Fibrillar Elements Research Articles

METHODS AND RESULTS OF STUDYING THE STRUCTURE AND TINCTORIAL PROPERTIES OF CATGUT THREAD

Suture material used in abdominal surgery must have more or less antigenic properties so that during wound healing it could be destroyed by an immune response to rejection, which is known to involve cytotoxic T-lymphocytes and macrophages. This, in turn, can lead to the development of aseptic peritonitis. Therefore, while planning an experimental simulation of aseptic peritonitis, we decided to use catgut thread as an antigenic stimulation of the local immune system. The aim of our research was to study the morphological criteria of the microscopic structure and tinctorial properties of catgut thread. The material of the research was the sterile catgut thread sized 2/0, 0.3 mm thick, of equal lengths, which we twisted arbitrarily into flat compact globules occupying an area of approximately 1 cm2. In order to carry out a histological examination, some catgut globules were subjected to total staining with a hematoxylineosin solution and subsequent embedding of these globules in a paraffin block and making appropriate sections from it. The other half of the catgut globules were studied using the method of epoxy plastination with the production of grinds with staining by a 1% solution of methylene blue on a 1% borax and without any staining. After the total staining of the catgut globules with a hematoxylin-eosin solution, a clear acidophilic reaction was revealed, which was manifested in the intense pink color. Catgut thread has a non-uniform fibrous structure, in which dark streaks stand out, dividing it into a series of longitudinal, lighter, layered fascicles, which consist of a dense collection of thin fibrillar elements. These fibrillar elements are orderly fascicles of collagen fibers, while the dark streaks are layers of loose fibrous connective tissue. These structures can objectively be indicative of morphological criteria in the microscopic analysis of destructive changes of a catgut implant in the peritoneal cavity in an experimental animal.

Attachment ability of combined biomimetic adhesive micro-textures of different shapes

There are various potential applications of biomimetic adhesive solutions including climbing robotic systems, mobile sensor platforms, and biomedical applications such as patches for external use. Achieving resistance to both normal and tangential loads, however, is a critical issue that still needs to be addressed. Some animals have developed exceptional attachment mechanisms based on combined fibrillar elements of different shapes and functions. Experimental investigation of combined biomimetic adhesive micro-textures on tribological performances such as adhesion, friction, and peeling resistance is needed to apply this idea to the design of an artificial texture having similar “biomimetic” properties. In the present study, we demonstrate that combinations of different shapes of biomimetic adhesive micro-textures show increased efficiency under different contact environments and enable long-term adhesive solutions. Our work sheds light on combinations of different element shapes inspired by nature and their adhesive efficiency as a function of the ratio of each biomimetic element, as well as their spatial repartition.

Ultrastructural behavior of cell wall polysaccharides

Studies on the behavior of wood polysaccharides inside and outside the cell wall were carried out under varying conditions using the electron microscope in combination with physico-chemical methods to gain new information on the super- molecular structure of the polysaccharides. Several fractions containing different polyoses were obtained by fractionation of the alkali extract of holocellulose. Isolated polyoses are at least partially able to arrange themselves in fibrillar elements. After delignification, the cellulose is recognizable within the cell wall as microfibrils with an average diameter of about 250 À. However, these fibrillar units seem to be rather unstable as they can easily be split into units 120 À in diameter by chemical treatment. After partial hydrolysis, isolated cellulose shows more subunits about 30 A in diameter at higher magnification. Moreover, the more sensitive portions of the fibrils in the longitudinal direction are attacked by partial hydrolysis, and these portions seem to grow with increasing duration of the hydrolysis. Fractionation of cellulose isolated from thermally treated wood shows that the chain fragments are multiples about 300 À in length, A tentative model of the supermolecular arrangement of the cellulose in relation to the other cell wall components is presented.

Helical fibrillar microstructure of tendon using serial block-face scanning electron microscopy and a mechanical model for interfibrillar load transfer.

