Double Basement Membrane Research Articles

An atypical basement membrane forms a midline barrier during left-right asymmetric gut development in the chicken embryo.

Correct intestinal morphogenesis depends on the early embryonic process of gut rotation, an evolutionarily conserved program in which a straight gut tube elongates and forms into its first loops. However, the gut tube requires guidance to loop in a reproducible manner. The dorsal mesentery (DM) connects the gut tube to the body and directs the lengthening gut into stereotypical loops via left-right (LR) asymmetric cellular and extracellular behavior. The LR asymmetry of the DM also governs blood and lymphatic vessel formation for the digestive tract, which is essential for prenatal organ development and postnatal vital functions including nutrient absorption. Although the genetic LR asymmetry of the DM has been extensively studied, a divider between the left and right DM has yet to be identified. Setting up LR asymmetry for the entire body requires a Lefty1+ midline barrier to separate the two sides of the embryo, without it, embryos have lethal or congenital LR patterning defects. Individual organs including the brain, heart, and gut also have LR asymmetry, and while the consequences of left and right signals mixing are severe or even lethal, organ-specific mechanisms for separating these signals are poorly understood. Here, we uncover a midline structure composed of a transient double basement membrane, which separates the left and right halves of the embryonic chick DM during the establishment of intestinal and vascular asymmetries. Unlike other basement membranes of the DM, the midline is resistant to disruption by intercalation of Netrin4 (Ntn4). We propose that this atypical midline forms the boundary between left and right sides and functions as a barrier necessary to establish and protect organ asymmetry.

Repositioning the Early Pathology of Type 1 Diabetes to the Extraislet Vasculature.

Type 1 diabetes (T1D) is a prototypic T cell-mediated autoimmune disease. Because the islets of Langerhans are insulated from blood vessels by a double basement membrane and lack detectable lymphatic drainage, interactions between endocrine and circulating T cells are not permitted. Thus, we hypothesized that initiation and progression of anti-islet immunity required islet neolymphangiogenesis to allow T cell access to the islet. Combining microscopy and single cell approaches, the timing of this phenomenon in mice was situated between 5 and 8 wk of age when activated anti-insulin CD4 T cells became detectable in peripheral blood while peri-islet pathology developed. This "peri-insulitis," dominated by CD4 T cells, respected the islet basement membrane and was limited on the outside by lymphatic endothelial cells that gave it the attributes of a tertiary lymphoid structure. As in most tissues, lymphangiogenesis seemed to be secondary to local segmental endothelial inflammation at the collecting postcapillary venule. In addition to classic markers of inflammation such as CD29, V-CAM, and NOS, MHC class II molecules were expressed by nonhematopoietic cells in the same location both in mouse and human islets. This CD45- MHC class II+ cell population was capable of spontaneously presenting islet Ags to CD4 T cells. Altogether, these observations favor an alternative model for the initiation of T1D, outside of the islet, in which a vascular-associated cell appears to be an important MHC class II-expressing and -presenting cell.

PS-R05-3: SEVERE GLOMERULAR MICROANGIOPATHY IN A PATIENT WITH NEPHROTIC SYNDROME FOLLOWING TAKAYASU ARTERITIS

Introduction: Takayasu arteritis (TA) is a large vessel inflammation that predominantly involves aorta and its main arteries. TA-caused kidney injury is known to be mainly due to renal artery stenosis. However, glomerulonephritis including membranous proliferative glomerulonephritis, focal segmental sclerosis, or crescentic nephritis has been histologically identified in some TA cases. Case description: A 69-year-old female presented with leg edema and refractory hypertension with nephrotic range proteinuria (17.0 g/gCr). She exhibited > 10 mmHg blood pressure discrepancy between left and right upper limbs and severe aortic regurgitation. Lab test showed high level of C-reactive protein and high erythrocyte sedimentation rate. Contrast-enhanced computed tomography identified wall thickening of the left subclavian artery, which led to the diagnosis of TA. Renal biopsy demonstrated that double basement membrane, enlarged subendothelial space, and mesangiolysis with no immunoglobulin deposition. Although azilsartan 40 mg QD and nifedipine 30 mg BID had been prescribed, systolic blood pressure (sBP) was still > 160 mmHg. Thus, while oral prednisolone was started to suppress TA-induced inflammation, azilsartan was replaced with sacubitril valsartan, an angiotensin receptor-neprilysin inhibitor (ARNI), for treating refractory hypertension. After a few weeks, sBP was decreased to 130 mmHg with sacubitril valsartan 200 mg QD and proteinuria was dramatically improved to 0.5 g/gCr. Discussion: TA commonly involves the renal artery, leading to stenosis and subsequent ischemic nephropathy. However, no stenosis and occlusion were observed in renal artery of this case. In addition, small vessel inflammation in the kidney such as vessel wall necrosis and immune cells infiltration around arterioles was not found. Serum levels of pro-inflammatory cytokines including vascular endothelial growth factor and interleukin-6, a well-known contributors for progression of TA-related inflammation, were within the normal range. The results indicate that TA-caused hypertension, but not vessel inflammation induced by TA, seems to play a pathological role for development of glomerular microangiopathy in this case. A previous study showed that 70% of TA patients presenting with glomerular lesion has severe hypertension and intimal thickening in renal arterioles was observed more frequently in TA patients presenting with glomerular lesion when compared to those without glomerulonephritis, which supports our hypothesis. In the present case, ARNI significantly reduced sBP which might directly contribute to the improvement of proteinuria. ARNI can be potent to control hypertension more effectively than conventional hypertensive agents in TA patients.

