Fine Collagen Fibrils Research Articles

The structure and properties of natural sheep casing and artificial films prepared from natural collagen with various crosslinking treatments

The structure and properties of natural sheep casing and collagen films with various crosslinking treatments have been investigated in detail to develop satisfied artificial casings prepared from collagen. The sheep casing consists of large number of thick collagen fibers oriented at ±45° from longitudinal direction with high-density interwoven network structure. The structural feature of sheep casing gave the special mouthfeel of ‘cracking bite’ of sausages. Whereas, layered structure filled with fine collagen fibrils and large gaps in collagen film results in poor mechanical properties and higher swelling ratio in water. Furthermore, a degree of denaturation of collagen during extraction process also lead to poor mechanical properties. After glutaraldehyde (GTA) and dehydrothermal (DHT) treatments, the formation of crosslinking improved mechanical properties of collagen films significantly and the tensile strength and tensile modulus increased more than three times compared with those of untreated collagen film in wet before and after boiling. The swelling ratio of treated collagen films also decreased dramatically. No obvious effects on denaturation of collagen film after GTA treatment, but the degree of denaturation of DHT treated collagen film increased slightly.

Changes in collagens and chondrocytes in the temporomandibular joint cartilage in growing rats fed a liquid diet

The temporomandibular joint (TMJ) of growing rats fed a soft diet is reported to be smaller in size and to have thinner condyle and glenoid fossa cartilage than rats fed a solid diet. The aim of this study was to determine the effect of a soft diet on the collagens and chondrocytes in the growing TMJ cartilage. Forty-eight male Wistar rats were divided into a control group fed a solid diet and an experimental group fed a liquid diet for 1–8 weeks. After the experimental period, the TMJs were harvested and examined histologically, immunohistochemically for collagen types I, II, and X, and with transmission electron microscopy. The condylar cartilage in the experimental rats showed weak immunoreactions for three types of collagens compared with the controls. The ultrastructure had fewer fine collagen fibrils in the experimental rats compared with that of the controls. The glenoid fossa cartilage in the experimental rats showed narrower Alcian blue-positive areas than the control staining. The immunoreactions for three types of collagen in the experimental rats were also weaker than those of the controls. The chondrocytes in the experimental rats appeared dark, had extended thin cytoplasmic processes, and had formed gap junctions, as assessed by transmission electron microscopy. Fewer fine collagen fibrils, but thick bands of collagen fibrils were observed in the glenoid fossa of the experimental cartilage. The results of the present study showed that a liquid diet had deleterious effects on the quality and quantity of collagens and chondrocytes in the TMJ cartilage in growing rats.

Scale structure in the Australian lungfish, Neoceratodus forsteri (Osteichthyes: Dipnoi).

Scales of the Australian lungfish, Neoceratodus forsteri, are secreted within the dermis by a capsule of scleroblasts, and enclosed in a pouch made of collagen fibers, in contact with the epidermis over the posterior third of the scale. Each scale grows from a focus, which represents the first formed part of the scale. On the internal surface of the scale is elasmodin, made of collagen fiber bundles arranged in layers. Elasmodin, unmineralized in N. forsteri, contains cells in the living animal, and the number of layers increases as the scales grow. Squamulin, on the thin external part of the scale, is also laid down in layers, and based on a matrix of fine collagen fibrils, mineralized with a poorly crystalline biogenic calcium hydroxylapatite. Squamulin is divided into separate sections called squamulae, and contains long tubules with cells applied to the wall of the tubule. The anterior and lateral surfaces of the squamulin are ornamented with pediculae, and the posterior surface has longitudinal ridges, from which collagen fibers extend to anchor the scale within the pouch. Elasmodin and squamulin are linked by unmineralized collagen fibrils. The layers, formed at irregular intervals, are connected around the margin of the scale, effectively converting the whole scale into a flat structure resembling a pearl, with the first formed tissues deeply embedded inside the scale, and the youngest on the outer surface. Incremental lines in the hard tissue, and the number of layers in the elasmodin, do not reflect the chronological age of the fish.