Tendon's hierarchical structure allows for load transfer between its fibrillar elements at multiple length scales. Tendon microstructure is particularly important, because it includes the cells and their surrounding collagen fibrils, where mechanical interactions can have potentially important physiological and pathological contributions. However, the three-dimensional (3D) microstructure and the mechanisms of load transfer in that length scale are not known. It has been postulated that interfibrillar matrix shear or direct load transfer via the fusion/branching of small fibrils are responsible for load transfer, but the significance of these mechanisms is still unclear. Alternatively, the helical fibrils that occur at the microstructural scale in tendon may also mediate load transfer; however, these structures are not well studied due to the lack of a three-dimensional visualization of tendon microstructure. In this study, we used serial block-face scanning electron microscopy to investigate the 3D microstructure of fibrils in rat tail tendon. We found that tendon fibrils have a complex architecture with many helically wrapped fibrils. We studied the mechanical implications of these helical structures using finite-element modelling and found that frictional contact between helical fibrils can induce load transfer even in the absence of matrix bonding or fibril fusion/branching. This study is significant in that it provides a three-dimensional view of the tendon microstructure and suggests friction between helically wrapped fibrils as a mechanism for load transfer, which is an important aspect of tendon biomechanics.

Fissures and pores in the capsule walls and hydrophobic elaters in Haplomitrium: a transmission and cryo-scanning electron microscope study

ABSTRACTIntroduction. Whereas the capsule wall in liverworts is usually described as smooth, our discovery of deep schizogenous fissures in the unistratose walls in Haplomitrium appears unique to this earliest diverging liverwort genus and may have important implications for understanding the evolution of the stomatal toolkit in land plants.Methods. Sporophytes of Haplomitrium gibbsiae and H. mnioides were examined by light, transmission and cryo-scanning electron microscopy.Key results. In striking contrast to cell breakage along the dehiscence grooves in the capsule walls, development of the schizogenous fissures is predetermined by a middle lamellar region of the radial walls comprising globular and fibrillar elements. The capsule wall cells remain alive, even after dehiscence, and have many cytological features in common with food-conducting cells in bryophytes including highly differentiated plasmodesmata. Whereas the highly ornamented spore exine retains water, the elaters are hydrophobic due to a coating of lipid droplets secreted by these cells.Conclusions. The development of regular thickened bands together with the schizogenous splitting of the capsule walls suggests that Haplomitrium may contain a possible primeval structural toolkit facilitating capsule desiccation and spore discharge. Thus we may be seeing, in this earliest extant liverwort lineage, the basic features needed for the assembly of stomata. There is no cytological evidence for a possible nutritive tapetal function for the capsule walls in Haplomitrium and the hydrophobic envelopes of the elaters probably enhance their effectiveness in spore dispersal.

ELECTRONMICROSCOPIC FEATURES OF STRUCTURAL ELEMENTS IN THE BRAIN MENINGS OF WHITE RATS

The aim of the work was to study the electron microscopic features of the structure of cellular and fibrillar elements in brain meninges of white rats. The main focus was on clarifying the relationship between cells that make up the barrier cell layer of the arachnoid (BCAL), and the so-called dural border cell layer (DBCL). Araldite-Epone blocks adopted in electron microscopy were made from fragments of the cerebral cortex and meninges taken from white rat. Subsequently, ultrathin sections were obtained using a Leica EM UC7 ultratome (Germany). After staining, the sections were examined with an electron microscope (JEM-1400-Japan) and photographed. Ultrastructurally, only cellular and fibrillar elements characteristic of connective tissue were revealed in the composition of the dura mater. Electron micrographs demonstrate that in leptomeninx as a result of a high degree of heterochromatin condensation in the nucleus and the nucleolus in the central part and the predominance of cytoskeleton elements in the peripheral part of cellular elements, the cells darken as they approach the BCDM. The presence of communicating, desmosomal and especially tight junctions between cells of so-called BCDM, which are the morphological indicators of the arachnoid barrier, shows that it is part of leptomeninx. The detection of the accumulation of cytoskeleton elements, the condensation of chromatin in nucleus, the presence of only individual ribosomes and mitochondria with destroyed cristae starting from the first layer, as well as the absence of a nucleus in the cells of the surface layer can be seen and identified as signs of keratinization in cells of BCAL which have the epithelial structure.