Unique basement membrane structure of human pancreatic islets: implications for β‐cell growth and differentiation

Basement membranes (BMs) are an important part of the physiological microenvironment of pancreatic islet cells. In mouse islets, beta-cells interact directly with BMs of capillary endothelial cells. We have shown that in the human islets, the capillaries are surrounded by a double BM both in foetal and adult tissues. The endocrine islet cells are facing a BM that is separate from the endothelia. Laminins are the functionally most important component of BMs. The only laminin isoform present in the human endocrine islet BM is laminin-511 (previously known as laminin 10). The islet cells facing this BM have a strong and polarized expression of Lutheran glycoprotein, which is a well-known receptor for the laminin alpha 5 chain. Dispersed human islet cells adhere to purified human laminin-511 and the binding is equally effectively blocked by a soluble form of Lutheran as by antibody against integrin beta1. Our results reveal unique features of the BM structure of human islets, different from rodents. This information has potentially important implications for the generation of an optimal microenvironment for beta-cell function, proliferation and differentiation.

Blood vessels of human islets of Langerhans are surrounded by a double basement membrane

Based on mouse study findings, pancreatic islet cells are supposed to lack basement membrane (BM) and interact directly with vascular endothelial BM. Until now, the BM composition of human islets has remained elusive. Immunohistochemistry with specific monoclonal and polyclonal antibodies as well as electron microscopy were used to study BM organisation and composition in human adult islets. Isolated islet cells and function-blocking monoclonal antibodies and recombinant soluble Lutheran peptide were further used to study islet cell adhesion to laminin (Lm)-511. Short-term cultures of islets were used to study Lutheran and integrin distribution. Immunohistochemistry revealed a unique organisation for human Lm-511/521 as a peri-islet BM, which co-invaginated into islets with vessels, forming an outer endocrine BM of the intra-islet vascular channels, and was distinct from the vascular BM that additionally contained Lm-411/421. These findings were verified by electron microscopy. Lutheran glycoprotein, a receptor for the Lm alpha5 chain, was found prominently on endocrine cells, as identified by immunohistochemistry and RT-PCR, whereas alpha(3) and beta(1) integrins were more diffusely distributed. High Lutheran content was also found on endocrine cell membranes in short-term culture of human islets. The adhesion of dispersed beta cells to Lm-511 was inhibited equally effectively by antibodies to integrin and alpha(3) and beta(1) subunits, and by soluble Lutheran peptide. The present results disclose a hitherto unrecognised BM organisation and adhesion mechanisms in human pancreatic islets as distinct from mouse islets.

喉頭異型上皮の癌化における血管新生と細胞外マトリックスの変化

The malignant progression mechanism of precancerous whitish lesions of the vocal cords is still unclear. In this study, the differences of angiogenesis and extracellular matrices between keratosis, dysplasia and invasive carcinoma of the vocal cords were investigated using immunohistochemical staining with anti-CD34, which discerns endothelial cells, and type IV collagen monoclonal antibodies.In vocal cord keratosis, only a few vessels with small diameters were observed under the basement membrane of the epithelium. An increased number of vessels were recognized around the dysplastic epithelium, downward papillary grow to the stroma. Interestingly, the basement membranes under the dysplastic epithelium and around the vessels were located very closely, suggesting some interaction between the two basement membranes. The double basement membranes were also observed in invasive carcinoma with exophytic growth. In addition, a type IV collagen staining was also seen around nests of carcinoma cells which had infiltrated into the stroma. These results suggest the important roles of the basement membrane and the extracellular matrix in the malignant progression and angiogenesis of the precancerous lesion of the vocal cords.

