Anatomy and Pathology Morphological Alterations Within the Peripheral Fixation of the Iris Dilator Muscle in Eyes With Pigmentary Glaucoma Cassandra M. Flu¨gel-Koch,1 Ozan Y. Tektas,2 Paul L. Kaufman,3 Friedrich P. Paulsen,1 and Elke Lu¨tjen-Drecoll1 1Department of Anatomy II, Friedrich-Alexander-University of Erlangen-Nu¨rnberg, Erlangen, Germany 2Department of Psychiatry and Psychotherapy, Friedrich-Alexander-University of Erlangen-Nu¨rnberg, Erlangen, Germany 3Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, Wisconsin, United States Correspondence: Cassandra M. PURPOSE. To analyze the peripheral fixation of the iris dilator muscle in normal eyes and in Flu¨gel-Koch, Department of Anatomy eyes with pigmentary glaucoma (PG). II, Friedrich-Alexander-University of Erlangen-Nu¨rnberg, Universit¨atsstr. METHODS. Using 63 control eyes (age 18 months–99 years), the peripheral iris dilator was 19, 91054 Erlangen, Germany; investigated by light microscopy, immunohistochemistry, and electron microscopy. Develop- cassandra.fluegel-koch@anatomie2. ment was studied using 18 differently aged fetal eyes stained immunohistochemically against med.uni-erlangen.de. a-smooth muscle (SM) actin. The peripheral iris dilator muscle in PG was analyzed using Submitted: December 13, 2013 semithin and ultrathin sections of six glutaraldehyde-fixed eyes from three donors aged 38, Accepted: June 6, 2014 62, and 74 years. Citation: Flu¨gel-Koch CM, Tektas OY, RESULTS. In normal eyes, the peripheral end of the iris dilator muscle is arranged in a sphincter- Kaufman PL, Paulsen FP, Lu¨tjen-Dre- like manner. Arcade-shaped tendinous connections associated with myofibroblasts (iridial coll E. Morphological alterations strands) anchor the iris dilator within the elastic–fibromuscular ciliary meshwork that also within the peripheral fixation of the serves as fixation area for the elastic tendons of the inner ciliary muscle portions. The iridial iris dilator muscle in eyes with pig- strands are innervated and can adapt their length during accommodation. The PG eyes show mentary glaucoma. Invest Ophthal- incomplete circular bundles and iridial strands that are mainly anchored to the iris stroma and mol Vis Sci. 2014;55:4541–4551. DOI:10.1167/iovs.13-13765 the flexible uveal parts of the trabecular meshwork. CONCLUSIONS. The normal anchorage of the peripheral iris dilator and its presumably neuronally regulated length adaptation stabilize the peripheral iris during accommodation. Insufficient fixation in PG could promote posterior bowing of the iris with rubbing against the zonular fibers and pigment liberation from the iris pigmented epithelium. Keywords: iris dilator muscle, anchorage, pigmentary glaucoma, morphology, immunohistochemistry n pigment dispersion syndrome (PDS), accommodation state, suggesting PDS/PG-related structural changes in this I causes rubbing of the pigmented iris epithelium against the area.17 zonular fibers, resulting in accumulation of pigment granules A major influence on the morphology of the iridiocorneal within the aqueous humor and the outflow tissue.1–5 If the angle during accommodation could be exerted by the aqueous outflow is sufficiently obstructed and intraocular peripheral fixation of the iris dilator muscle, which appears pressure is elevated, secondary open-angle glaucoma (pigmen- to have connections to the ciliary muscle.18,19 Studies on the tary glaucoma, PG) with optic neuropathy may occur.6–8 peripheral fixation of the iris dilator muscle in PDS/PG are The reason for the iridiozonular contact and the increased lacking, and it is not even described in detail in normal eyes. pigment dispersion is unknown. A genetic etiology with The most comprehensive review article dates back to 1926.20 multifactorial pattern of inheritance or a possible autosomal- The existence of filaments from the dilator muscle extending dominant inheritance pattern with incomplete penetrance and up to the venous vascular walls, pectinate ligament, ciliary expressivity has been suggested.9–11 It has been proposed that muscle, or connective tissue of the ciliary body was described, theirisistoolargefortheeye,1,12,13 and ultrasound but affirmation or further evaluations could not be made due to biomicroscopy (UBM) measurements have shown a deeper the lack of more specific investigative methods. anterior chamber and a larger distance between iris insertion In this study we had a unique collection of six well- and trabecular meshwork in PDS/PG eyes compared to normal preserved postmortem eyes of human donors aged 38, 62, and age-matched control eyes.14–16 Moreover, UBM studies evaluat- 74 years who had suffered from PG. The morphological ing iridiocorneal architecture in the relaxed and stimulated changes of the peripheral