Extracting Subcellular Fibrillar Alignment with Error Estimation: Application to Microtubules

The order and orientation of cortical microtubule (CMT) arrays and their dynamics play an essential role in plant morphogenesis. To extract detailed CMT alignment structures in an objective, local, and accurate way, we propose an error-based extraction method that applies to general fluorescence intensity data on three-dimensional cell surfaces. Building on previous techniques to quantify alignments, our method can determine the statistical error for specific local regions, or the minimal scales of local regions for a desired accuracy goal. After validating our method with synthetic images with known alignments, we demonstrate the ability of our method to quantify subcellular CMT alignments on images with microtubules marked with green fluorescent protein in various cell types. Our method could also be applied to detect alignment structures in other fibrillar elements, such as actin filaments, cellulose, and collagen.

Antibiotic Susceptibility in Biofilms of Mycobacterium smegmatis

In the present article we observed the quantification and morphological, ultrastructural features of biofilms of fast growing clinical isolates M. smegmatis in presence of first line antibacterial drug streptomycin, isoniazid rifampicin, ethambutol and pyrazinamide. Biofilm of M. smegmatis was found to be unaffected at concentration of drugs that inhibited growth of planktonic bacilli .Thus, the biofilm growth modus appears to be a strategy for replicating bacilli to evade the trap of antibacterials. Planktonic and biofilm cells had similar intrinsic antibiotic susceptibility. Electron microscopy revealed that control (no drug) biofilms consisted primarily of bacterial clusters and fibrillar elements. The extracellular polymeric substance (EPS) material was less abundant in antibiotic-treated than in control biofilms beacause in the presence of high antibiotic concentrations at MIC level. The study is explored that the effect of drug on biofilm is time dependent means if the drugs were added at initial phase of biofilm, significant inhibitory effect were observed.Int J Appl Sci Biotechnol, Vol 3(4): 635-641

Structural Heterogenity of Intraluminal Content of The Prostate: A Histochemical and Ultrastructural Study

Intraluminal contents of benign and malignant prostatic tissue are associated with varying forms of acellular structures. These include corpora amylacea, prostatic calculi, and prostatic crystalloids. There are relatively few microscopy studies about the characterization of intraluminal structures from benign and malignant prostatic glands and little is known about their chemical composition. In the present study, we used a combination of special histochemical methods, immunohistochemistry, and transmission electron microscopy to characterize intraluminal contents of benign and malignant prostate glands. The study was done on 33 radical prostatectomy and four transurethral resections of prostate specimens. Histochemical methods such as von Kossa, autometallography (AMG), as well as PSA immunohistochemistry and transmission electron microscopy were performed to characterize intraluminal contents of benign and malignant prostate glands. Von Kossa staining was observed in acellular structures, corpora amylacea, prostatic calculi, and calcified blood vessels. AMG staining was observed in the lumen of small glands, in the epithelium lining prostate glands, and corpora amylacea. PSA staining showed prostatic glands with both positive and negative corpora amylacea and epithelial cells. Ultrastructural observation revealed the presence of a variety of highly heterogeneous aggregates composed of fibrillar elements that were similar to those of amyloid.

Stability of early-stage amyloid-β(1–42) aggregation species

Accumulation of aggregated amyloid-β protein (Aβ) is an important feature of Alzheimer's disease. There is significant interest in understanding the initial steps of Aβ aggregation due to the recent focus on soluble Aβ oligomers. In vitro studies of Aβ aggregation have been aided by the use of conformation-specific antibodies which recognize shape rather than sequence. One of these, OC antiserum, recognizes certain elements of fibrillar Aβ across a broad range of sizes. We have observed the presence of these fibrillar elements at very early stages of Aβ incubation. Using a dot blot assay, OC-reactivity was found in size exclusion chromatography (SEC)-purified Aβ(1–42) monomer fractions immediately after isolation (early-stage). The OC-reactivity was not initially observed in the same fractions for Aβ(1–40) or the aggregation-restricted Aβ(1–42) L34P but was detected within 1–2weeks of incubation. Stability studies demonstrated that early-stage OC-positive Aβ(1–42) aggregates were resistant to 4M urea or guanidine hydrochloride but sensitive to 1% sodium dodecyl sulfate (SDS). Interestingly, the sensitivity to SDS diminished over time upon incubation of the SEC-purified Aβ(1–42) solution at 4°C. Within 6–8days the OC-positive Aβ42 aggregates were resistant to SDS denaturation. The progression to, and development of, SDS resistance for Aβ(1–42) occurred prior to thioflavin T fluorescence. In contrast, Aβ(1–40) aggregates formed after 6days of incubation were sensitive to both urea and SDS. These findings reveal information on some of the earliest events in Aβ aggregation and suggest that it may be possible to target early-stage aggregates before they develop significant stability.