Anin VivoModel of Human Proliferative Scar

Background.Many aspects related to the biology and the effective therapy of proliferative scars have remained undefined, in part due to a lack of an accurate and reproducible animal model with which to systematically study them. This report describes a new model for investigating the pathophysiology and manipulation of human proliferative scars.Materials and methods.Human proliferative scars (n= 86) were explanted into flaps based on isolated vascular pedicles in congenitally athymic rats. Serial analysis of the structural and functional integrity of the explanted scars was performed by microscopy and by measurement of human procollagen type III peptide (PIIIP) production, human factor VIII immunostaining, andin vitrocellular proliferation.Results.By these methods, both fibroblastic and epithelial components of explanted scar specimens retained the histologic characteristics of original human scar specimens, for up to 12 months. Over the same duration, scar explants continued to have high levels of human PIIIP, comparable to those found in original surgical specimens. The microvasculature of scar explants demonstrated a double basement membrane, with no staining of human factor VIII in the inner capillary endothelial layer, suggesting that host vessels were growing into ghost vessels of the human donor scar. Human factor VIII staining decreased over time. Fibroblasts cultured from explanted scar demonstrated less aggressive growth characteristics than those from original surgical specimens.Conclusions.This new model is the first to allow such long-term maintenance and serial evaluation of human proliferative scar on an accessible, isolated vasculature. It may prove useful in further defining the biology and therapy of this widespread pathologic process.

Glomerular basement membrane outpockets and glomerular growth in the postnatal development of the rat kidney.

Distribution of glomerular basement membrane (GBM) outpockets and dimensional growth of glomeruli were studied in the maturing stage of rat glomeruli after completion of nephrogenesis. We observed the postnatal rat glomeruli from 5 to 60 days of age by transmission electron microscopy and estimated the structural development of glomeruli by computerized morphometry. On day 5, the GBM was double in structure, possessing an epithelial and endothelial lamina densa. After day 10, the lamina densa of the GBM was single and sent branches toward the epithelial side making outpockets. There is no change in the distributional pattern of the outpockets, at least from day 10 to day 60, although they decreased considerably in number between days 20 and 40. They were found almost exclusively on the peripheral surface of the glomerulus. The rat glomeruli increased in volume constantly in this period, and the capillary volume increased more significantly than the mesangial volume. The GBM surface area increased in parallel with the glomerular tuft volume. The growing mode of capillaries was different before and after day 40; namely before day 40 elongation was predominant, whereas after day 40 widening was more pronounced. These results indicate that if the outpockets are the other site of GBM assembly after fusion of double basement membranes, the GBM must be redistributed from the peripheral to the paramesangial site to enable elongation and branch formation of capillaries during the growth of glomeruli.

Ultrastructure of developing kidney glomerular basement membranes: temporal changes in binding of anti-laminin IgG and cationized ferritin.

In vivo labeling of infant rat and mouse glomerular basement membranes (GBMs) with polyclonal anti-laminin IgGs results in binding across the full widths of GBMs at all stages of development. These stages include the pre-fusion, double basement membranes found beneath endothelial cells and podocytes in early glomeruli, and the subepithelial matrix outpockets where newly synthesized GBM is spliced into fused basement membrane during glomerular maturation. Identical binding results are obtained either with peroxidase or post-embedding immunogold techniques. Although injected cationized ferritin also binds abundantly to all developing GBMs, it quickly disappears and, 24 hours after injection, is generally absent from GBMs but remains within mesangial matrices. Injection of newborn mice with monoclonal anti-laminin IgGs results in dense labeling of pre-fusion GBMs but post-fusion GBMs and subepithelial outpockets are weak-negative. Although masking can not be excluded, these results indicate that laminin epitopes are removed during GBM fusion and splicing, either by isoform substitution or proteolytic processing. The loss of bound cationized ferritin is believed to occur mainly through rapid turnover of GBM proteoglycans.

Experimental cerebral infarction in the dog. Ultrastructural study of microvessels in subacute cerebral infarction.