Formation and structure of scales in the Australian lungfish, neoceratodus forsteri (Osteichthyes: Dipnoi)

The large elasmoid scales of the Australian lungfish, Neoceratodus forsteri, are formed within the dermis by unpigmented scleroblasts, growing within a collagenous dermal pocket below a thick glandular epidermis. The first row of scales, on the trunk of the juvenile lungfish, appears below the lateral line of the trunk, single in this species, at around stage 53. The scales, initially circular in outline, develop anteriorly and posteriorly from the point of initiation in the mid-trunk region, and rows are added alternately below the line, and above the line, until they reach the dorsal or ventral midline, or the margins of the fins. Scales develop later on the ventral surface of the head, from a separate centre of initiation. Scales consist of three layers, all produced by scleroblasts of dermal origin. The outermost layer of interlocking plates, or squamulae, consists of a mineralised matrix of fine collagen fibrils, covered by unmineralised collagen and a single layer of cells. Squamulae of the anterior and lateral surfaces are ornamented with short spines, and the mineralised tissue of the posterior surface is linked to the pouch by collagen fibrils. The innermost layer, known as elasmodin, consists of bundles of thick collagen fibrils and cells arranged in layers. An intermediate layer, made up of collagen fibrils, links the outer and inner layers. The elasmoid scales of N. forsteri can be compared with scale types among other osteichthyan groups, although the cellsand canaliculi in the mineralised squamulae bear little resemblance to typical bone.

Ultrastructure of submandibular salivary glands of mouse: TEM and HRSEM observations

The fine structure of submandibular glands of mouse were analyzed using light microscopy (LM), high resolution scanning electron microscopy (HRSEM), and transmission electron microscopy (TEM) methods. For LM, the specimens were embedded in Spurr resin, stained by toluidin blue solutions. For TEM, the tissues of submandibular salivary glands were fixed with modified Karnovsky solution and postfixed with osmium tetroxide. For HRSEM, the tissues were fixed with 2% osmium tetroxide solution in 1/15M sodium phosphate buffer (pH 7.4). The samples were immersed successively in dymethylsulphoxide and freeze cracked. The maceration was made in diluted osmium tetroxide for 24-48 h. The samples were examined by high resolution scanning electron microscopy. The intracellular components of acinar and ductal cells revealed clearly the Golgi apparatus, rough endoplasmic reticulum, secretory granules, and mitochondria. The end bulbs of Golgi lamellae and flattened cisterns of rough endoplasmic reticulum showed the luminal surface. A few mitochondria were identified intermingling between the rough endoplasmic reticulum and the mitochondriales cristae in three-dimensional HRSEM images. Secretory granules were numerous and presented different sizes. Small granules of ribosomes were attached on cistern surface, measuring 20-25 nm in diameter. Numerous arranged microvilli were found on the luminal surface of secretory canaliculus. The contact surfaces of acinar cells revealed complicated interdigitations by cytoplasmic processes. The mitochondria of duct cells were disposed vertically and surrounded by basal infoldings of plasma membranes. Basement membrane showed a spongy-like structure having an irregular surface with various strands and meshes of fine collagen fibrils.

Fibroblast expression of α-smooth muscle actin, α2β1 integrin and αvβ3 integrin: Influence of surface rigidity

Open wound contraction necessitates cell and connective tissue interactions, that produce tension. Investigating fibroblast responses to tension utilizes collagen coated polyacrylamide gels with differences in stiffness. Human foreskin fibroblasts were plated on native type I collagen-coated polyacrylamide gel cover slips with different rigidities, which were controlled by bis-acrylamide concentrations. Changes in alpha smooth muscle actin (αSMA), α2β1 integrin (CD49B) and αvβ3 integrin (CD-51) were documented by immuno-histology and Western blot analysis. Cells plated on rigid gels were longer, and expressed αvβ3 integrin and αSMA within cytoplasmic stress fibers. In contrast, cells on flexible gels were shorter, expressed α2β1 integrin and had fine cytoskeletal microfilaments without αSMA. Increased tension changed the actin makeup of the cytoskeleton and the integrin expressed on the cell's surface. These in vitro findings are in agreement with the tension buildup as an open wound closes by wound contraction. It supports the notion that cells under minimal tension in early granulation tissue express α2β1 integrin, required for organizing fine collagen fibrils into thick collagen fibers. Thicker fibers create a rigid matrix, generating more tension. With increased tension cytoskeletal stress fibers develop that contain αSMA and αvβ3 integrin that replaces α2β1 integrin, consistent with cell switching from collagen to non-collagen proteins interactions.

