Rudolf Mallinger

Interlacing and cross-angle distribution of collagen lamellae in the human cornea.

Purpose The interlacing and cross angles between the collagen lamellae within the human corneal stroma were studied by means of scanning electron microscopy (SEM). Methods For SEM, cells and noncollagenous extracellular matrix were removed with 10% sodium hydroxide. Transmission electron microscopy (TEM) preparations were performed according to standard procedures. The interlacing of lamellae was studied within the limbal, paracentral, and central regions of five different layers. The cross angles between the longitudinal axes of adjacent lamellae were measured. The distribution of these angles within defined layers and regions was compared. Special attention was paid to the interlacing of the lamellae. Results Lamellae split in an anteroposterior direction as well as horizontally into branches and are interlaced by crossing the fissures between the branches. Smaller lamellae cross through clefts of neighboring lamellae. The cross angles show a high variability of 1°–90°. With the exception of the limbal region of the layer adjacent to Descemets membrane, the distribution of cross angles is similar. A frequent occurrence of cross angles <30° (68%) in this limbal layer can be explained by a pseudo-circular orientation (ligamentum circulare corneae) of the lamellae. Conclusion The present study shows that the three-dimensional organization of the collagen lamellae is characterized by a greater extent of lamellar interlacing than has been assumed until now.

ALTERED ORGANIZATION OF COLLAGEN IN THE APEX OF KERATOCONUS CORNEAS

Purpose: In 15 keratoconus corneas, the three-dimensional arrangement of collagen lamellae was investigated by means of scanning electron microscopy. Methods: Keratoconus corneas without visible scars were obtained during perforating keratoplasty. The noncollagenous matrix of the stroma was removed with sodium hydroxide. Descemet’s membrane was removed mechanically and deeper layers of the stroma were exposed by cutting the tissue tangentially to the corneal surface with an ultramicrotome. The apical and the para-apical regions of keratoconus were compared to the central regions of normal corneas. Results: In the apical regions of 11 out of the 15 keratoconus corneas (73%), the arrangement of the collagen lamellae differs from those of the para-apical regions and normal corneas. Their collagen fibrils form uniform layers and no delimited collagen lamellae can be differentiated. Interlacing between adjacent layers is extremely decreased or even absent. In the para-apical region of keratoconus corneas the three-dimensional arrangement of collagen lamellae does not differ from that in normal corneas. Conclusion: Stromal thinning and conical ectasia in the apex of keratoconus corneas alters the organization of collagen. This will certainly affect the biomechanical properties of the cornea and further lead to a progression of keratoconus irrespective of its primary pathogenesis.

Interlacing of collagen lamellae in the midstroma of the human cornea.

Purpose. To investigate by means of scanning electron microscopy (SEM) the interlacing of collagen lamellae in the midstroma of the human cornea after opening the interlamellar spaces. Material and Methods. For SEM, cells and noncollagenous extracellular matrix were removed with 10% sodium hydroxide. Specimens were dehydrated in a series of graded tertiary butanols, frozen at −24°C and dried in an exsiccator by sublimation of the frozen butanol. Dried corneas were cut vertically with a razor blade, and the interlamellar spaces were exposed by stretching the stroma along its anterior-posterior axis by pulling apart the inner (endothelial) and outer (epithelial) edges. Specimens were sputtered with gold and examined with a Cambridge Stereoscan 90 microscope. Results. The opened interlamellar spaces gave the stroma at the cutting edge the appearance of a polymorphic honeycomb. The stromal openings differed in size, from approximately 10 &mgr;m to over 150 &mgr;m length and up to 80 &mgr;m in height. Adjacent lamellae remained connected at several interconnecting regions, either through an exchange of short merging sublamella or single fibrils. Interlacing lamellae crossed through fissures between the branches of splitting lamellae. Others crossed clefts of neighboring lamellae, and other lamellae tunneled crosswise through a horizontally split lamellae hanging in the inferior branch as in a hammock. Large interweaving zones in which a mixture of several types of interlacing was localized close together could also be found. Conclusion. The current study indicates that interlacing is a distinct and important feature of the human cornea, and it gives new insights into the stromal morphology by demonstrating various types of interlacing that occur between collagen lamellae.

