-step detergent-enzymatic extraction method involving sodium dodecyl sulfate in combination with RNase and DNase. Cellular and acellular allogeneic (2x, n = 4) and syngeneic valve grafts (2x, n = 3) were grafted infrarenally into the descending aorta for 21 days. Immunohistochemical techniques were used to study extracellular matrix constitution (elastin, collagen, fibronectin, and chondroitin sulfate) and cellular infiltration. RESULTS The decellularization procedure resulted in a complete loss of all cellular structures from the entire valve conduit with minimal damage to the extracellular matrix. All transplanted cellular allografts became deformed, swollen, and acellular with major changes in extracellular matrix structure. The transplanted decellularized allografts, however, retained normal preserved valve leaflets comparable to transplanted cellular and acellular syngeneic grafts. With the exception of cellular syngeneic grafts, all other grafts showed retrovalvular thrombi. CONCLUSIONS Damage to the valves caused by decellularization technique is much less than the damage caused by the recipients immune response. In vitro removal of viable cells in (cryopreserved) homografts may decrease graft failure. Seeding with autologous or major histocompatibility complex-matched donor endothelial cells will be necessary to diminish damage induced by an absent blood-tissue barrier.

HNK-1 expression patterns in the embryonic rat heart distinguish between sinuatrial tissues and atrial myocardium

HNK-1 expression was studied by immunohistochemistry in serial sections of embryonic and fetal rat hearts from 11.5 to 16.5 embryonic days. Graphic reconstructions were made to obtain detailed 3D information on the localization of immunoreactive tissues. The antibody used appeared to stain most parts of the venous sinus and the sinuatrial transitional zone as well as the atrioventricular transitional zone, but the patterns varied through the different developmental stages. At 11.5 days, positive myocardium was found in the right atrium and on top of the ventricular septal primordium. At 13.5 days, the left venous valve and the posterior atrial wall containing the orifice of the pulmonary vein were immunoreactive, and so were the right venous valve, the septum spurium and the superior, right-lateral and inferior parts of the atrioventricular canal. From the latter, immunoreactivity continued onto the crest of the ventricular septum. At 15.5 days, HNK-1 positivity in the two venous valves had become continuous, whereas the right-lateral part of the atrioventricular canal had lost its positivity, thus making the positive areas in the superior and inferior parts of this canal discontinuous. From the venous valves immunoreaction continued into the venous sinus septum but this area remained discontinuous with the inferior part of the atrioventricular canal. It is concluded that the entirety of venous sinus and sinuatrial transitional zone expresses the HNK-1 antigen and that the orifice of the pulmonary vein belongs to this complex, rather than to the embryonic atrium proper, which is HNK-1 negative. Extrapolation of these data to the adult human atrium leads to the conclusion hat most ’’atrial septal structures’’ are of sinuatrial origin, leaving the flap valve of the oval fossa (atrial septum primum) as the only really atrial structure. It is suggested that the atrioventricular node is derived from the inferior portion of the atrioventricular canal, and that two expansions of sinuatrial tissue form the substrate for anterior and posterior atrionodal inputs which in the literature have been described as internodal tracts.

New insights in the neuroanatomy of the human adult superior hypogastric plexus and hypogastric nerves.

BACKGROUND The superior hypogastric plexus (SHP) is an autonomic plexus, located ventrally to the abdominal aorta and its bifurcation, innervating pelvic viscera. It is classically described as being composed of merely sympathetic fibres. However, post-operative complications after surgery damaging the peri-aortic retroperitoneal compartment suggest the existence of parasympathetic fibres. This immunohistochemical study describes the neuroanatomical composition of the human mature SHP. MATERIAL AND METHODS Eight pre-determined retroperitoneal localizations including the lumbar splanchnic nerves, the SHP and the HN were studied in four human cadavers. Control tissues (white rami, grey rami, vagus nerve, splanchnic nerves, sympathetic ganglia, sympathetic chain and spinal nerve) were collected to verify the results. All tissues were stained with haematoxylin and eosin and antibodies S100, tyrosine hydroxylase (TH), vasoactive intestinal peptide (VIP) and myelin basic protein (MBP) to identify pre- and postganglionic parasympathetic and sympathetic nerve fibres. RESULTS All tissues comprising the SHP and hypogastric nerves (HN) showed isolated expression of TH, VIP and MBP, revealing the presence of three types of fibres: postganglionic adrenergic sympathetic fibres marked by TH, unmyelinated VIP-positive fibres and myelinated preganglionic fibres marked by MBP. Analysis of control tissues confirmed that TH, VIP and MBP were well usable to interpret the neurochemical composition of the SHP and HN. CONCLUSION The human SHP and HN contain sympathetic and most likely postganglionic parasympathetic fibres. The origin of these fibres is still to be elucidated, however surgical damage in the peri-aortic retroperitoneal compartment may cause pelvic organ dysfunction related to both parasympathetic and sympathetic denervation.