Chemical visualization of individual chondrocytes in articular cartilage by attenuated-total-reflection Fourier Transform Infrared Microimaging

Fourier transform infrared imaging (FTIRI) and the attenuated total reflection Fourier transform infrared microimaging (ATR-FTIRM) were used to study the chemical and structural distributions of cellular components surrounding individual chondrocytes in canine humeral cartilage, at 6.25µm pixel resolution in FTIRI and 1.56µm pixel resolution in ATR-FTIRM. The chemical and structural distributions of the cellular components in chondrocytes and tissue can be successfully imaged in high resolution ATR-FTIRM. One can also study the territorial matrix of fine collagen fibrils surrounding the individual chondrocytes by the polarization experiments using the absorption ratio of amide I to amide II bands.

Are the cytoplasmic microstructures within GBM pathologically significant?

The current issue of CEN features the so-called podocytic infolding glomerulopathy, which is characterized by the emergence of spherical or tubular cytoplasmic microstructures in association with cytoplasmic projections of podocytes within the thickened glomerular basement membrane (GBM) in electron micrographs as well as nonargyrophilic holes in the GBM at light microscopic levels [1]. This type of glomerular change has been recently proposed as a possible new pathological entity in Japan. The three original articles and 15 case reports in this issue are the first survey of the cases with this type of glomerular pathology. The GBM and other basement membranes are made up by self-assembly of constituent molecules, such as collagen type IV and laminin, which are secreted by the adjacent parenchymal cells. The established basement membranes are thought to be a stable structure, providing a mechanical support and constant milieu for the parenchymal cells. Reflecting the structural and functional stability of basement membranes, cytoplasmic microstructures and cellular projections have been only rarely reported within the basement membranes. One of those rare observations includes the basement membranes of the proximal convoluted tubule, which contains occasional cytoplasmic vesicles at the concavity of the tubular bend [2]. These vesicles are thought to be torn off from the basal cytoplasm of the proximal tubular cells due to higher mechanical stress at this location, and preserved within the basement membranes which shield the vesicles from disposal by interstitial cells. In this issue, Fujigaki et al. [3] showed clearly that the microstructures in the GBM are connected with the cellular processes of podocytes by three dimensional analysis. Another example of heterogeneity of the basement membrane was found around the ductal epithelium of excretory glands, including the liver, pancreas, lacrimal glands and salivary glands [4, 5], where the cellular projections of ductal epithelial cells were associated multilaminar basement membranes and fine collagen fibrils. The multilaminar lamina densa together with fine collagen fibrils were thought to be produced by the epithelial cells. The functional significance of cellular projection of ductal epithelial cells are unknown, but may be correlated the extremely high mechanical demand of this epithelium. The 25 cases reported in this issue revealed clearly that the podocyte infolding glomerulopathy represents not solitary unreproducible observations, but constantly reproducible ones, although the incidence is extremely low. The unique microscopic appearance suggests the common pathological process of these cases, but the etiology is obviously diverse. In this issue, Masuda et al. [6] reported a different type of glomerulopathy with cytoplasmic microstructures within GBM, and suggested that they represented cellular debris trapped during the remodeling of the GBM. What does the morphology of podocyte infolding glomerulopathy indicate? It would be to some extent rationalized to suppose that the podocytes in this condition are surrendered to more mechanical demand to produce more GBM materials and make basal projections which are torn off to deliver cytoplasmic microstructures to be remained for considerable period in GBM. The paucity of the cases and the diversity of the etiology at present prevent further analysis of the pathology. T. Sakai (&) Department of Anatomy and Life Structure, Graduate School of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan e-mail: tatsuo@juntendo.ac.jp

