Retinal Detachment after Treatment of Retinopathy of Prematurity with Laser versus Intravitreal Anti-Vascular Endothelial Growth Factor.

PURPOSE: To compare rates of short-term retinal detachment (RD) of infants treated for type 1 retinopathy of prematurity (ROP) with intravitreal anti-vascular endothelial growth factor (VEGF) therapy with infants treated with laser therapy. The choice between these 2 treatments remains controversial. Comparative data are limited and describe re-treatment rates rather than retinal structural outcomes predictive of long-term vision. Anti-vascular endothelial growth factor acts faster than laser therapy, which may be beneficial for more aggressive ROP. DESIGN: Nonrandomized, comparative cohort study. PARTICIPANTS: The study included 1167 eyes of 640 infants treated for type 1 ROP. Among these, 164 eyes received anti-VEGF therapy and 1003 eyes received laser therapy. METHODS: Pretreatment and posttreatment examinations and treatments were completed by ophthalmologists with expertise in ROP. The study was a secondary analysis of data from the retrospective Postnatal Growth and Retinopathy of Prematurity Study (G-ROP) 1 study (2006-2012) and the prospective G-ROP 2 study (2015-2017). MAIN OUTCOME MEASURES: Rate of RD (ROP stages 4A, 4B, or 5) within 8 weeks of initial treatment, an end point predictive of poor long-term vision. The results were stratified by postmenstrual age (PMA) at treatment as occurring before versus at or after 36 weeks and 0 days, because earlier disease may be considered more aggressive. RESULTS: Among 458 eyes treated before PMA 36 weeks and 0 days, the short-term RD rate was higher after laser therapy (29/368 eyes [7.9%]) than after anti-VEGF therapy (0/90 eyes [0%]; P < 0.001). Of 709 eyes treated at or after PMA 36 weeks and 0 days, short-term RD risk did not differ between groups (laser [20/635 eyes], 3.1%; anti-VEGF [1/74 eyes], 1.4%; P = 0.27). CONCLUSIONS: Anti-vascular endothelial growth factor therapy results in better short-term structural outcomes than laser therapy when type 1 ROP is treated before 36 weeks' PMA. After this age, both treatments have very low rates of short-term RD. The faster action of anti-VEGF agents likely is responsible for these findings.

Elucidating the framework for specification and determination of the embryonic retina.

How cell determination is regulated remains a major unsolved problem in developmental biology. The early embryonic rudiments of many tissues and organs are difficult or impossible to identify, isolate and study at the time when determination occurs. We have examined the commitment process leading to retina formation in Xenopus laevis, where presumptive eye tissue can be identified and studied to assay its biological properties during the events leading up to determination. We find that for the retina, specification, the point at which a tissue placed in neutral culture medium can first properly differentiate, occurs during mid-gastrulation. By late gastrulation, determination, the final, irreversible step in commitment, has occurred. At this stage, the presumptive retina will differentiate and cannot be reprogrammed even if exposed to other active inducers, e.g. when challenged by transplantation to ectopic sites in the embryo. Key eye regulatory genes are initially expressed in the retinal field during specification and/or determination (e.g. rax, pax6, lhx2, and fzd5) potentially linking them, or genes that regulate them, to these processes. This study provides essential groundwork for defining the mechanisms for how these important developmental transitions occur.

no privacy, a Xenopus tropicalis mutant, is a model of human Hermansky-Pudlak Syndrome and allows visualization of internal organogenesis during tadpole development.

We describe a novel recessive and nonlethal pigmentation mutant in Xenopus tropicalis. The mutant phenotype can be initially observed in tadpoles after stage 39/40, when mutant embryos display markedly reduced pigmentation in the retina and the trunk. By tadpole stage 50 almost all pigmented melanophores have disappeared. Most interestingly, those embryos fail entirely to make pigmented iridophores. The combined reduction/absence of both pigmented iridophores and melanophores renders these embryos virtually transparent, permitting one to easily observe both the developing internal organs and nervous system; accordingly, we named this mutant no privacy (nop). We identified the causative genetic lesion as occurring in the Xenopus homolog of the human Hermansky-Pudlak Syndrome 6 (HPS6) gene, combining several approaches that utilized conventional gene mapping and classical and modern genetic tools available in Xenopus (gynogenesis, BAC transgenesis and TALEN-mediated mutagenesis). The nop allele contains a 10-base deletion that results in truncation of the Hps6 protein. In humans, HPS6 is one of the genes responsible for the congenital disease HPS, pathological symptoms of which include oculocutaneous albinism caused by defects in lysosome-related organelles required for pigment formation. Markers for melanin-producing neural crest cells show that the cells that would give rise to melanocytes are present in nop, though unpigmented. Abnormalities develop at tadpole stages in the pigmented retina when overall pigmentation becomes reduced and large multi-melanosomes are first formed. Ear development is also affected in nop embryos when both zygotic and maternal hsp6 is mutated: otoliths are often reduced or abnormal in morphology, as seen in some mouse HPS mutations, but to our knowledge not described in the BLOC-2 subset of HPS mutations nor described in non-mammalian systems previously. The transparency of the nop line suggests that these animals will aid studies of early organogenesis during tadpole stages. In addition, because of advantages of the Xenopus system for assessing gene expression, cell biological mechanisms, and the ontogeny of melanosome and otolith formation, this should be a highly useful model for studying the molecular mechanisms underlying the acquisition of the HPS phenotype and the underlying biology of lysosome-related organelle function.

