Frank L. Kretzer

Free Radicals and Ocular Disease

Ames, Shigenaga, and Hagen recently published a thorough review of the relationship between oxidants, antioxidants, and degenerative diseases of ageing. They point out that only 9% of Americans daily consume the two fruits and three vegetables recommended by the National Cancer Institute and the National Research Council/National Academy of Science. In addition to antioxidants, these foodstuffs contain many essential micronutrients. To date, specific recommendations for antioxidant supplementation have not been made by any governmental agency or professional association. A number of clinical, basic, and epidemiological studies have implicated free radical induced lipid peroxidation in various ocular disorders. It would seem prudent that those persons at greatest risk for these disorders take some precautions, which could include sunglasses that filter ultraviolet light; hats that shield the eyes from direct sunlight; and the ingestion of fruits, vegetables, and antioxidants.

A new freeze-drying technique applied to the analysis of the molecular structure of mitochondrial and chloroplast membranes.

For the analysis of the molecular structure of cellular components by means of electron microscopy, a resolution of at least 10 A was considered required. Only very thin sections of material embedded in plastic offer sufficiently thin specimens for attaining this resolution. The problem of transferring cells and tissues from an aqueous environment to a plastic without producing extensive conformational changes requires an elaborate use of means to stabilize protein conformation and to reduce the tendency for unfolding of peptide chains until the material is locked in in a solid plastic. The method that was developed involved freeze-drying and embedding at a low temperature. The material was first stabilized by means of brief cross-linking with glutaraldehyde (minimum time), partially dehydrated with 30% ethylene glycol to prevent ice crystallization, rapidly frozen at −180° in propane, dried through vacuum distillation at −80°C and infiltrated by an embedding medium, hydroxypropyl methacrylate, at −30°C followed by polymerization by means of uv irradiation at −27°C. All steps in the procedure are discussed in detail in the light of present physical chemical and biochemical knowledge. Further improvements of the technique are proposed. The development of a new embedding medium is of crucial importance. Some results from applying the method to an analysis of mitochondrial and chloroplast membranes are reported. These results confirm those obtained earlier by Sjostrand and Barajas (46) and by Kretzer (16,) with cross-linking, ethylene glycol dehydration and Vestopal embedding. The observations are related to the membrane model proposed originally by Sjostrand in 1965 and further developed by Sjostrand and Barajas (46, 47), and to the models proposed later by Singer and Nicholson (36) and Packer (23).

A small foveal avascular zone may be an historic mark of prematurity.

OBJECTIVE To compare in children the area and diameter of the foveal avascular zone (FAZ) of former preterm infants, when no significant retinopathy of prematurity (ROP) developed, to the area and diameter of the FAZ of former term infants. DESIGN Retrospective observational case series and literature review. PARTICIPANTS Forty-nine children (39 former preterm infants and 10 former term infants) between the ages of 1 and 17 years had fluorescein angiograms. All of these children had been appropriate weight for gestational age at birth and had no genetic disorders. Neither eye of any of these children had any macular ectopia or vessel traction, had been treated for active ROP, had developed active ROP >stage 3 mild, or had any refractive error > +/- five diopters. Every child had a visual acuity of 20/40 or better in both eyes. METHODS The area and greatest diameter of the FAZ were measured using digital image analysis of masked fundus fluorescein angiograms. Variables of gender, race, multiple birth, gestational age, birth weight, ROP stage, age, and refraction at the time of fluorescein angiography, and final visual acuity were recorded. RESULTS Increasing FAZ area and greatest diameter correlated significantly with increasing gestational age and birth weight: FAZ area (microm2) versus gestational age (weeks) (R/F/P = 0.88/166.70/<0.0001); FAZ greatest diameter (microm) versus gestational age (weeks) (R/F/P = 0.87/151.10/<0.0001); FAZ area (micro/m2) versus birth weight (g) (R/F/P = 0.88/167.06/<0.0001); and FAZ greatest diameter (microm) versus birth weight (g) (R/F/P = 0.87/148.74/ <0.0001). A small or absent FAZ was found in all former preterm infants who had been < or = 30 weeks gestational age or had weighed < or = 1100 g at birth. A normal FAZ was present in all children who had been > or = 36 weeks gestational age or had weighed > or = 2650 g at birth. None of the other parameters studied correlated with FAZ area or greatest diameter. CONCLUSION This study provides evidence that the FAZ in developing humans is initially densely vascularized with a fine meshwork of inner retinal vessels during vasculogenesis. This vascular meshwork undergoes regression by apoptosis in all infants > or = 36 weeks gestational age at birth to form a normal FAZ, but apoptosis almost never occurs in preterm infants < or = 30 weeks gestational age at birth. Although there is no effect on final visual acuity, a small or absent FAZ may be an historic mark of prematurity.