Adult vitreous structure and postnatal changes

This review will focus on the molecular organisation of the adult vitreous and how it undergoes ageing changes throughout life that result in vitreous liquefaction and a predisposition towards posterior vitreous detachment and retinal break formation. At birth, the vitreous humour is in a gel state due to the presence of a network of fine collagen fibrils. With ageing, these collagen fibrils progressively aggregate due to a loss of type IX collagen from their surfaces. The aggregation of collagen fibrils may cause vitreous liquefaction which, when combined with an age-related weakening of postbasal vitreoretinal adhesion, predisposes to posterior vitreous detachment. Throughout postnatal life, the posterior border of the vitreous base migrates posteriorly from the ora serrata into the peripheral retina. This is due to new collagen synthesis by the peripheral retina. This new collagen intertwines with pre-existing cortical vitreous collagen to create new adhesions and thereby extends the vitreous base posteriorly. If irregularities in the posterior border of the vitreous base arise from this process, there is a predisposition towards retinal break formation during posterior vitreous detachment and subsequent rhegmatogenous retinal detachment.

Effect of Collagen Oligopeptide Injection on Rabbit Tenositis

Effects of the collagen oligopeptide (COP) were examined by its repeat injection into the inflammatory rabbit Achilles tendon (Tendo calcaneus communis), in which tenositis was induced by injection of bacterial collagenase. COP was evaluated 5 times over a period of 3 weeks to 1 month after injection of collagenase. At 1 month after treatment, the therapeutic effect of COP was evaluated by examining the structure of collagen fibrils, amount and components of glycosaminoglycans (GAGs) and matrix metalloproteinases (MMPs), and compared with the saline injection, control, and normal groups. The Achilles tendon of rabbit in the control group (no COP injection) and saline injection group showed a notable increase in the number of fine collagen fibrils, a change in GAG composition and increase in the amount of pro-MMP-2, indicating the weakening of the tendon. In contrast, the size distribution of collagen fibrils, GAG composition and the amount of pro-MMP-2 was similar to those in the normal group. These results suggest that COP injection promotes healing processes of the tendon injury.

Growth changes of the collagen content and architecture in the pectoralis and iliotibialis lateralis muscles of cockerels

1. Growth changes of the collagen content and architecture in the pectoralis (PT) and iliotibialis lateralis (ITL) muscles were examined using cockerels from 1 to 14 weeks of age. 2. Total collagen content in PT muscle showed little change, but in ITL muscle reached a maximum at 5 weeks and thereafter decreased slightly until 14 weeks. The collagen content was markedly larger in ITL muscle after 5 weeks. Pyridinoline content of collagen increased abruptly from 5 to 14 weeks in both muscles, but no difference between muscle types was detected. 3. The cell size of the endomysial honeycombs increased with the development of myofibres, and the mesh size of the perimysium around the honeycombs enlarged. 4. In both muscles endomysia were an incomplete network of collagen fibrils with many foramina at one week, became a very thin membrane of felt-like fabric in 2 to 5 weeks and thereafter increased in thickness until 11 to 14 weeks. 5. Perimysial width around the secondary fasciculus differed between the muscle types after 5 weeks. In the wider perimysium of ITL muscle, the collagen fibres increased in number and size to make a stack of collagen bands around the fasciculus. In the narrower perimysium of PT muscle, a few platelets of collagen fibres also developed. 6. The perimysial collagen fibre at 1 to 2 weeks had a smooth surface and appeared to be composed of fine collagen fibrils. The fibre at 11 to 14 weeks showed a rugged surface and was composed of coarser collagen bundles that combined with each other into a net-like configuration with very slim meshes. 7. Our results showed that the collagenous components of chicken intramuscular connective tissue changed markedly during the early period of muscle growth in distribution, architecture and quality but with little difference in quantity.