iris dilator and its fixation in the accommodative state have found that the iridiocorneal angle ciliary body of these eyes were compared with the normal and iris concavity are the most discriminatory and statistically morphology of these structures. The latter were studied with different parameters between PDS/PG and controls. These immunohistochemical and ultrastructural methods in a large differences were especially prominent in the accommodative group of control eyes and included the development of the Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc. www.iovs.org j ISSN: 1552-5783 4541 Downloaded from jov.arvojournals.org on 09/25/2021 Peripheral Fixation of Iris Dilator Muscle in PG IOVS j July 2014 j Vol. 55 j No. 7 j 4542 TABLE. Source and Concentration of the Primary Antibodies alcohols, the specimens were embedded in paraffin and serial 7-lm thin sections were cut in sagittal, tangential (parallel to Antibody Host Dilution Source the iris surface), frontal, and oblique-frontal planes. a-Smooth muscle actin Mouse 1:300 Sigma-Aldrich, St. Louis, The sections were placed on 0.1% poly-L-lysine–coated glass MO, USA slides, deparaffinized, and stained with hematoxylin-eosin Collagen type VI Rabbit 1:200 Rockland, Gilbertsville, (HE), Azan, Crossmon, or Weigert’s stain for elastin or for PA, USA immunohistochemistry as described below. Collagen type IV Mouse 1:200 Dako, Hamburg, Additional PFA-fixed and rinsed specimens from each Germany quadrant were deep frozen in isopentane precooled with Elastin Rabbit 1:400 Chemicon (Millipore), liquid nitrogen, and series of 10-lm thin sections were cut in Billerica, MA, USA midsagittal, tangential, and oblique-frontal planes. The sections Pan neurofilament Mouse 1:200 Zymed, San Francisco, were also placed on slides that had been coated with 0.1% CA, USA poly-L-lysine and were stained immunohistochemically. Protein gene product Biotrend, Cologne, Immunohistochemistry. (PGP) 9.5 Rabbit 1:100 Germany Staining of Paraffin and Frozen Sections. Deparaffinized Synaptophysin Mouse 1:20 Dako, Glostrup, sections as well as frozen sections that had been allowed to dry Denmark for several hours were preincubated for 15 minutes in 1% dry Calretinin Rabbit 1:1000 Swant, Bellinzona, milk solution to prevent nonspecific staining, then incubated Switzerland overnight at room temperature with the primary antibodies Neuropeptide Y (NPY) Rat 1:1000 Biotrend, Cologne, (Table) diluted according to the manufacturer’s recommenda- Germany tions. Following rinsing the sections were incubated with Tyrosine hydroxylase Chemicon, Hofheim, Alexa Fluor 488– or Alexa Fluor 555–labeled immunoglobulin (TH) Rabbit 1:40 Germany G secondary antibody (MoBiTec, Goettingen, Germany). For Substance P (SP) Rabbit 1:200 Peninsula Laboratories, several sections, various double stainings were also performed, San Carlos, CA, USA for example, combining a-smooth muscle (SM) actin antibody Vesicular acetylcholine Goat 1:1000 Bioscience, Heidelberg, with anti-type VI collagen, anti-calretinin, anti-tyrosine hydrox- transporter (VAChT) Germany ylase, anti-vesicular acetylcholine transporter (VAChT), or Calcitonin gene-related Rabbit 1:600 Biotrend, Cologne, calretinin with anti-neurofilament. The sections were rinsed peptide (CGRP) Germany again, mounted in Kaiser’s glycerine (Merck, Darmstadt, Germany), and viewed with a fluorescence microscope (Aristoplan; Ernst Leitz, Wetzlar, Germany). peripheral iris dilator and its adaptation during accommodation Whole Mounts. As all serial sections through different planes and disaccommodation. could not reveal a complete picture of the connection between The results point to an altered peripheral iris fixation in PG iris and ciliary body, whole mounts from this area were versus normal control eyes that could predispose to the prepared that were stained in a free-floating manner following disease. immunohistochemical staining protocols as described above, but with longer periods of incubation and rinsing time. For this purpose, 10 eyes ranging in age from 38 to 91 years were chosen. From each quadrant 0.5- to 1.0-cm-wide MATERIALS AND METHODS specimens were prepared containing parts of the ciliary body Control Eyes and peripheral iris. In order to reduce the thickness of the whole mounts, the tips of the ciliary processes and parts of the To investigate the peripheral iris dilator fixation in normal eyes, outermost layers of the ciliary muscle were removed, taking 63 eyes from 45 human donors with an age range from 18 care to preserve the intactness of the transitional zone of iris months to 99 years were investigated. The eyes had been and ciliary body. In some whole mounts, iris pigment was obtained from the Department of Anatomy, University of scraped away