Variable Expressivity of Autosomal Dominant Anterior Segment Mesenchymal Dysgenesis in Six Generations

Of 58 members in a six-generation family with anterior segment mesenchymal dysgenesis with variable expressivity, 21 of 35 members (60%) at risk were affected. Of the 15 living affected family members, nine (60%) had visual acuities of 6/12 (20/40) or better in at least one eye, five (33%) had visual acuities between 6/15 and 6/60 (20/50 and 20/200) in at least one eye, and one (7%) had a visual acuity of less than counting fingers at one foot in both eyes. All nine affected patients who underwent slit-lamp examinations had corneal abnormalities with and without synechiae. All 15 affected patients also had cataracts, and three of the 15 (20%) had optic nerve abnormalities. In a corneal button from the severely affected proband, Descemets layer and endothelial cells were absent even in the periphery. Other corneal and lenticular changes were secondary to the primary endothelial defect. Anterior segment mesenchymal dysgenesis in this family appeared to be caused by an aberration of the first wave of mesenchyme from the rim of the optic cup.

Oculocutaneous Albinoidism as a Manifestation of Reduced Neural Crest Derivatives in the Prader-Willi Syndrome

Nine patients with Prader-Willi syndrome (five female and four male; one Oriental and eight white), all of whom had interstitial deletions of the proximal long arm of one chromosome 15 (q11-q13) were found to have decreased tyrosinase activity in isolated hair bulbs. As infants, all patients had light hair and skin coloring, both of which darkened with age. Light and electron microscopic analysis of skin and hair bulbs disclosed a reduced number of melanocytes in the basal epidermis and hair bulbs. Each patient demonstrated decreased pigmentation of the iris stroma, which was accentuated peripherally and manifested clinically as iris translucency. There was no foveal hypoplasia, nystagmus, or photophobia, and ocular function was normal. Oculocutaneous albinoidism is thus a component of del(15q) Prader-Willi syndrome with reduction of melanocytes of neural crest origin (skin, hair, and iris stroma) and retention of normal retinal and iris pigment epithelia of neuroectodermal origin.

Suppression of severe retinopathy of prematurity with vitamin E supplementation. Ultrastructural mechanism of clinical efficacy

Three clinical trials enrolling 418 infants (less than or equal to 1500 g birth weight) and an ultrastructural data base of 71 pairs of whole eye donations have elucidated the efficacy of vitamin E in suppressing the development of severe retrolental fibroplasia (ROP). Only continuous vitamin E supplementation to adult physiologic levels from the first hours of life suppresses the development of severe ROP. Supplementation does not increase the incidence of necrotizing enterocolitis, sepsis, intraventricular hemorrhage, or mortality. Only multivariate analysis, which considers all risk factors simultaneously, is appropriate when appraising the efficacy of supplementation since all the clinical risk factors uniquely impinge on the oxygen dynamics of the developing retina. Mesenchymal spindle cells are the cellular mediators of the induction of ROP by oxygen in which increased oxygen tension triggers extensive gap junction formation between adjacent spindle cells. This cellular event, which occurs as early as four days of life, halts the normal vasoformative process and triggers neovascularization, which becomes clinically evident some 8 to 12 weeks later.

An interpretation of retinopathy of prematurity in terms of spindle cells: relationship to vitamin E prophylaxis and cryotherapy

Spindle cells in the hyperoxygenated, avascular, vanguard retina are proposed to be the peripheral inducers of the neovascularization associated with retinopathy of prematurity (ROP). The induction of ROP is conceptualized in terms of three basic events. First, activation of spindle cells results initially in the increase in gap junctions between adjacent spindle cells, secondarily in the increase in cytoplasmic volume of rough endoplasmic reticulum, and ultimately in the synthesis and secretion of angiogenic factors. Second, maturation of spindle cells is associated with a decrease in gap junctions, a diminished cytoplasmic volume of rough endoplasmic reticulum, and a cessation of synthesis and secretion of angiogenic factors. Third, myofibroblasts invade the vitreous concomitantly with spindle cell maturation and provide the tractional force that can produce retinal separation. The extent of interstitial retinol binding protein within the subretinal space explains the gestational-age-dependent efficacy of vitamin E in suppressing the development of severe ROP. The kinetics of both spindle cell activation/maturation and myofibroblast invasion predict the efficacy of appropriately timed and placed transretinal cryotherapy.