Morphogenetic differences of secundum atrial septal defects

It is generally considered that the development of secundum atrial septal defect (ASDII) or oval fossa defect is the result of excessive resorption of the embryological atrial septum primum, but this does not seem to explain all defects. We investigated 58 postmortem hearts with an ASDII and 22 normal hearts from patients ranging in age from 1 day to 49 years. The different structures of the oval fossa were examined. In 86% of the specimens, the defects were the result of a malformation of the valvula foraminis ovalis or embryological atrial septum primum, and in 14% an absent superior limbus (septum secundum) was the cause of the interatrial communication. The “septum primum” ASDs were divided into four subgroups based on the degree of deficiency of the septum primum and position of the ostium secundum within the septum primum. We conclude that the morphogenesis of ASDII is variable and both septum primum and septum secundum defects occur, which may be relevant in view of genetic studies that may lead to further differentiation of patients with and without genetically determined ASDIIs.

Lymphatic drainage pathways from the cervix uteri: Implications for radical hysterectomy?

OBJECTIVE Radical hysterectomy with pelvic lymphadenectomy is the treatment of choice for early-stage cervical cancer. Wertheims original technique has been often modified, mainly in the extent of parametrectomy. Okabayashis technique is considered as the most radical variant regarding removal of the ventral parametrium and paracolpal tissues. Surgical outcome concerning recurrence and survival is good, but morbidity is high due to autonomic nerve damage. While the autonomic network has been studied extensively, the lymphatic system is less understood. This study describes the lymphatic drainage pathways of the cervix uteri and specifically the presence of lymphatics in the vesico-uterine ligament (VUL). METHODS A developmental series of 10 human female fetal pelves was studied. Paraffin embedded blocks were sliced in transverse sections of 8 or 10 μm. Analysis was performed by staining with antibodies against LYVE-1 (lymphatic endothelium), S100 (Schwann cells), alpha-Smooth Muscle Actin (smooth muscle cells) and CD68 (macrophages). The results were three-dimensionally represented. RESULTS Two major pathways drained the cervix uteri: a supra-ureteral pathway, running in the cardinal ligament superior to the ureter, and a dorsal pathway, running in the utero-sacral ligament towards the rectal pillars. No lymph vessels draining the cervix uteri were detected in the VUL. In the paracolpal parametrium lymph vessels draining the upper vagina fused with those from the bladder. CONCLUSIONS The VUL does not contain lymphatics from the cervix uteri. Hence, the favorable survival outcomes of the Okabayashi technique cannot be explained by radical removal of lymphatic pathways in the ventrocaudal parametrium.

Dlx1 and Rgs5 in the Ductus Arteriosus: Vessel-Specific Genes Identified by Transcriptional Profiling of Laser-Capture Microdissected Endothelial and Smooth Muscle Cells

Closure of the ductus arteriosus (DA) is a crucial step in the transition from fetal to postnatal life. Patent DA is one of the most common cardiovascular anomalies in children with significant clinical consequences especially in premature infants. We aimed to identify genes that specify the DA in the fetus and differentiate it from the aorta. Comparative microarray analysis of laser-captured microdissected endothelial (ECs) and vascular smooth muscle cells (SMCs) from the DA and aorta of fetal rats (embryonic day 18 and 21) identified vessel-specific transcriptional profiles. We found a strong age-dependency of gene expression. Among the genes that were upregulated in the DA the regulator of the G-protein coupled receptor 5 (Rgs5) and the transcription factor distal-less homeobox 1 (Dlx1) exhibited the highest and most significant level of differential expression. The aorta showed a significant preferential expression of the Purkinje cell protein 4 (Pcp4) gene. The results of the microarray analysis were validated by real-time quantitative PCR and immunohistochemistry. Our study confirms vessel-specific transcriptional profiles in ECs and SMCs of rat DA and aorta. Rgs5 and Dlx1 represent novel molecular targets for the regulation of DA maturation and closure.

