Frank J.R. Rietveld

A surgical technique for posterior lamellar keratoplasty.

PURPOSE To design a surgical technique for transplantation of posterior corneal tissue, while leaving the recipient anterior cornea intact. METHODS In human cadaver eyes, and in a cat and monkey model, recipient eyes had an 8.0-mm limbal incision made with a diamond blade set to 50% of central pachymetry. A stromal pocket was created across the cornea, and a 6.0-mm diameter posterior lamellar disc was excised. A donor posterior disc was implanted into the recipient opening, and the limbal incision was sutured. The procedure was evaluated with keratometry, biomicroscopy, endothelial (supra)vital staining, and light microscopy. RESULTS In human cadaver eyes, post-operative astigmatism averaged 1.2 D (SD, +/- 0.6 D). Posterior transplants showed an intact endothelial cell layer with 1.0% (SD, +/- 1.2%) of cell death. In the animals, six (75%) eyes had clear transplants 2 weeks after surgery; one of these eyes later developed an allograft rejection. Two (25%) eyes showed corneal decompensation, because of inverted implantation of the donor disc. Microscopy showed minimal scarring at the donor-to-host interface and a normal wound-healing response at the posterior stromal wound edges. CONCLUSION In experimental models, posterior lamellar keratoplasty can be performed through a limbal incision and a mid-stromal pocket. The procedure may be a potential alternative in the surgical management of corneal endothelial disorders.

Transplantation of Descemet's membrane carrying viable endothelium through a small scleral incision.

Purpose. To design a technique for transplantation of the Descemets membrane (DM) as a carrier for its endothelium, while maintaining the low-astigmatic recipient anterior corneal curvature. Methods. In a human eye bank eye model, recipient eyes (n = 15) had a 5.0-mm scleral tunnel incision made, extending 1.0 mm into the peripheral cornea. A 9.0-mm-diameter Descemeto rhexis was created, i.e., a circular portion of DM was stripped from the posterior stroma. With use of a custom-made inserter, a 9.0-mm-diameter donor DM carrying autologous donor endothelium was brought into the anterior chamber and positioned against the recipient posterior stroma. The procedure was evaluated by keratometry, endothelial vital and supravital staining, and light microscopy. Results. Mean postoperative astigmatism was 1.0 D (±0.6 D). Implanted donor DM showed an intact endothelial cell layer, with 3.4% (±1.1%) dispersed focal cell death. Microscopy showed normal endothelial cell morphology and complete apposition of the donor DM against the recipient posterior stroma. Conclusions. DM can be transplanted in vitro with acceptable damage to the donor endothelium and with little induced astigmatism.

Early and extensive contribution of pericytes/vascular smooth muscle cells to microvascular proliferation in glioblastoma multiforme: An immuno-light and immuno-electron microscopic study

Although florid microvascular proliferation (MVP) in glioblastoma multiforme (GBM) has long been considered as proliferation of endothelial cells (EC), recent immuno-light microscopic studies demonstrated many α-smooth muscle actin (α-sm actin)-positive cells in this MVP, suggesting a major contribution of pericytes and/or vascular smooth muscle cells (VSMC). Under certain culture conditions, however, α-sm actin expression has also been described in EC. In order to further investigate to what extent pericytes/VSMC participate in MVP in GBM, we performed an immunohistochemical study at both the light and electron microscopic levels with anti-α-sm actin, with an antibody against EC (EN-4) and with an antibody recently described to react with “activated” pericytes in conditions with neovascularization (anti-high molecular weight-melanoma associated antigen). In this detailed study of MVP in GBM, two distinct cell types could be recognized on the basis of a consistent ultrastructural localization and immunophenotype: EC and pericytes/VSMC; no transitional forms were found between these two cell types. The contribution of pericytes/VSMC to MVP in GBM was extensive and already present in many delicate tumor capillaries, suggesting not only an essential but also an early role of these cells in this type of tumor angiogenesis.

Transition of horizontal to vertical growth phase melanoma is accompanied by induction of vascular endothelial growth factor expression and angiogenesis

Melanoma progression in general is characterized by an increase in both metastatic frequency and the vascular density of the tumour tissue. Although a direct correlation between these two parameters in individual cases seems to be lacking, it is clear that metastasis is invariably preceded by angiogenesis. One of the angiogenic factors that is produced by human melanoma cells is vascular endothelial growth factor (VEGF). To investigate the role of this factor in the angiogenic process in primary cutaneous melanoma we determined the mean vascular density and the presence of VEGF protein in biopsies of human lesions. The results were compared with those found in normal skin or uninvolved skin from melanoma patients. In addition, we studied morphological and antigenic features of the proliferating neovasculature. We show that (1) the mean vascular density gradually rises along with melanoma progression, (2) transition of horizontal to vertical growth phase melanoma is accompanied by induction of VEGF protein expression and accumulation of this factor in the stroma, (3) vertical growth phase melanoma is often organized in nodules separated by septa containing blood vessels, but without lymphatics, and (4) blood vessel lumina in vertical growth phase melanoma are separated from tumour nodules by two basal lamina containing collagen type IV and the endothelium shows activated morphology and focal expression of the adhesion molecule E-selectin. Our findings indicate that VEGF is a prominent angiogenic factor in melanoma angiogenesis. Although its expression is induced during progression, the effect of VEGF on the incidence of metastasis is probably indirect.