CxGELSIX: A Novel Preparation of Type VI Collagen with Possible Use as a Biomaterial

This study was initiated to evaluate tissue acceptance and stability of a novel type VI collagen preparation (CxGelsix) as a biomaterial in the rabbit corneal stroma. We hypothesized that CxGelsix, embedded intrastromally, does not have any adverse affect on surrounding corneal tissues, and remains intact in the presence of an acute inflammatory reaction during corneal wound healing. Type VI collagen was extracted and purified from rabbit corneal stroma under nondenaturing conditions. This preparation, Gelsix, was concentrated and cross-linked with polyethylene glycol to produce a transparent film (CxGelsix). Discs of CxGelsix, 4.0-mm diameter, 9- to 35-microm thick were implanted intrastromally and clinically examined periodically for 4 months. In another experiment, implantation of CxGelsix, 2.0-mm-diameter, was followed by corneal wounding adjacent to the implant and examined clinically for 30 weeks. At the end of these periods, the tissues from these experiments were processed for light and transmission electron microscopy. An intralamellar 4.0-mm-diameter disc of CxGelsix does not alter the structure of corneal epithelium above the implant, suggesting normal transport of nutrients through CxGelsix. Moreover, no structural abnormalities were seen in the rest of the cornea, and the cornea remains transparent. Although the cornea accepts the presence of CxGelsix disc as judged by clinical criteria, gradual degradation of the implant is seen ultrastructurally. CxGelsix is remarkably stable despite its exposure to endogenous enzymes during inflammation and wound healing. Partial degradation of the implant occurs only after many months, and it is gradually replaced with bundles of fine collagen fibrils reminiscent of normal cornea. The results of this study suggest that CxGelsix is potentially useful as a biomaterial.

Assessment of cell alignment by fibronectin multi-fibre cables capable of large scale production

The repair of damaged human tissue will be enhanced greatly by a capacity to organise the arrangement of the cells. We have demonstrated an approach to quantifying the capacity of fibronectin multi-fibre cables to align human cells. It is based on staining and subsequent image analysis of cells in the neighbourhood of the cables, and the application of an Orientation Index (S), to give a quantitative measure of alignment of the cells. Alignment of human dermal fibroblasts, parallel to the fibronectin cable axis, was observed by light microscopy after 3 days in culture. At 7 days, alignment was determined by image analysis after hematoxylin and eosin staining. This showed that the lateral extent of cell alignment i.e. the number of cell layers lined up beside a cable, termed the cell docking band width, was independent of cable diameter. Scanning electron microscopy confirmed cell alignment and the deposition of fine collagen fibrils by the cells. Orientation of cells to the cable, as measured by an orientation index (S), was S = 0.97 ± 0.05 in cell docking bands denoting almost perfect alignment. Cell seeding levels ranging from 14 K cells/cm2 up to 54 K cells/cm2 resulted in cell docking band widths of aligned cells from 335 μm to 890 μm respectively, increasing as a function of cell seeding levels. This method represents a quantitative measure of quality for potential contact guidance materials. The guidance system is capable of large-scale manufacture.

Fibrosis and inflammatory cells in human chronic chagasic myocarditis: scanning electron microscopy and immunohistochemical observations