Toward a Highly-Detailed 3D Pelvic Model: Approaching an Ultra-Specific Level for Surgical Simulation and Anatomical Education

The surgical anatomy of the pelvis is highly complex. Anorectal and urogenital dysfunctions occur frequently after pelvic oncological surgery and are mainly caused by surgical damage of the autonomic nerves. A highly‐detailed 3D pelvic model could increase the anatomical knowledge and form a solid basis for a surgical simulation system. Currently, pelvic surgeons still rely on the preoperative interpretation of 2D diagnostic images. With a 3D simulation system, pelvic surgeons could simulate and train different scenes to enhance their preoperative knowledge and improve surgical outcome. To substantially enrich pelvic surgery and anatomical education, such a system must provide insight into the relation between the autonomic network, the lymphatic system, and endopelvic fasciae. Besides CT and MR images, Visible Human Datasets (VHDs) are widely used for 3D modeling, due to the high degree of anatomical detail represented in the cryosectional images. However, key surgical structures cannot be fully identified using VHDs and radiologic imaging techniques alone. Several unsolved anatomical problems must be elucidated as well. Therefore, adequate analysis on a microscopic level is inevitable. The development of a comprehensive anatomical atlas of the pelvis is no straightforward task. Such an endeavor involves several anatomical and technical challenges. This article surveys all existing 3D pelvic models, focusing on the level of anatomical detail. The use of VHDs in the 3D reconstruction of a highly‐detailed pelvic model and the accompanying anatomical challenges will be discussed Clin. Anat., 2013.

New aspects of development of the cardiac conduction system, clinical relevance

Summary Electrophysiological and new immunohistochemical markers have resulted in a better understanding of the development of the cardiac conduction system. In the primary heart tube the blood is pumped forward by peristaltic contractions as a result of alternating slow and fast conducting segments. At later stages a more specialized conduction system develops. The contribution of the primary fold and the sinus venosus to the formation of the cardiac conduction system is discussed as well as the role of extracardiac cells such as neural crest and epicardial derived cells. These new developmental aspects of the cardiac conduction system could provide an explanation of the position conduction system in heart malformations and certain arrhythmias in patients with structurally normal hearts.

What Is the Anatomical Relationship of the Nerves in the Pelvis

The pelvis is often considered to be a complex anatomical region. Due to the funnel-shaped pelvic cavity, there is a close anatomical relationship of the rectum to vital structures such as blood vessels, lymphatics and somatic and autonomic nerves. This, taken together with the relatively inaccessible location of the rectum, makes total mesorectal excision (TME) a challenging surgical procedure. The principles of TME surgery involve removal of the diseased rectum with the surrounding mesorectum as an en bloc specimen with an intact mesorectal fascia and preservation of the pelvic autonomic nerves [1]. Over the last decades, both oncological and functional outcomes of TME surgery have gained much attention. It is well known that surgical disruption of the autonomic and somatic nerves may result in postoperative urogenital and anorectal dysfunction, affecting the patient’s functional outcome [2]. If the oncological safety is not in danger, surgeons must preserve the autonomic and somatic nerves in order to warrant sufficient postoperative pelvic function.

What Is Cancer of the Rectum

Rectal cancer constitutes one-third of all colorectal cancers, representing the cancer with the second highest incidence and the second cause of cancer death in the western society [1, 2]. An estimated 100,000 new cases of rectal cancer are diagnosed each year in Europe. The incidence is increasing, mainly due to earlier detection and increasing age of the population, as the highest incidence of rectal cancer is found in the sixth and seventh decades. High incidence rates are found especially in western world populations, i.e. Western Europe, North America and Australia. This can probably be explained by a combination of factors, including dietary patterns with high amounts of red meat, obesity and smoking [3]. The United States is the only country with significantly decreasing incidence rates in both males and females in the most recent time period, which largely reflects detection and removal of precancerous lesions through colorectal cancer screening [4]. Next to dietary and life-style factors, risk factors for rectal cancer include inflammatory bowel disease and primary sclerosing cholangitis. Also genetic predisposition plays a role; however, rectal cancer most commonly occurs sporadically and is inherited in only 5% of the cases. Five-year survival rate of rectal cancer is about 60% and depends to a large extent on disease stage at diagnosis [5].