Xenopus pax6 mutants affect eye development and other organ systems, and have phenotypic similarities to human aniridia patients.

Mutations in the Pax6 gene cause ocular defects in both vertebrate and invertebrate animal species, and the disease aniridia in humans. Despite extensive experimentation on this gene in multiple species, including humans, we still do not understand the earliest effects on development mediated by this gene. This prompted us to develop pax6 mutant lines in Xenopus tropicalis taking advantage of the utility of the Xenopus system for examining early development and in addition to establish a model for studying the human disease aniridia in an accessible lower vertebrate. We have generated mutants in pax6 by using Transcription Activator-Like Effector Nuclease (TALEN) constructs for gene editing in X. tropicalis. Embryos with putative null mutations show severe eye abnormalities and changes in brain development, as assessed by changes in morphology and gene expression. One gene that we found is downregulated very early in development in these pax6 mutants is myc, a gene involved in pluripotency and progenitor cell maintenance and likely a mediator of some key pax6 functions in the embryo. Changes in gene expression in the developing brain and pancreas reflect other important functions of pax6 during development. In mutations with partial loss of pax6 function eye development is initially relatively normal but froglets show an underdeveloped iris, similar to the classic phenotype (aniridia) seen in human patients with PAX6 mutations. Other eye abnormalities observed in these froglets, including cataracts and corneal defects, are also common in human aniridia. The frog model thus allows us to examine the earliest deficits in eye formation as a result of pax6 lesions, and provides a useful model for understanding the developmental basis for the aniridia phenotype seen in humans.

Xenopus mutant reveals necessity of rax for specifying the eye field which otherwise forms tissue with telencephalic and diencephalic character.

The retinal anterior homeobox (rax) gene encodes a transcription factor necessary for vertebrate eye development. rax transcription is initiated at the end of gastrulation in Xenopus, and is a key part of the regulatory network specifying anterior neural plate and retina. We describe here a Xenopus tropicalis rax mutant, the first mutant analyzed in detail from a reverse genetic screen. As in other vertebrates, this nonsense mutation results in eyeless animals, and is lethal peri-metamorphosis. Tissue normally fated to form retina in these mutants instead forms tissue with characteristics of diencephalon and telencephalon. This implies that a key role of rax, in addition to defining the eye field, is in preventing alternative forebrain identities. Our data highlight that brain and retina regions are not determined by the mid-gastrula stage but are by the neural plate stage. An RNA-Seq analysis and in situ hybridization assays for early gene expression in the mutant revealed that several key eye field transcription factors (e.g. pax6, lhx2 and six6) are not dependent on rax activity through neurulation. However, these analyses identified other genes either up- or down-regulated in mutant presumptive retinal tissue. Two neural patterning genes of particular interest that appear up-regulated in the rax mutant RNA-seq analysis are hesx1 and fezf2. These genes were not previously known to be regulated by rax. The normal function of rax is to partially repress their expression by an indirect mechanism in the presumptive retina region in wildtype embryos, thus accounting for the apparent up-regulation in the rax mutant. Knock-down experiments using antisense morpholino oligonucleotides directed against hesx1 and fezf2 show that failure to repress these two genes contributes to transformation of presumptive retinal tissue into non-retinal forebrain identities in the rax mutant.

A Multifunctional, Low Cost and Sustainable Neonatal Database System.