The present study deals with both pathologic fibrosis and matrix connective tissue in chronic chagasic myocarditis. A total of 12 hearts were obtained at autopsy. Eight cases of chronic chagasic myocarditis were selected. Four cases without evidence of cardiac disease were used as controls. The diagnosis of chronic Chagas' heart disease was based on previously established criteria. A cell-maceration method was utilized to evaluate the spatial organization of the fibrillar collagen accumulation after removal of the myocardial tissue non-fibrous elements. The relationship between inflammatory cells identified by monoclonal antibodies and interstitial fibrosis stained with picrosirius red was assessed. Striking structural alterations of the collagen matrix in the perimysium were detected: increase in number and thickness of tendon-like structures, and markedly thickened and aggregated collagen strands. Besides, a diffuse increase in the thickness of collagen fibers surrounding individual myocytes, consisting of the endomysial matrix, mainly adjacent to the perimysium, could be observed. The dense-weave endomysial meshwork was composed of fine collagen fibrils, and it was continuous with those of adjacent myocytes, obscuring the lateral struts. Sometimes, thicker struts tethering myocytes to myocytes could be seen. These changes were associated with scattered dense scar-like foci, probably reflecting repair fibrosis associated with myocyte necrosis. Furthermore, the present results clearly showed the colocalization of foci of myocyte necrosis and degeneration and associated fibrosed areas and fibroblasts with T lymphocytes and macrophages. The accumulation of interstitial collagen fibers in chronic chagasic myocarditis may be expected to decrease myocardial compliance and disrupt synchronous contraction of the ventricles during systole, contributing to a spectrum of ventricular dysfunction that involve either the diastolic or systolic phase of the cardiac cycle or both. Myocardial fibrosis can be also implicated in the genesis of malignant ventricular tachyarrhythmias, major causes of sudden death among chronic chagasic patients. The increase in myocardial fibrosis could be directly related to an inflammatory reaction mainly composed of T lymphocytes and macrophages.

Neural architecture in transected rabbit sciatic nerve after prolonged nonreinnervation.

Observations have been made on the rabbit sciatic nerve distal to a transection, with survival periods of up to 26 mo and prevention of reinnervation. It was confirmed that the nerve becomes compartmented by fibroblast processes and that a zone of fine collagen fibrils develops around the Schwann cell columns that constitute the Büngner bands. The Schwann cells become progressively more atrophic but after 6 mo of denervation still expressed low affinity p75 nerve growth factor receptor (NGFR), the latest stage at which this was examined. NGFR was also expressed by the processes of the fibroblasts producing the endoneurial compartmentation. By 26 mo after transection the site of previous nerve fibres was indicated by sharply demarcated domains of approximately circular outline in transverse section consisting of densely packed longitudinally oriented collagen fibrils. Some of these domains still possessed centrally situated Schwann cells or residual basal lamina but many were acellular. The central collagen fibrils in these domains were of smaller diameter than those situated peripherally but were of larger size than those that form around the Büngner bands during wallerian degeneration. The peripherally located fibrils in the domains were of the same calibre as for normal endoneurial collagen. The collagen domains were encircled by fibroblast processes or at times enclosed in a perineurial cell ensheathment. Long-standing axonal loss therefore leads to a striking reorganisation of the internal architecture of peripheral nerve trunks. The findings may be relevant for the interpretation of the appearances in chronic peripheral neuropathies in man.

Ultrastructural Study of the Cornea in a Bone Marrow-Transplanted Hurler Syndrome Patient

This case report describes a 14-year-old girl with Hurler syndrome, who had received a successful bone marrow-transplant at the age of two. Corneal clouding was present at the time of transplant and has only partially cleared. A right penetrating keratoplasty was performed and the corneal specimen was examined by light microscopy, transmission electron microscopy with Cuprolinic blue staining for proteoglycans, and low-angle X-ray diffraction. The results show the corneal stroma to be disrupted by vacuolated stromal cells. There is abnormal accumulation of proteoglycans in the vacuolated stromal cells and nearby stroma. These proteoglycans mainly contain chondroitin/dermatan sulphate glycosaminoglycans since they are susceptible to chondroitinase ABC. There are a large range of fibril diameters (12.5–50.1nm) and there is an abnormal distribution of the fibril diameters measured from micrographs. Both are confirmed by X-ray diffraction results (the mean collagen fibril diameters are in a range between 29.7 and >51.1nm). X-ray diffraction also shows that the mean centre-to-centre distance of the fibrils slightly increases. These findings suggest that proteoglycans play a role in modelling the stromal structure and can also explain the corneal clouding. Many long-spacing collagen structures with a mean periodicity of 91.8nm are observed in the corneal stroma. The finding that the long-spacing collagen consists of fine collagen fibrils and that very few proteoglycans filaments bind to them suggests that some change in the interaction of proteoglycans and collagen is responsible for the formation of long-spacing collagen. To our knowledge, this is the first ultrastructural study of the cornea from a bone marrow-transplant patient with Hurler syndrome. The structural features documented here relate to a cornea incompletely corrected by bone marrow transplantation.