Large-sized infarctions of the cerebral hemisphere (LSICH) were experimentally induced in 20 dogs by injecting two silicone cylinders through the right cervical internal carotid artery. The LSICH models were perfused with heparinized saline and fixed with 1.5% glutaraldehyde solution through the contralateral carotid artery 7 days after the embolization. Mercox® was then injected manually. The brains were immediately removed, and specimens were obtained from the basal ganglia and the ectosylvian gyrus. Regenerated microvessels were observed within the necrosis in the basal ganglia. The regenerated microvessels had an irregular, “bead-like” surface, and formed anastomosed networks. The regenerated capillaries had double basement membranes and endothelia with abundant ribosome and increased pinocytotic vesicles. It is suggested that the regenerated capillaries had immature blood-brain barriers of increased permeability. It is thought that the brain functions of this area are reversible. The microvessels within the vasogenic edema in the corticomedullary junctional area of the ectosylvian gyrus had smooth surfaces, and formed normal capillary networks, but their perivascular spaces were dilated with edematous fluids.

Evidence for splicing new basement membrane into old during glomerular development in newborn rat kidneys.

Tannic acid in glutaraldehyde fixatives greatly enhanced the visualization of two developmentally and morphologically distinct stages in glomerular basement membrane (GBM) formation in newborn rat kidneys. First, in early stage glomeruli, double basement membranes between endothelial cells and podocytes were present and, in certain areas, appeared to be fusing. Second, in maturing stage glomeruli, elaborate loops and outpockets of basement membrane projected into epithelial, but not endothelial, sides of capillary walls. When Lowicryl thin sections from newborn rat kidneys were sequentially labeled with rabbit anti-laminin IgG and anti-rabbit IgG-colloidal gold, gold bound across the full width of all GBMs, including double basement membranes and outpockets. The same distribution was obtained when sections from rats that received intravenous injections of rabbit anti-laminin IgG 1 h before fixation were labeled directly with anti-rabbit IgG-colloidal gold. When kidneys were fixed 4 d after anti-laminin IgG injection, however, loops beneath the podocytes in maturing glomeruli were usually unlabeled and lengths of unlabeled GBM were interspersed with labeled lengths. In additional experiments, rabbit anti-laminin IgG was intravenously injected into newborn rats and, 4-14 d later, rats were re-injected with sheep anti-laminin IgG. Sections were then doubly labeled with anti-rabbit and anti-sheep IgG coupled to 10 and 5 nm colloidal gold, respectively. Sheep IgG occurred alone in outpockets of maturing glomeruli and also in lengths of GBM flanked by lengths containing rabbit IgG. These results indicate that, after fusion of double basement membranes, new segments of GBM appear beneath developing podocytes and are subsequently spliced into existing GBM. This splicing provides the additional GBM necessary for expanding glomerular capillaries.

Distribution of intravenously injected cationized ferritin within developing glomerular basement membranes of newborn rat kidneys.

To label heparan sulfate proteoglycans and other strong anions within glomerular basement membranes (GBM) during assembly, cationized ferritin (CF), with a narrow isoelectric range of 7.7 to 8.2, was intravenously injected into newborn rats. Kidneys were then fixed and processed for electron microscopy at intervals ranging from 1 to 72 h after CF injection. One hour after injection, CF bound extensively to the lamina rara interna and externa of developing GBM and mesangial matrix and to tubular basement membranes (TBM). In double basement membranes of early stage glomeruli, large amounts of CF were also seen in central areas between the endothelial and epithelial basement membranes. In maturing-stage glomeruli, CF bound throughout interior regions of GBM outpockets projecting into the epithelial side of capillary walls as well as to the laminae rarae. Because in adult rats CF binds only to the laminae rarae, the abundant anionic sites seen here in newborns between double basement membranes and within GBM outpocket interiors may be subsequently neutralized or removed during the GBM assembly process. In addition to basement membranes, CF was also located intracellularly within endocytic vesicles and lysosomes of glomerular endothelial, mesangial, and epithelial cells 1 h postinjection. CF was also present in similar structures within the tubular epithelium. In contrast to these findings, CF was gradually lost from developing GBM 5, 15, and 24 h after injection and was essentially cleared from all GBM, mesangial matrices, and TBM after 48 h. Large CF aggregates were progressively accumulated within mesangial lysosomes, however. The transient binding of CF to GBM anionic sites seen here was most likely due to its endocytic removal by developing glomerular endothelial, mesangial, and epithelial cells. Anions in the circulation probably also competed effectively with the GBM and TBM for bound CF.