Continuous improvement in the clinical performance of neonatal intensive care units (NICU) depends on the use of locally relevant, reliable data. However, neonatal databases with these characteristics are typically unavailable in NICUs using paper-based records, while in those using electronic records, the inaccuracy of data and the inability to customize commercial data systems limit their usability for quality improvement or research purposes. We describe the characteristics and uses of a simple, neonatologist-centered data system that has been successfully maintained for 30 years, with minimal resources and serving multiple purposes, including quality improvement, administrative, research support and educational functions. Structurally, our system comprises customized paper and electronic components, while key functional aspects include the attending-based recording of diagnoses, integration into clinical workflows, multilevel data accuracy and validation checks, and periodic reporting on both data quality and NICU performance results. We provide examples of data validation methods and trends observed over three decades, and discuss essential elements for the successful implementation of this system. This database is reliable and easily maintained; it can be developed from simple paper-based forms or used to supplement the functionality and end-user customizability of existing electronic medical records. This system should be readily adaptable to NICUs in either high- or limited-resource environments.

Simple and efficient CRISPR/Cas9-mediated targeted mutagenesis in Xenopus tropicalis.

We have assessed the efficacy of the recently developed CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system for genome modification in the amphibian Xenopus tropicalis. As a model experiment, targeted mutations of the tyrosinase gene were verified, showing the expected albinism phenotype in injected embryos. We further tested this technology by interrupting the six3 gene, which is required for proper eye and brain formation. Expected eye and brain phenotypes were observed when inducing mutations in the six3 coding regions, as well as when deleting the gene promoter by dual targeting. We describe here a standardized protocol for genome editing using this system. This simple and fast method to edit the genome provides a powerful new reverse genetics tool for Xenopus researchers.

Defining progressive stages in the commitment process leading to embryonic lens formation.

The commitment of regions of the embryo to form particular tissues or organs is a central concept in development, but the mechanisms controlling this process remain elusive. The well-studied model of lens induction is ideal for dissecting key phases of the commitment process. We find in Xenopus tropicalis, at the time of specification of the lens, i.e., when presumptive lens ectoderm (PLE) can be isolated, cultured, and will differentiate into a lens that the PLE is not yet irreversibly committed, or determined, to form a lens. When transplanted into the posterior of a host embryo lens development is prevented at this stage, while ~ 3 h later, using the same assay, determination is complete. Interestingly, we find that specified lens ectoderm, when cultured, acquires the ability to become determined without further tissue interactions. Furthermore, we show that specified PLE has a different gene expression pattern than determined PLE, and that determined PLE can maintain expression of essential regulatory genes (e.g., foxe3, mafB) in an ectopic environment, while specified PLE cannot. These observations set the stage for a detailed mechanistic study of the genes and signals controlling tissue commitment.

Special Considerations for Making Explants and Transplants with Xenopus tropicalis.

Although Xenopus laevis is an important model organism for embryological experimentation, the smaller, more genetically tractable, and faster developing Xenopus tropicalis provides advantages for using genetic approaches to understand developmental mechanisms. Explant cultures and transplants of X. tropicalis embryonic tissues present unique opportunities to examine embryonic tissue determination in a simplified setting. Here we demonstrate preparation of explants and transplants of preplacodal head ectoderm in order to illustrate these approaches; however, these methods apply broadly to tissues throughout the embryo. We focus on technical adjustments to accommodate the differences in size, tissue character, and rate of development between X. laevis and X. tropicalis With only modest modifications, X. tropicalis embryos are quite amenable to the same kinds of experimental manipulations as X. laevis.

Short-term retinal detachment risk after treatment of type 1 retinopathy of prematurity with laser photocoagulation versus intravitreal bevacizumab.

PURPOSE: To perform a stratified comparison of the short-term risk of retinal detachment after treatment of type 1 retinopathy of prematurity treated with panretinal photocoagulation laser versus intravitreal bevacizumab. METHODS: The medical records of consecutive infants treated for type 1 ROP between 2010 and 2018 were retrospectively reviewed. An a priori decision was made to divide infants into two groups, those treated before postmenstrual age (PMA) of 36 0/7 weeks and those treated at or after PMA of 36 0/7 weeks. The primary outcome was presence of any retinal detachment (stage 4A, 4B, or 5) during the 8 weeks following treatment. RESULTS: A total of 222 eyes of 115 infants were included. In eyes treated before 36 0/7 weeks' PMA, retinal detachment occurred in 0 of 34 eyes treated initially with bevacizumab compared with 9 of 56 (16%) treated with laser (P = 0.0112); in eyes treated at or after 36 0/7 weeks, in 0 of 2 eyes treated with bevacizumab and 1 of 130 eyes (0.8%) treated with laser. CONCLUSIONS: The short-term risk of retinal detachment among infants requiring treatment for type 1 ROP prior to 36 0/7 weeks' PMA was lower in eyes treated with intravitreal bevacizumab than in eyes treated with laser, presumably due to the faster effect of bevacizumab in eyes that have more aggressive ROP.

Screen Shots: When Patients and Families Publish Negative Health Care Narratives Online.