Ultrastructure of clear corneal incisions

Purpose: To examine the ultrastructure of clear corneal incisions (CCls) performed with diamond keratomes and steel blades as well as the corneal trauma after implantation of a foldable intraocular lens (IOL) through two incision widths. Setting: University Eye Clinic and Institute of Histology and Embryology II, University of Vienna, Austria. Methods: Twenty‐four human cadaver eyes without prior ocular surgery were obtained from the University Eye Bank, Vienna. Single‐plane CCIs were performed with 3.0 and 3.2 mm Alcon steel blades and with a 3.0 mm Huco diamond keratome. The AVIO PhacoFlex II lens was implanted with a Fine II folder. During the entire procedure, the eye pressure was kept between 26 and 30 mm Hg by infusing balanced salt solution into the anterior chamber. Specimens were prepared for light microscopy, transmission electron microscopy, and scanning electron microscopy according to standard procedures. Results: The diamond keratome produced cleaner cuts than the steel blade. After IOL implantation, 3.0 mm steel blade incisions exhibited extensions at their lateral ends. Within these extensions, the collagen lamellae were displaced and torn. This was not true with 3.2 mm tunnels. Because of the thickness of a 3.0 mm diamond keratome, the extent of corneal trauma was between that found with 3.0 and 3.2 mm steel keratome tunnels. Conclusions: Implantation of the SI‐30 through 3.0 mm CCls produced by the steel blade led to more severe corneal trauma than implantation through 3.2 mm steel blade incisions or 3.0 mm diamond keratome incisions. Thus, IOL implantation through incisions that are too small intensifies corneal trauma.

Ultrastructure of clear corneal incisions: Part II: Corneal trauma after lens implantation with the Microstaar injector system

Purpose: To examine the ultrastructure of clear corneal incisions (CCls) after implantation of a plate‐haptic intraocular lens (IOL) with the Microstaar™ injector system through two incision widths. Setting: University Eye Clinic and Institute of Histology and Embryology II; University of Vienna, Austria. Methods: Fourteen human cadaver eyes without prior ocular surgery were obtained from the University Eye Bank, Vienna. Single‐plane CCls were performed with 3.0 and 3.2 mm steel keratomes. Using the Microstaar injector system, a foldable silicone plate‐haptic IOL (23 diopters) was implanted in the anterior chamber. During the entire procedure, the eye pressure was kept between 26 and 30 mm Hg by infusing balanced salt solution into the anterior chamber. Specimens for light microscopy and scanning electron microscopy were prepared according to standard procedures. Results: After IOL implantation, the 3.0 mm steel blade incisions exhibited distinct distensions at their lateral ends. Adjacent to these distensions, the collagen lamellae were pressed apart, displaced, and torn. In 3.2 mm tunnels, the corneal trauma at both lateral ends was considerably less severe. These incisions also showed a better primary adaptation of the wound lips after implantation. Conclusions: Implantation performed with the Microstaar injector system through 3.0 mm steel blade CCIs led to considerably more severe corneal trauma than implantation through 3.2 mm incisions.

Zur dreidimensionalen Anordnung der kollagenen Lamellen im posterioren Stroma der menschlichen Hornhaut

ZusammenfassungDie dreidimensionale Anordnung der kollagenen Lamellen in der menschlichen Hornhaut ist bis heute nicht restlos geklärt. In einer Vorversuchsreihe haben wir eine spezielle Präparationstechnik zur optimalen Darstellung des Kollagens in der Cornea entwickelt und untersuchten mittels Raster- und Transmissionselektronenmikroskopie die Anordnung der subendothelialen Lamellen in verschiedenen Regionen (limbal, parazentral, zentral) der menschlichen Hornhaut.Die kollagenen Lamellen verlaufen nicht nur in einer Ebene, sondern erreichen, indem sie sich aufteilen und durchkreuzen, auch benachbarte Hornhautschichten. Die Teilungswinkel der Lamellen betragen in der gesamten Cornea etwa 25 °. Auffällig ist, daß die Kreuzungswinkel in den verschiedenen Regionen unterschiedlich sind. Sie betragen in der limbalen Region durchschnittlich 25 °; parazentral und zentral variieren sie zwischen 40° und 75°. Eine limbusparallele Hauptverlaufsrichtung läßt sich für die limbale und parazentrale Region ermitteln.SummaryThe discussion about the three-dimensional arrangement of collagen lamellae in the human cornea is equivocal. In the present study we investigated the limbal, paracentral and central region of the posterior layer of human corneal stroma by scanning and transmission electron microscopy.In all regions lamellae split into branches at angles of about 25 ° and are interlaced by crossing each other. By doing so lamellae reach different layers. In all regions we found splitting angles of about 25 °. However, different angles of criss-crossing were found — in the limbal region of about 25 °; in the paracentral and central cornea the angles vary between 40 ° and 75 °. In the limbal and paracentral region we could demonstrate a main course of the lamellae which is parallel to the limbus.