Relation between Antibiotic Susceptibility and Ultrastructure of Staphylococcus aureus Biofilms on Surgical Suture

Infectious biofilms are recalcitrant to antimicrobial therapy, but the mechanism(s) responsible for the greater resistance are unclear. Experiments were designed to clarify the association between antibiotic resistance and biofilm ultrastructure. Staphylococcus aureus was cultivated for 24 h on silk suture, where robust biofilms formed. Initial experiments compared the susceptibilities of planktonic (free-living) cells and mechanically dispersed biofilm cells to ampicillin, oxacillin, and vancomycin. Antibiotics in bactericidal concentrations were then incubated overnight with 24-h biofilms, and subsequent assays determined the viability of cells in mechanically dispersed biofilms, biofilm metabolic capacity and biomass, and biofilm ultrastructure (scanning electron microscopy). Planktonic and biofilm cells had similar intrinsic antibiotic susceptibility. Nonetheless, a stable population of bacteria remained viable after biofilms were incubated with inhibitory drug concentrations, although biofilm metabolic capacity often was not detected, and biomass generally was reduced. Electron microscopy revealed that control (no drug) biofilms consisted primarily of bacterial clusters amid fibrillar elements. Antibiotic-treated biofilms had some staphylococci with smooth cells walls similar to planktonic cells, but other cocci were encased in extracellular material. This material was more abundant in antibiotic-treated than in control biofilms. In the presence of high antibiotic concentrations, dense extracellular material may inhibit interaction of antibiotics with their bacterial targets.

Mechanosensitive peptidegelation: mode of agitation controls mechanical properties and nano-scale morphology

In this study we demonstrate the sensitivity of fibrillar self-assembly and gelation of aromatic peptide amphiphiles to different modes of mechanical agitation. In particular, we show how different homogenization techniques (vortex vs. manual or orbital agitation) during the gelation process can dramatically influence the mechanical properties and the nano-scale organization of self-assembled materials. The pH-induced self-assembly of mixtures of fluorenylmethoxycarbonyl-diphenylalanine (Fmoc-FF) and Fmoc-diglycine (Fmoc-GG) to gel phase materials was performed under different agitation conditions (vortex vs. manual or orbital agitation). We demonstrate that mechanically induced differences of up to one order of magnitude in shear modulus could be obtained for chemically identical samples as measured using oscillatory rheology and in creep and recovery experiments. AFM imaging and nanoindentation were then employed to highlight morphological, mechanical and physico-chemical differences in the fibrillar elements (ribbons). Furthermore, we have demonstrated that although Fmoc-diglycine alone does not undergo fibrillar aggregation, nor is capable of gelation at neutral pH, its presence influences the properties of gels at both fibrillar and macroscopic level; in particular, a Fmoc-diglycine/Fmoc-diphenylalanine 1 : 1 molar ratio appeared to provide the highest moduli and the most regular fibers, suggesting a specific mode of intercalation of Fmoc-diglycine in Fmoc-diphenylalanine fibrils.

Bacterial Contamination of Surgical Suture Resembles a Biofilm

Although much attention is currently directed to studying microbial biofilms on a variety of surfaces, few studies are designed to study bacterial growth on surgical suture. The purpose of this study was to compare the kinetic development of Staphylococcus aureus and Enterococcus faecalis on five surgical suture materials and to clarify factors that might influence this growth. Pure cultures of S. aureus and E. faecalis were incubated with five types of suture for four days using either tissue culture medium or a bacterial growth medium. Suture-associated bacteria were quantified daily. In selected experiments, the bacterial growth medium was supplemented with heparin, a substance known to promote S. aureus biofilm formation. The ultrastructure of S. aureus biofilm developing on braided suture was studied with scanning electron microscopy. Staphylococcus aureus and E. faecalis were recovered in greater numbers (typically p < 0.01) from braided than from monofilament suture, and the numbers of bacteria were greater (often p < 0.01) on sutures incubated in bacterial growth medium rather than tissue culture medium. Addition of heparin 1,000 U/mL to silk or braided polyglactin 910 suture incubated three days with S. aureus resulted in greater numbers of bacteria on day one but not on subsequent days. Scanning electron microscopy showed a maturing S. aureus biofilm that developed from small clusters of cells among amorphous material and fibrillar elements to larger clusters of cells that appeared covered by more consolidated extracellular material. Bacterial growth was favored on braided vs. monofilament suture, and heparin enhanced bacterial adherence after day one, but not at subsequent times. Staphylococcus aureus adhered to suture material and formed a structure consistent with a bacterial biofilm.