Social media sites and their relationship to health care is a subject of intense debate. Common discussions regarding social media address patient privacy, or e-professionalism. This case study explores the tensions that arise for health care providers when negative patient statements surface in social media and blog forums. Recognizing that patients and families often find relief in sharing personal illness narratives, we contemplate if, and how, individual health care professionals and institutions should address complaints aired in public, unmoderated media. Our discussion begins by presenting a case of a family blogging on the Internet to share grievances (to deidentify the case, we have changed some details). Next, we offer an exploration of the impact on health care delivery when professionals become aware of specific criticisms published online. Strategies for managing electronic criticisms are then proposed. We conclude by proposing a novel E-THICS approach to address negative patient expressions via electronic word of mouth (eWOM). Our examination of this evolving issue focuses on maintaining satisfactory relationships between health care providers and patients/families when dealing with health care narratives published in open online media.

A gynogenetic screen to isolate naturally occurring recessive mutations in Xenopus tropicalis.

In the rapidly developing, diploid amphibian Xenopus tropicalis, genetics can be married to the already powerful tools of the amphibian system to overcome a disability that has hampered Xenopus laevis as a model organism: the difficulties inherent in conducting genetic analyses in a tetraploid organism with a longer generation time. We describe here a gynogenetic screen to uncover naturally occurring recessive mutations in wild X. tropicalis populations, a procedure that is both faster and easier than conventional genetic screens traditionally employed in model organisms to dissect early developmental pathways. During the first round of our screen, gynogenetic diploids from over 160 females comprising four different wild-caught populations were examined. Forty-two potential mutant phenotypes were isolated during this round of gynogenesis. From this group, we describe 10 lines that have genetically heritable recessive mutations. A wide range of developmental defects were obtained in this screen, encompassing effects limited to individual organs as well phenotypes characterized by more global changes in tadpole body morphology. The frequency of recessive mutations detected in our screen appears lower than that seen in other vertebrate genetic screens, but given constraints on the screening procedure used here, is likely to be consistent with rates seen in other animals, and clearly illustrates how wild-caught animals can be a productive source of developmental mutations for experimental study. The development of genetic strategies for the Xenopus system, together with new genomic resources, existing technologies for transgenesis, and other means for manipulating gene expression, as well as the power of performing embryonic manipulations, will provide an impressive set of tools for resolving complex cell and developmental phenomena in the future.

Non-invasive Respiratory Support and Severe Retinopathy of Prematurity.

The authors describe two premature infants who developed stage 3, zone I retinopathy of prematurity (ROP) with plus disease in both eyes, despite limited exposure to supra-ambient oxygen. Both infants received noninvasive respiratory support for several weeks. Both cases are notable because the ROP was more posterior and aggressive than is typical for the gestational ages or birth weights. These cases are insufficient to make definitive conclusions regarding the factors that cause ROP. Further investigation is required to determine if there is an association between the use of non-invasive respiratory support, even in the absence of supra-ambient oxygen, and severe ROP development. [J Pediatr Ophthalmol Strabismus. 2016;53:e47-e50.].

Surgical site infections in the NICU.

BACKGROUND: Surgical site infections (SSI) increase morbidity and mortality. In adult and pediatric populations, the incidence ranges from 1.5-12%. Studies in neonates have shown an association between preoperative stay in an intensive care unit and development of SSI. To date, there has only been a single study looking exclusively at SSI in the Neonatal Intensive Care Unit (NICU). Additionally, there has been a suggestion that prematurity may be a risk factor for SSI, but this has come from studies looking at all neonates less than 28days, rather than only neonates hospitalized in a NICU. OBJECTIVE: Primary outcome variable was to calculate the incidence of SSI in a NICU population. Secondary outcome variables were to determine if SSI is more common in premature infants and to identify additional risk factors for the development of SSI. METHODS: An IRB-approved retrospective chart review of all patients undergoing surgical procedures in a level IIIC NICU over a 2-year period was used. We utilized the CDC's definitions of surgical procedures and SSI. An epidemiologist reviewed charts if the diagnosis of SSI was questionable. Statistical analysis was done with t test and Fisher's exact test. RESULTS: We identified 165 patients who underwent 264 surgical procedures. Incidence of SSI was 11.7%. There were 31 SSI that occurred in 29 neonates over the 2-year period, with no mortality in that group. In patients who developed an SSI, 34.5% occurred after the 1st procedure, 41.4% occurred after a 2nd procedure, and 24.1% occurred after the 3rd or later procedure. There was no difference in perioperative antibiotic usage. CONCLUSIONS: This study describes SSI in a strictly neonatal population in a large academic NICU. Prematurity does not appear to be a risk factor for SSI. SSI is more common in neonates who have undergone an abdominal procedure or multiple procedures. Perioperative antibiotics are not significantly associated with prevention of SSI.