Tissue damage at anterior capsule edges after continuous curvilinear capsulorhexis, high-frequency capsulotomy, and erbium:YAG laser capsulotomy

Purpose: To compare the effect of erbium:YAG laser photocapsulotomy (ELC), continuous curvilinear capsulorhexis (CCC), and high‐frequency capsulotomy (HFC) on anterior capsule edges using light microscopy (LM) and transmission electron microscopy (TEM). Setting: Department of Ophthalmology, Department of Histology and Embryology II, University of Vienna, Vienna, Austria. Methods: Five anterior capsule membranes were obtained experimentally by ELC, which was performed with the Oertli MicroLaser photoemulsification tip in eyes from 5 human cadavers. Thirty anterior capsule membranes were obtained during cataract surgery by CCC (n = 15) or, in cases with poor or missing red fundus reflexes, by HFC (n = 15). Continuous curvilinear capsulorhexis was performed conventionally with a manual, bent, 27‐gauge cannula, and HFC was performed according to Klöti. Membranes were processed and examined by LM and TEM according to standard procedures. Results: The edges obtained by ELC showed mild signs of denaturation and mechanical disruption, tears, and micro‐tears. Lens capsule edges obtained by CCC were smooth with no irregularities in any specimen; the edges were beveled anteriorly to posteriorly with no evidence of tearing. Edges produced by HFC were considerably wider; the surfaces showed distinct denaturation, preformed tears, and micro‐tears. The edges were surrounded by a nonhomogeneous mass, which consisted mainly of denatured lens capsule material. Conclusions: Of the 3 techniques, CCC produced the mildest tissue damage. The histological damage at ELC edges was relatively mild and intermediate compared with that seen at CCC and HFC edges. These observations suggest that ELC is an appropriate option for anterior capsulotomy in cataract surgery.

Effect of trephination technique on the ultrastructure of corneal transplants: guided trephine system v posterior punch technique.

AIM Different trephination methods may lead to differences in degree of tissue damage and endothelial cell loss, which both influence the outcome of penetrating keratoplasty. Light, transmission, and scanning electron microscopy were used to compare the ultrastructural appearance of the cut edges and the endothelial cell loss in 26 human corneal donor buttons obtained by trephination with the suction fixated guided trephine system (GTS) and with the free hand posterior punch technique (PPT). METHODS Human corneas were stored between 5 and 14 days in Optisol. One cornea from each pair was used for each technique. Trephinations (7.5 mm) were performed either from the anterior direction with the GTS (n=13) or from the posterior direction with the PPT (n=13) using Pharmacia Superblade trephines. Light microscopy, transmission electron, and scanning electron microscopy were performed according to standard procedures. Widening of the cut edges and the extent of endothelial cell loss were measured at three different areas per corneal button and analysed statistically. RESULTS In contrast with the PPT, the GTS trephine produced considerable fibrillar disorder at the cut edges of the corneal buttons. The distance to which the endothelial cell loss extended from the edges of the cuts was significantly (p<0.001) lower for the GTS (42.2 (SD 50.8) μm from the edge) than for the PPT (109.3 (68.1) μm). Stromal widening at the edges (measured as percentage increase in stromal thickness, compared with the thickness of the central cornea) was observed with both techniques. However, the mean stromal widening produced by the GTS was significantly greater than that produced by PPT (106% (24%)v 69% (21%); p<0.002). CONCLUSION Both trephination techniques produced only minor tissue damage. Nevertheless, there were distinct differences in the fine appearance of the cuts produced by the GTS and the PPT techniques. The extent of the fibrillar dislocation and stromal widening was greater at the edges of the GTS buttons. The GTS technique produced significantly less endothelial cell loss at the cut edges than did the free hand punching technique, PPT.