Matrix Metalloproteinase 2 (MMP-2) Degrades Soluble Vasculotropic Amyloid-β E22Q and L34V Mutants, Delaying Their Toxicity for Human Brain Microvascular Endothelial Cells

Patients carrying mutations within the amyloid-beta (Abeta) sequence develop severe early-onset cerebral amyloid angiopathy with some of the related variants manifesting primarily with hemorrhagic phenotypes. Matrix metalloproteases (MMPs) are typically associated with blood brain barrier disruption and hemorrhagic transformations after ischemic stroke. However, their contribution to cerebral amyloid angiopathy-related hemorrhage remains unclear. Human brain endothelial cells challenged with Abeta synthetic homologues containing mutations known to be associated in vivo with hemorrhagic manifestations (AbetaE22Q and AbetaL34V) showed enhanced production and activation of MMP-2, evaluated via Multiplex MMP antibody arrays, gel zymography, and Western blot, which in turn proteolytically cleaved in situ the Abeta peptides. Immunoprecipitation followed by mass spectrometry analysis highlighted the generation of specific C-terminal proteolytic fragments, in particular the accumulation of Abeta-(1-16), a result validated in vitro with recombinant MMP-2 and quantitatively evaluated using deuterium-labeled internal standards. Silencing MMP-2 gene expression resulted in reduced Abeta degradation and enhanced apoptosis. Secretion and activation of MMP-2 as well as susceptibility of the Abeta peptides to MMP-2 degradation were dependent on the peptide conformation, with fibrillar elements of AbetaE22Q exhibiting negligible effects. Our results indicate that MMP-2 release and activation differentially degrades Abeta species, delaying their toxicity for endothelial cells. However, taking into consideration MMP ability to degrade basement membrane components, these protective effects might also undesirably compromise blood brain barrier integrity and precipitate a hemorrhagic phenotype.

The ultrastructure and contractile properties of a fast-acting, obliquely striated, myosin-regulated muscle: the funnel retractor of squids.

We investigated the ultrastructure, contractile properties, and in vivo length changes of the fast-acting funnel retractor muscle of the long-finned squid Doryteuthis pealeii. This muscle is composed of obliquely striated, spindle-shaped fibers ~3 mum across that have an abundant sarcoplasmic reticulum, consisting primarily of membranous sacs that form 'dyads' along the surface of each cell. The contractile apparatus consists of 'myofibrils' approximately 0.25-0.5 microm wide in cross section arrayed around the periphery of each cell, surrounding a central core that contains the nucleus and large mitochondria. Thick myofilaments are approximately 25 nm in diameter and approximately 2.8 microm long. 'Dense bodies' are narrow, resembling Z lines, but are discontinuous and are not associated with the cytoskeletal fibrillar elements that are so prominent in slower obliquely striated muscles. The cells approximate each other closely with minimal intervening intercellular connective tissue. Our physiological experiments, conducted at 17 degrees C, showed that the longitudinal muscle fibers of the funnel retractor were activated rapidly (8 ms latent period following stimulation) and generated force rapidly (peak twitch force occurred within 50 ms). The longitudinal fibers had low V(max) (2.15 +/-0.26 L(0) s(-1), where L(0) was the length that generated peak isometric force) but generated relatively high isometric stress (270+/-20 mN mm(-2) physiological cross section). The fibers exhibited a moderate maximum power output (49.9 W kg(-1)), compared with vertebrate and arthropod cross striated fibers, at a V/V(max) of 0.33+/-0.044. During ventilation of the mantle cavity and locomotion, the funnel retractor muscle operated in vivo over a limited range of strains (+0.075 to -0.15 relative to resting length, L(R)) and at low strain rates (from 0.16 to 0.91 L(R) s(-1) ), corresponding to a range of V/V(max) from 0.073 to 0.42. During the exhalant phase of the jet the range of strains was even narrower: maximum range less than +/-0.04, with the muscle operating nearly isometrically during ventilation and slow, arms-first swimming. The limited length operating range of the funnel retractor muscles, especially during ventilation and slow jetting, suggests that they may act as muscular struts.