Convergence of a head-field selector Otx2 and Notch signaling: a mechanism for lens specification.

Xenopus is ideal for systematic decoding of cis-regulatory networks because its evolutionary position among vertebrates allows one to combine comparative genomics with efficient transgenic technology in one system. Here, we have identified and analyzed the major enhancer of FoxE3 (Lens1), a gene essential for lens formation that is activated in the presumptive lens ectoderm (PLE) when commitment to the lens fate occurs. Deletion and mutation analyses of the enhancer based on comparison of Xenopus and mammalian sequences and in vitro and in vivo binding assays identified two essential transcriptional regulators: Otx2, a homeodomain protein expressed broadly in head ectoderm including the PLE, and Su(H), a nuclear signal transducer of Notch signaling. A Notch ligand, Delta2, is expressed in the optic vesicle adjacent to the PLE, and inhibition of its activity led to loss, or severe reduction, of FoxE3 expression followed by failure of placode formation. Ectopic activation of Notch signaling induced FoxE3 expression within head ectoderm expressing Otx2, and additional misexpression of Otx2 in trunk ectoderm extended the Notch-induced FoxE3 expression posteriorly. These data provide the first direct evidence of the involvement of Notch signaling in lens induction. The obligate integration of inputs of a field-selector (Otx2) and localized signaling (Notch) within target cis-regulatory elements might be a general mechanism of organ-field specification in vertebrates (as it is in Drosophila). This concept is also consistent with classical embryological studies of many organ systems involving a ;multiple-step induction'.

PIGMENTATION IN THE ORANGE TUNICATE, ECTEINASCIDIA TURBINATA.

Some chemical properties of the orange pigment from eggs and embryos and the mantle tissue of adult Ecteinascidia turbinata, as well as ultrastructural characteristics of pigment masses in the mantle, have been examined. The diffuse egg and embryo pigment has solubility and absorbence characteristics of carotenoids, which are solubilized within yolk platelets. The granular mantle pigment, however, is not carotenoid. It is soluble only in organic-aqueous systems, stable to boiling and gross changes in pH, and absorbs strongly in the UV region with a single major peak at 360 nm. Mantle pigment is found in pigment bodies which ultrastructurally are composed of apparently straight and continuous tubular subunits, approximately 500 to 600 Å in diameter, and oriented parallel to the long axis of the body. The pigment bodies are not localized in obvious cells but found within large areas of fibrillar material that are bound by cytoplasmic membranes and occasionally contain an apparently degenerate nucleus. It may be that mantle pigment is derived from pigment cells of the blood which lose their typical cellular appearance as they become permanent features of the mantle tissue.

Xenopus tropicalis transgenic lines and their use in the study of embryonic induction.

For over a century, amphibian embryos have been a source of significant insight into developmental mechanisms, including fundamental discoveries about the process of induction. The recently developed transgenesis for Xenopus offers new approaches to these poorly understood processes, particularly when undertaken in the quickly maturing species Xenopus tropicalis, which greatly facilitates establishment of permanent transgenic lines. Several X. tropicalis transgenic lines have now been generated, and experiments demonstrating the value of these lines to study induction in embryonic tissue recombinants and explants are presented here. A revised protocol for transgenesis in X. tropicalis resulting in a significant increase in the percentage of transgenic animals that reach adulthood is presented, as well as improvements in tadpole and froglet husbandry, which have facilitated the raising of large numbers of adults. Working transgenic populations have been rapidly expanded, and some transgenes have been bred to homozygosity. Established lines include those bearing the promoter regions of Pax-6, Otx-2, Rx, and EF1alpha coupled to fluorescent reporter genes. Multireporter lines combining, in a single animal, up to three gene promoters coupled to different fluorescent reporters have also been established. The value of X. tropicalis transgenic lines for the study of induction is demonstrated by showing activation of Pax-6 by noggin treatment of Pax-6/GFP transgenic animal caps, illustrating how reporter lines allow a rapid, in vivo assay for an inductive response. An experiment showing lens induction in gamma-crystallin/GFP transgenic lens ectoderm when it is recombined with mouse optic vesicle demonstrates conservation of inducing signals from amphibians and mammals. It also shows how the warmer culture temperatures tolerated by X. tropicalis embryos can be used in assays of factors produced by mammalian cells and tissues. The many applications of transgenic reporter lines and other lines designed to target gene expression in particular tissues promise to bring significant new insights to the classic issues first defined in amphibian systems.