Three-Dimensional Visualization of the Aorta and Elastic Cartilage after Removal of Extracellular Ground Substance with a Modified NaOH Maceration Method

A modified NaOH maceration method for removing extracellular ground substance was applied to scanning electron microscopic (SEM) studies of the mammalian aorta and elastic cartilage. Fixed tissues were freeze-fractured in liquid nitrogen and then immersed in 2N NaOH solution for 4-6 hr at room temperature. Since this method selectively digested proteoglycans, cellular and fibrous elements were observable clearly under the SEM. The elastic laminae of smooth muscle cells of the aortic tunica media were concealed by fine collagen fibrils, while chondrocytes in the elastic cartilage were encapsulated by a dense network of fine collagen fibrils and branching elastin fibers.

Ultrastructural arrangement of collagen fibrils in the rat facial nerve.

We studied the collagen fibril arrangement in the connective tissue sheaths (epineurium, perineurium, and endoneurium) of the intratemporal and extra-temporal portion of the rat facial nerve by transmission electron microscopy after fixing the nerves with tannic acid, and by scanning electron microscopy after digesting cellular elements by sodium hydroxide treatment. These studies revealed that the epineurium consists of thick bundles of collagen fibrils and a coarse meshwork of collagen fibrils, the perineurium consists of a lacework sheet of bundles of collagen fibrils, and the endoneurium consists of a meshwork of fine collagen fibrils in the inner layer and of longitudinally oriented bundles of collagen fibrils in the outer layer. There was little difference in the three-dimensional organization of collagen fibrils between the extra-temporal segment and vertical and horizontal segments of the facial nerve. However, the facial nerve lost the epineurium and perineurium in the central portion of the horizontal segment near the geniculate ganglion and in the labyrinthine portion, i.e. the proximal portion of the intratemporal segment. In these segments, the endoneurium consisted of a meshwork of fine collagen fibrils.

The chemical morphology of the vitreous.

The water of the vitreous is stabilised by an ordered meshwork of very fine collagen fibrils that are tied together in loosely parallel bundles or sprays by bridges of sulphated glycosaminoglycan. Some fibril bundles run at right angles to other bundles. In these respects vitreous resembles a very dilute corneal stroma. The glycosaminoglycans of the vitreous (hyaluronan and chondroitin sulphate) aggregate with themselves and with each other in solution. The protein cores of the proteoglycans are attached to collagen fibrils. Thus, a three-component cross-linked structure is formed: collagen fibril-->proteoglycan protein core-->glycosaminoglycan chain-->glycosaminoglycan chain-->protein core-->collagen fibril. Hyaluronan can aggregate directly with chondroitin-6-sulphate, or with aggregated glycan cross-links, thus entering into an infinite meshwork.

Venous Microvasculature of the Pineal Body and Choroid Plexus in the Rat

Rats were perfused from aorta with Ringer's solution and with Karnovsky's fixative, and injected from the right atrium with Mercox resin. Specimens were properly taken, observed under LM, TEM, SEM, and stereo-photographed. Fenestrated endothelial cells of the pineal capillary were observed to contain plenty of microtubules running parallel to the long axis of the vessel. The endothelial basal lamina appeared anchored by microfibrils onto fine collagen fibrils and onto the basal lamina of the perivascular cells. These findings indicate resistance of the capillary endothelium against compression by perivascular hydrostatic pressure, and strongly suggest that the perivascular cerebrospinal fluid is absorbed into the capillary lumen. Resin cast of the pineal venous system looked like a glomerule with two longer collecting veins on the ventral surface and with shorter ones on the dorsal side all emptying into the straight sinus and the confluence of sinuses. Resin cast of the choroid plexus venous system tended to lobulate and looked like a vine with a spiral collecting vein emptying into the great vein. Two veins were found connecting the great vein to the confluence and to the transverse sinuses. These could be effective collaterals in case of occlusion of Galen's vein.