A TECHNIQUE TO VISUALIZE CORNEAL INCISION AND LAMELLAR DISSECTION DEPTH DURING SURGERY

PURPOSE To describe a surgical technique to visualize the depth of corneal incisions and lamellar stromal dissections during surgery. METHODS In porcine cadaver eyes, the aqueous was exchanged by air. Thus an air-to-endothelium interface (i.e., a useful optical surface) was created at the posterior corneal surface. The air-to-endothelium interface was used as a reference plane to visualize the corneal thickness and the relative depth of corneal incisions and dissections. Freehand peripheral corneal incisions, tangential keratotomy incisions, and lamellar stromal dissections were made at an intended corneal depth of 60, 80, and 99%. Light microscopy was used to measure the relative depth of the incisions and dissections. RESULTS Achieved depth for peripheral corneal incisions averaged 65.2+/-5.3%, 78.8+/-5.1%, and 93.4+/-6.0%, respectively (p<0.05); and for tangential keratotomy incisions, 68.2+/-7.3%, 83.2+/-4.4%, and 95.8+/-3.6%, respectively (p<0.05). Achieved depth for lamellar stromal dissections averaged 58.3+/-9.4%, 81.1+/-3.4%, and 94.4+/-1.5%, respectively (p<0.05). Microperforations occurred with three incisions made at 99% intended depth. CONCLUSION During surgery, the depth of incisions and lamellar dissections relative to the corneal thickness can be visualized by filling the anterior chamber with air (i.e., by creating an optical interface at the posterior corneal surface).

Aminopeptidase A is a constituent of activated pericytes in angiogenesis

Monoclonal antibody (MAb) RC38 recognizes a human renal antigen of 160 kD recently identified as human aminopeptidase A (APA; EC 3.4.11.7). This ectoenzyme is able to hydrolyse selectively N‐terminal glutamyl and aspartyl residues from oligopeptides. By enzyme histochemistry, APA activity has also been localized in the microvessels of all organs in animals and man. The purpose of this study was to investigate the distribution of human APA as recognized by MAb RC38 in the microvasculature of normal human tissues and pathological conditions associated with neovascularization. Unexpectedly, in normal tissues vascular staining with MAb RC38 was generally weak and often absent, while in tumours, granulation tissue, and chronic synovitis, marked microvascular staining was demonstrated. By immuno‐electron microscopy, the antigen was found on the cell membrane of activated pericytes and their processes in the tumour vasculature. RC38 expression could not be detected on cultured human endothelial cells or pericytes. These observations suggest that pericyte expression of a subtype of APA (as recognized by MAb RC38) is markedly enhanced in the vasculature of tumours and wound healing tissue as compared with normal resting tissues. This provides further evidence of the altered state of pericytes in these conditions. Pericyte APA may be involved in the metabolism of biologically active oligopeptides during neovascularization, supporting a regulatory role of pericytes in this process. In addition, MAb RC38 may be useful as a marker of pericyte activation in tissue sections.

Healing of reopened-and-sutured radial keratotomy wounds

Abstract We designed a study to evaluate healing in reopened‐and‐sutured (RAS) keratotomy wounds to determine the efficacy of reoperations in treating radial keratotomy overcorrections. Using light and transmission electron microscopy, we compared stromal scar tissue organization (transverse fibroblast orientation and collagen fiber continuity across the wound) in RAS wounds and in sutured and unsutured control wounds in 18 monkey eyes one to nine weeks after surgery. Wound healing morphology of RAS wounds varied with the interval between reoperation and termination of the experiment. Scar tissue organization was sagittal at one week postoperatively, transverse in the anterior and mid regions after four weeks, and transverse over the entire wound after nine weeks. Sutured wounds showed a similar pattern of healing, although transverse scar tissue organization was restricted to the anterior and mid regions in the late healing phases. In contrast, unsutured wounds showed a temporary, transverse scar tissue organization over the entire wound depth at two to four weeks and a progressive reorientation of the mid and posterior scar tissue sagittal to the wound at later intervals. The results suggest that reopening and suturing keratotomy incisions to treat radial keratotomy overcorrections may be effective through a myopic shift induced by sutured wound apposition and long‐term wound remodeling, contraction, or both.

Expression of the high molecular weight melanoma-associated antigen by percytes during antigionesis in tumors and in healing wounds

In the course of immunohistochemical characterization of murine monoclonal antibodies recognizing the human high molecular weight-melanoma associated antigen (HMW-MAA), a striking reactivity with blood vessels in the tumor stroma was noted. Immunocytochemical analysis by light and electronmicroscopy of a panel of tissues and cell lines showed that the staining of microvessels by anti-HMW-MAA monoclonal antibodies was restricted to pericytes. Correspondingly, anti-HMW-MAA monoclonal antibodies were found to react with cultured pericytes from human brain, but not with endothelial cells in serologic assays, and to immunoprecipitate from biosynthetically labeled pericytes an antigen with the characteristic structural profile of HMW-MAA. At the subcellular level, the expression of HMW-MAA in cultured pericytes was mainly restricted to microspikes that are localized in clusters on the cellular membrane. Staining by anti-HMW-MAA monoclonal antibodies of pericytes was not only found in the tumor stroma, but also in other lesions associated with angiogenesis, such as granulation tissue of wound healing and synovitis. In these lesions, microvascular staining for another marker of pericytes, ie, alpha-smooth muscle actin, also was observed. These results suggest that, in conditions associated with vascular proliferation, 1) pericytes acquire HMW-MAA and 2) the number of pericytes may be increased as compared with normal tissues.