Origin of the glomerular basement membrane visualized after in vivo labeling of laminin in newborn rat kidneys.

To examine the origin and assembly of glomerular basement membranes (GBMs), affinity purified anti-laminin IgG was directly coupled to horseradish peroxidase (HRP) and intravenously injected into newborn rats. Kidneys were then processed for peroxidase histochemistry and microscopy. Within 1 h after injection, anti-laminin bound to basement membranes of nephrons in all developmental stages (vesicle, comma, S-shaped, developing capillary loop, and maturing glomeruli). In S-shaped and capillary loop glomeruli, anti-laminin-HRP labeled a double basal lamina between the endothelium and epithelium. Sections incubated with anti-laminin in vitro showed labeling within the rough endoplasmic reticulum of endothelium and epithelium, indicating that both cell types synthesized laminin for the double basement membrane. In maturing glomeruli, injected anti-laminin-HRP bound throughout the GBMs, and double basement membranes were rarely observed. At this stage, however, numerous knobs or outpockets of basement membrane material extending far into the epithelial side of the capillary wall were identified and these were also labeled throughout their full thickness. No such outpockets were found in the endothelial cell layer of newborn rats (and they normally are completely absent in fully mature, adult glomeruli). In contrast with these results, in kidneys fixed 4-6 d after anti-laminin IgG-HRP injection, basement membranes of vesicle, comma, and S-shaped nephrons were unlabeled, indicating that they were assembled after injection. GBM labeling was seen in maturing glomeruli, however. In addition, the outpockets of basement membrane extending into the epithelium were often completely unlabeled whereas GBMs lying immediately beneath them were labeled intensely, which indicates that the outpockets were probably assembled by the epithelium. Injections of sheep anti-laminin IgG followed 8 d later with injections of biotin-rabbit anti-laminin IgG and double-label immunofluorescence microscopy confirmed that GBM formation continued during individual capillary loop expansion. GBM assembly therefore occurs by at least two different processes at separate times in development: (a) fusion of endothelial and epithelial basement membranes followed by (b) addition of new basement membrane from the epithelium into existing GBMs.

Thymic Stromal Alterations in Mice Undergoing a Chronic Graft-Versus-Host Reaction

Graft-versus-host induced immunosuppression has previously been shown to be accompanied by severe morphological changes in the thymus; furthermore chronic GvH could become acute by grafting a normal syngeneic thymus, suggesting a functional defect in the autologous thymus. In this work, we monitored the changes occuring in two biologically active thymic stromal fractions during a state of chronic GvH reaction. It was thus observed that a soluble thymic factor (STF), normally found in the reticuloepithelial cells, was lost, and that an insoluble thymic fraction (ITF) found in a double basement membrane surrounding medullary blood vessels, became markedly hypertrophied. These changes are interpreted as being possibly related to the state of immunosuppression by interfering with normal T cell differentiation and traffic through the thymus.

Ultrastructure of the neurohypophysis of the teleost Poecilia latipinna in relation to neural control of the adenohypophysial cells

The structure of the neurohypophysis of Poecilia latipinna (green molly, sailfin molly) was studied with the electron microscope. Profile diameters of neurosecretory granules in the non-myelinated neurohypophysial nerve fibres were measured and mathematically corrected for error due to section thickness. Six different types of nerve fibres could be distinguished by statistical classification of their granules and by other ultrastructural features. One fibre-type (type B) contained granules with a mean diameter of 85 nm, and the other five types (types Ala, Alb, A2, A3 and A4) all contained granules with mean diameters greater than 100 nm. Synaptic contacts were observed between type B fibres and all the adenohypophysial cell-types, although in the case of the ACTH cells the synapses were separated from the cell membrane by a continuous double basement membrane. Type A fibres were observed to contact the cells of the proximal pars distalis and pars intermedia, but did not form synapses. However, synapses occurred between type A fibres and pituicytes, and between type A fibres and the pericapillary basement membrane in the interior of the neurohypophysis. The possible roles of the different types of nerve fibres in controlling the adenohypophysial cells are discussed in the context of evidence from other teleosts.