Zur Anordnung der kollagenen Lamellen beim Keratokonus

ZusammenfassungDie Pathogenese des Keratokonus ist bis heute nicht geklärt. So findet sich neben einer normalen Verteilung der Kollagentypen auch eine normale posttranslationale Modifikation des Kollagens. Biochemische Untersuchungen weisen beim Keratokonus jedoch auf Veränderungen der Grundsubstanz hin. Zudem unterscheidet sich das biomechanische Verhalten gegenüber Zug-, Druck- und Scherkräften von jenem der normalen Hornhaut.Da die Kollagenlamellen in licht- und elektronenmikroskopischen Querschnitten zahlreiche morphologische Veränderungen aufweisen, untersuchten wir mit dem Rasterelektronenmikroskop (REM) die Anordnung der kollagenen Lamellen im Apex sowie in der para-apikalen Region des Keratokonus und verglichen diese mit der normalen Hornhaut.Im Gegensatz zur para-apikalen Region lassen sich im Apex keine einzelnen Lamellen abgrenzen. Die Fibrillen bilden großflächige Lagen und scheinen entsprechend der Hornhautverformung verdrängt zu sein. Weiters finden sich keine Lamellen, die diese Fibrillenlagen durchkreuzen und miteinander verbinden. In der para-apikalen Region des Keratokonus entspricht die Lamellenanordnung jener der normalen Hornhaut.Als Erklärung für diese Veränderungen kommen sowohl eine gestörte Biosynthese der extrazellulären Matrix als auch degenerative Prozesse in Frage. Jedoch dürfte die veränderte Lamellenanordnung im Apex des Keratokonus selbst zu Veränderungen der biomechanischen Eigenschaften der Hornhaut führen und so unabhängig von der primären Pathogenese zum Fortschreiten der Erkrankung beitragen.SummaryThe pathogenesis of keratoconus is obscure. The distribution of different collagen types as well as the posttranslational modification of the collagen were found to be within normal ranges. However, alterations of glycosaminoglycan components within the ground substance have been postulated. Additionally, the biomechanical properties of keratoconus corneas differ from that of normal ones.Since light- and electron microscopy reveal distinct morphological alterations of the collagen fibers we investigated the three-dimensional organisation of collagen lamellae by means of scanning electron microscopy (SEM). The apical as well as the paraapical regions of corneas with keratoconus were compared to those of normal corneas.Within the apical region no distinct collagen lamellae can be differentiated. The fibrils are constituents of uniform layers and the arrangement of the fibrils seems to be related to the apical distorsion. No interlacing between these layers was found. In contrast to that the paraapical region shows no differences when compared to the normal cornea.Enzymatic defects of the keratocyts as well as degenerative processes might bring about these alterations. It seems evident that changes of the collagen arrangement leads to alterations in the biomechanical properties of keratoconus corneas. These alterations may further propagate the development of a kertoconus independent of it’s primary pathogenesis.

Diamond-tip versus blunt-tip caliper enlargement of clear corneal incisions

Purpose: To assess corneal trauma after clear corneal incision (CCI) enlargement with a diamond‐tip versus a blunt‐tip caliper. Setting: University of Vienna, Austria. Methods: Human bulbi obtained from the eye bank of the University of Vienna had a 3.0 mm single‐plane CCI enlarged to 5.0 mm using either a diamond‐tip or a blunt‐tip adjustable caliper. During the process, eye pressure was kept between 22 and 25 mm Hg by infusing balanced salt solution using an anterior chamber maintainer. Light, scanning electron, and transmission electron microscopy were performed according to standard procedures. Results: Incisions enlarged with the diamond‐tip caliper were well‐defined and allowed good closure of the wound lips. In contrast, the blunt‐tip caliper produced widened, drumstick‐shaped recesses at the lateral ends of the incisions. Adjacent to these recesses, the collagen lamellae were severely dislocated and torn. Conclusion: Cutting the corneal tissue with diamond tips caused less tissue damage than expanding it with blunt tips. It was not possible to temporarily widen incisions using a blunt‐tip caliper without significantly intensifying tissue damage.