Prostate carcinogenesis induced by N-methyl-N-nitrosourea (mnu) in gerbils: Histopathological diagnosis and potential invasiveness mediated by extracellular matrix components

In the present study prostate lesions were induced in gerbils ( Meriones unguiculatus) treated with a single N-methyl-N-nitrosourea (MNU) dose; thus, the incidence, latency and histology of these lesions were evaluated. Fibrillar elements of the extracellular matrix associated with microinvasive sites were also investigated. Animals were divided into 5 groups, including 2 control groups: (1) remained untreated; (2) received the corn oil vehicle (vehicle, 0.1 ml/application) and three different tumor induction regimens: (1) received MNU (30 mg/kg) and weekly testosterone (2 mg/kg) (MNU + testosterone); (2) received only MNU (30 mg/kg); (3) received weekly testosterone doses (2 mg/kg). After 3 and 6 months the animals were dissected and the prostates were evaluated morphologically, immunohistochemically and quantitatively. MNU plus androgen contributed to the development of prostatic intraepithelial neoplasia, microinvasive carcinoma and adenocarcinoma in gerbil prostate. However, these lesions occurred earlier in time in groups that received MNU and androgen compared to control animals as they over time also developed to a high extent microinvasive lesions. Cytochemistry and immunohistochemistry showed that these injuries were commonly associated with inflammatory cells whereas the epithelial cells presented proliferative activity. The α-methylacyl-CoA racemase (AMACR) expression in prostate cancer cells facilitated diagnosis of gerbil lesions. Testosterone, MNU and MNU + testosterone showed an increased epithelial volume, although the secretory activity was significantly suppressed mainly at neoplastic foci. In the prostatic stroma, reticular fibers increased significantly in MNU, MNU + testosterone and among the lesions found in these groups, while collagen fibers decreased at neoplastic sites. The disruption of the basement membrane was proven at malignant sites by ultrastructural analysis and type IV collagen and laminin degradation. The prostate carcinogenesis mediated by MNU and androgen stimulated the emergence of proliferative lesions in gerbils after short periods and showed the importance of a dynamic remodeling of stromal components for cellular invasiveness.

Preservation of structure and immunoreactivity at the vitreoretinal interface of the rabbit eye

The vitreous body is implicated in the etiology and pathology of a variety of retinal conditions. Many such conditions are treated surgically to remove the posterior vitreous from the inner limiting lamina (ILL) of the retina, and there is current interest in the adjunct use of enzymes for this purpose. To evaluate the efficacy of these agents in future preclinical studies, improved preservation and assessment methods were developed to establish a baseline histological profile of the vitreous and retina of the rabbit, to identify and distinguish artifactual vitreoretinal separation from authentic posterior vitreous detachment, and to preserve structural integrity while maintaining antigenicity for immunohistochemical analysis. Two pigmented rabbits each underwent perfusion with one of three fixatives, either: (1) 10% neutral buffered formalin + cetylpyridinium chloride (NBF/CPC), (2) acid-ethanolic formalin + CPC (AEF/CPC), or (3) formaldehyde-glutaraldehyde + CPC (FG/CPC). An eye fixed in NBF/CPC by immersion, from an additional rabbit, was also included for comparison. Eyes were processed whole through paraffin infiltration. Treatments were assessed by immunohistochemical labeling for retinal and cortical vitreous (CV) markers. In contrast to immersion fixation, perfusion with either NBF/CPC or AEF/CPC maintained vitreous adherence to the ILL during histological processing. NBF/CPC proved best for highlighting intralaminar structure and for labeling type II collagen in the CV, particularly with antigen retrieval. AEF/CPC caused condensation of fibrillar elements in the CV. Collagen XVIII in the ILL was observed with AEF/CPC exclusively. Only retinal vessels near the optic nerve head were labeled for type IV collagen. The labeling of glia was useful for distinguishing between cellular and extracellular elements. GF/CPC hindered detection of collagen II and disrupted posterior segment structure. Expression of type II collagen extended from the ONH directly to CV affiliated with the central canal of Cloquet, a feature characteristic of rabbit eyes. Careful tissue preservation and processing techniques minimize artifactual separation of the CV from the ILL. By optimizing the tissue architecture and antigenicity of the vitreoretinal complex, CV may be distinguished from the ILL immunohistochemically. The techniques described may be used to evaluate more effectively the utility of pharmacologic vitreolysis, using experimental animal models.