Peripheral retinopathy in offspring of carriers of Norrie disease gene mutations. Possible transplacental effect of abnormal Norrin.

BACKGROUND The Norrie disease (ND) gene (Xp11.3) (McKusick 310600) consists of one untranslated exon and two exons partially translated as the Norrie disease protein (Norrin). Norrin has sequence homology and computer-predicted tertiary structure of a growth factor containing a cystine knot motif, which affects endothelial cell migration and proliferation. Norrie disease (congenital retinal detachment), X-linked primary retinal dysplasia (congenital retinal fold), and X-linked exudative vitreoretinopathy (congenital macular ectopia) are allelic disorders. METHODS Blood was drawn for genetic studies from members of two families to test for ND gene mutations. Sixteen unaffected family members were examined ophthalmologically. If any retinal abnormality were identified, fundus photography and fluorescein angiography was performed. RESULTS Family A had ND (R109stp), and family B had X-linked exudative vitreoretinopathy (R121L). The retinas of 11 offspring of carrier females were examined: three of seven carrier females, three of three otherwise healthy females, and one of one otherwise healthy male had peripheral inner retinal vascular abnormalities. The retinas of five offspring of affected males were examined: none of three carrier females and none of two otherwise healthy males had this peripheral retinal finding. CONCLUSIONS Peripheral inner retinal vascular abnormalities similar to regressed retinopathy of prematurity were identified in seven offspring of carriers of ND gene mutations in two families. These ophthalmologic findings, especially in four genetically healthy offspring, strongly support the hypothesis that abnormal Norrin may have an adverse transplacental (environmental) effect on normal inner retinal vasculogenesis.

Postnatal Retinal Vascularization in Former Preterm Infants with Retinopathy of Prematurity

PURPOSE To study the postnatally vascularized retina in former preterm infants in whom retinopathy of prematurity (ROP) stages 2 to 4a developed and spontaneously regressed. METHODS Matched fundus photographs and fluorescein angiograms of the temporal peripheral retinas of 133 eyes (72 patients) were obtained after 2 years of age (mean, 7.7 years; range, 2-16.2 years) and were quantified by two masked observers with respect to the following parameters: (1) macular ectopia (in disc diameters); (2) vessel traction (in 30 degrees sectors); (3) radial length of postnatally vascularized retina (in disc diameters); and (4) capillary scaffolding of postnatally vascularized retina (as a density). These cicatricial outcomes were then compared with their active worst ROP stage. RESULTS Of the 133 retinal montages, the following active worst ROP stages had been documented: 30 with stage 2, 42 with stage 3 mild, 32 with stage 3 moderate, 20 with stage 3 severe, and 9 with stage 4a. As active worst ROP stage increased, macular ectopia and vessel traction increased, and radial length and capillary scaffolding of postnatal retinal vascularization decreased. Retinal holes were documented frequently in eyes with high myopia. CONCLUSIONS The peripheral retina in former preterm infants warrants close scrutiny for possible late rhegmatogenous retinal detachments. Prolonged retinal traction (by remnant shunt and extraretinal fibrovascular proliferation) between stable, posterior, prenatally vascularized retina, and unstable, postnatally vascularized retina may lead to the development of retinal holes characteristically located in the fragile, anterior, undifferentiated, nonvascularized retina.

The mitochondrion in dividing Leishmania tarentolae cells is symmetric and circular and becomes a single asymmetric tubule in non-dividing cells due to division of the kinetoplast portion

Kinetoplastid protozoa have a single mitochondrion that extends throughout the cell. The disk-shaped portion of the mitochondrion adjacent to the basal body of the flagellum contains the kinetoplast DNA nucleoid body which consists of thousands of catenated minicircles and a smaller number of catenated maxicircles. The maxicircles contain structural genes and cryptogenes, rRNA genes, and a few guide RNA genes The minicircles contain the majority of the guide RNA genes. The long slender non-dividing stationary phase Leishmania tarentolae cells in culture have an asymmetric mitochondrion that consists of a single tubule extending from one edge of the kinetoplast portion. This presents a problem for cell division, in that one daughter cell will receive significantly less mitochondrial membranes than the other cell. We show in this paper that the solution to this problem is that dividing cells, which are normally shorter and rounder than stationary phase cells, possess a symmetric circular mitochondrion that has mitochondrial tubules extending from both edges of the kinetoplast which are joined in the posterior region of the cell. This implies that growth of the mitochondrion occurs after cell division, either from elongation of the longitudinal tubule towards the anterior of the cell, or from elongation of the kinetoplast portion of the mitochondrion towards the posterior region and fusion of the tubules.