Hypothalamic Hamartoma

The presence of a hypothalamic hamartoma and precocious puberty in a 19-month-old boy provided an opportunity to study their relation. Excised tissue had the ultrastructural characteristics of an independent neuroendocrine unit -- i.e., neurons containing neurosecretory granules and blood vessels with fenestrated endothelium and double basement membranes. Immunofluorescence studies using specific antibody to luteinizing-hormone-releasing factor showed antigenicity to the factor in the hamartoma. The testicular-hypothalamic-pituitary axis was tested. Clomiphene unresponsiveness suggested a lack of maturation of central-nervous-system events characteristic of normal puberty. The negative feedback system between gonad and brain was intact but partially resistant to steroid suppression. These studies suggest that hypothalamic hamartomas may cause precocious puberty by autonomous production and release of luteinizing-hormone-releasing factor into vessels that communicate with the pituitary portal blood system.

The Pituitary Gland of the RoachLeuciscus rutilus

Abstract Two endocrine cell types, PbH+ and PAS+ cells, were recognized in the pars intermedia (PI) of the roach, Leuciscus rutilus, by light and electron microscopy. They can be distinguished by the appearance of their secretory granules. The PAS+ cells stain with toluidine blue in semithin sections as opposed to the PbH+ cells. After 5 days' treatment with metopirone the PbH+ cells are highly activated whereas the PAS+ cells show no response. The PbH+ cells are assumed to produce MSH. In roaches adapted to diluted sea water (10%) the transfer to fresh water for 1—1 1/2 hours or reserpine injections for the same period had no notable effect on neither cell type. Three kinds of axons innervate the PI. Two of these, which have granules averaging 111 nm and 151 nm in diameter, originate in the nucleus preopticus (NPO). The third type of axons has granules of an average size of 74 nm and is probably aminergic. The glia cells in this part of the neurohypophysis (NH) are granular pituicytes, which are innervated by all three axon types. The border between the NH and the PI is composed of a double basement membrane from which a network extends into the NH. This network, the basement membrane and the capillaries are the main sites for the terminations of the axons although nerve fibers also invade the endocrine tissue. Only axon type 3 was observed to synapse frequently with the PbH+ cells.

The Pituitary Gland of the RoachLeuciscus rutilus

Three different endocrine cell types were identified by electron microscopy in the rostral pars distalis (RPD) of the roach, Leuciscus rutilus. The cells which are characterized by the morphology and sizes of their secretory granules are assumed to secrete prolactin, ACTH and TSH. Prolactin cells occur in two forms which could be separated on account of the sizes of their granules. Intra-peritoneal injections of reserpine had a strong and persistent effect 24 hours after the treatment. The prolactin cells with small granules reacted with a marked release of secretion. The border of the RPD is composed of a so-called double basement membrane in contact with the perivascular spaces and forms extensive ramifications into the rostral neurohypophysis (RNH). Nerve fibers of different categories terminate at the border and its network in the RNH. Substances released from these terminals may influence the endocrine cells of the RPD. The ACTH cells and the prolactin cells are innervated by axons with granules less than 100 nm. The nomenclature of type ‘A’ fibers and ‘B’ fibers is discussed because it was difficult to make a reliable discrimination of the fibers and their nature by the sizes of their granules. However, four types of nerve fibers were distinguished in connection with the RPD although the presence of more types was indicated. In all four types the size variations of the granules are great and overlapping. The average sizes of the granules in these fibers are 157, 114, 119 and 81 nm. The origin of the nerve fibers innervating the RPD is discussed. All morphological data indicate a complex hypothalamic control of the RPD of the roach.

Area postrema: Chemoreceptor trigger zone for vomiting - is that all?

Publisher Summary This chapter discusses the role of area postrema (AP) as chemoreceptor trigger zone for vomiting. The area postrema of Retzius is the most caudal of the circumventricular organs in the brain. It is found that in all mammalian species, as well as in many other vertebrates, astride the opening of the spinal canal into the fourth ventricle. AP is one of an uncertain number of midline structures that are in contact with the cerebrospinal fluid and whose capillaries, like those in the choroid plexuses, are permeable to protein-bound vital dyes. The capillaries of AP are similar to those in choroid plexus in that they consist of a leaky fenestrated endothelium surrounded by a double basement membrane forming a perivascular space with which the capillary blood makes water and solute exchange. Pinocytotic vesicles are often found in the endothelial cells, and luminal microvilli have been observed in the capillaries. The peripapillary space is believed to represent an extension of the subarachnoid space, with the external basement membrane corresponding to the piamater. This arrangement should then permit direct chemical communication between blood and subarachnoid CSF.