Clinical and Histological Aspects of CNV Formation: Studies in an Animal Model

The purpose of the present thesis was to develop an animal model of CNV in order to study the early formation of CNV and to test the effects of an anti-angiogenic treatment. Porcine eyes were chosen as a substrate for CNV induction, since they are similar to human eyes in terms of both macroscopic and microscopic morphology. However, a major difference is that pigs lack a fovea; instead they have a visual streak, with a relatively stable and high concentration of cones. By surgical perforation of Bruch's membrane we were able to induce formation of CNV membranes. The morphology and cellular composition of these membranes varied with the surgical technique employed. When RPE cells were locally removed at the time of perforation, the resulting CNV was thinner, contained fewer blood vessels and was less prone to leak on fluorescein angiography than when RPE cells were left intact at induction. The neuroretina overlying the perforation site was not damaged by any of the surgical techniques, thus allowing the subsequent retinal damage to be ascribed to the actual process of CNV formation. Using this animal model allowed us to directly map histological findings onto fluorescein angiograms and thereby perform meaningful correlations between histopathologic and photographic features. Such correlations have been hampered in human subjects, since human eyes are not enucleated as a consequence of CNV and are therefore only available for post-mortem studies. In such studies there often is a considerable time-gap between the death of the patient and the latest available fluorescein angiogram, thereby allowing macular pathology to evolve in the interim. Histological examination of the porcine membranes demonstrated that they were composed of RPE cells, glial cells, macrophages, endothelial cells, collagen and smooth muscle fibres, which are the same cellular and fibrillar elements that dominate human CNV membranes. The porcine model was applied to test the effects, in a randomized and masked fashion, of intravitreally injected bevacizumab. Bevacizumab, a pan VEGF A antibody, was found to reduce both the proliferation of endothelial cells in CNV membranes and the propensity to leak in fluorescein angiograms. Immunohistochemically, bevacizumab was detected in the inner limiting membrane, in retinal blood vessels and binding uniformly to the entire CNV membrane without any cellular predisposition. Based on the above findings we believe that the porcine CNV model shows a bearing to human disease and therefore might be used as a tool to obtain improved treatments for this debilitating condition.

Chromatin structure in sites of DNA replication

Conformational changes in chromatin structure are nowadays the object of intensive research due to its importance for proper regulation of intranuclear processes. The fine structure of chromatin within the DNA replication sites was studied in in situ fixed cells and cells permebilized by low ionic strength solutions in the presence of divalent cations. The latter method provides visualization of higher level chromatin structures such as globular chromomeres and chromonema fibres. Nascent DNA was detected immunochemically using anti-BrdU antibodies on the surface of ultrathin sections prepared from Epon-embedded material. It was shown that newly replicated DNA preferentially localized within the zones filled with globular and fibrillar elements with characteristic diameter of 30 nm, and not in chromonema fibres, while after replication had been completed DNA became embedded into as thick as 60-80 nm chromonema elements. The results obtained are discussed in the context of conception of hierarchical folding of chromatin fibers.

Evidence for distinct leptomeningeal cell-dependent paracrine and EGF-linked autocrine regulatory pathways for suppression of fibrillar collagens in astrocytes

A unique and unresolved property of the central nervous system is that its extracellular matrix lacks fibrillar elements. In the present report, we show that astrocytes secrete triple helices of fibrillar collagens type I, III and V in culture, while no astroglial collagen expression could be detected in vivo. We discovered two inhibitory mechanisms that could underlie this apparent discrepancy. Thus, we uncover a strong inhibitory effect of meningeal cells on astrocytic collagen expression in coculture assays. Furthermore, we present evidence that EGF-receptor activation downregulates collagen expression in astrocytes via an autocrine loop. These investigations provide a rational framework to explain why the brain is devoid of collagen fibers, which is a unique feature that characterizes the structure of the neural extracellular matrix. Moreover, fibrillar collagens were found transiently upregulated in a laser-induced cortical lesion, suggesting that these could contribute to the glial scar that inhibits axonal regeneration.