Robert M. Petters

Oxidative damage is a potential cause of cone cell death in retinitis pigmentosa

Retinitis pigmentosa (RP) is a prevalent cause of blindness caused by a large number of different mutations in many different genes. The mutations result in rod photoreceptor cell death, but it is unknown why cones die. In this study, we tested the hypothesis that cones die from oxidative damage by performing immunohistochemical staining for biomarkers of oxidative damage in a transgenic pig model of RP. The presence of acrolein‐ and 4‐hydroxynonenal‐adducts on proteins is a specific indicator that lipid peroxidation has occurred, and there was strong immunofluorescent staining for both in cone inner segments (IS) of two 10‐month‐old transgenic pigs in which almost all rods had died, compared to faint staining in two 10‐month‐old control pig retinas. In 22‐ and 24‐month‐old transgenic pigs in which all rods and many cones had died, staining was strong in cone axons and some cell bodies as well as IS indicating progression in oxidative damage between 10 and 22 months. Biomarkers for oxidative damage to proteins and DNA also showed progressive oxidative damage to those macromolecules in cones during the course of RP. These data support the hypothesis that the death of rods results in decreased oxygen consumption and hyperoxia in the outer retina resulting in gradual cone cell death from oxidative damage. This hypothesis has important therapeutic implications and deserves rapid evaluation.

Ectopic synaptogenesis in the mammalian retina caused by rod photoreceptor-specific mutations

In addition to rod photoreceptor loss, many mutations in rod photoreceptor-specific genes cause degeneration of other neuronal types. Identifying mechanisms of cell–cell interactions initiated by rod-specific mutations and affecting other retinal cells is important for understanding the pathogenesis and progression of retinal degeneration. Here we show in animals with rod and cone degeneration due to mutations in the genes encoding rhodopsin and cGMP phosphodiesterase β-subunit (PDE-β) respectively, that rod bipolar cells received ectopic synapses from cones in the absence of rods. Thus, synaptic plasticity links certain rod-specific mutations to retina-wide structural alterations that involve different types of neurons.

In vitro culture of pig embryos

Abstract Culture of pig embryos obtained prior to the four-cell stage has been difficult to accomplish. The ‘in vitro developmental block’ at the four-cell stage can be overcome by a number of methods to allow complete development of pig embryos from the one-cell stage to the blastocyst stage in vitro. Mouse oviducts in organ culture have been shown to provide a suitable environment for pig embryo development. Co-culture of pig embryos with oviductal cells or supplementation of culture medium with pig oviductal fluid results in improved embryonic development in vitro. Modifications to simple culture media have demonstrated that glutamine can serve as the sole exogenous energy source for development from the zygote to the blastocyst stage in vitro. Glucose was not inhibitory to the development of pig embryos in vitro. Addition of taurine and hypotaurine to the medium further increased the degree of embryo development in vitro. A number of different media have been reported that support pig embryo development in vitro. Although the nature of the ‘in vitro developmental block’ is not known, a number of methods now exist to circumvent this problem in the pig.

Retinal Rod Photoreceptor-Specific Gene Mutation Perturbs Cone Pathway Development

Rod-specific photoreceptor dystrophies are complicated by the delayed death of genetically normal neighboring cones. In transgenic (Tg) swine with a rod-specific (rhodopsin) gene mutation, cone photoreceptor physiology was normal for months but later declined, consistent with delayed cone cell death. Surprisingly, cone postreceptoral function was markedly abnormal when cone photoreceptor physiology was still normal. The defect was localized to hyperpolarizing cells postsynaptic to the middle wavelength-sensitive cones. Recordings throughout postnatal development indicated a failure of cone circuitry maturation, a novel mechanism of secondary cone abnormality in rod dystrophy. The results have implications for therapy for human retinal dystrophies and raise the possibility that rod afferent activity plays a role in the postnatal maturation of cone retinal circuitry.

Transgenic animals as models for human disease.

Transgenic animals, especially mice, have been used quite extensively as models for various human diseases. At first, the level of scientific inquiry was driven by the need to establish the model. In many cases, these models may be considered quite crude because of their limitations. More recently, transgenic models of disease have become more refined and are currently being used to study the pathological mechanisms behind the disease rather than to just provide a model of the disease. Using some examples from the recent literature, we will document the current level and complexity of inquiry using transgenic animals. New techniques and techniques that may prove promising will be discussed.

Production of ELOVL4 transgenic pigs: a large animal model for Stargardt-like macular degeneration

Background Truncation mutations in the elongation of very long chain fatty acids-4 (AF277094, MIM #605512) (ELOVL4) gene cause Stargardt-like macular dystrophy type 3 (STGD3). Mice expressing truncated ELOVL4 develop rapid retinal degeneration, but are poor STGD3 models since mice lack a macula. Photoreceptor topography in the pig retina is more similar to that in humans as it includes the cone rich, macula-like area centralis. The authors generated transgenic pigs expressing human disease-causing ELOVL4 mutations to better model the pathobiology of this macular disease. Methods Pronuclear DNA microinjection and somatic cell nuclear transfer were used to produce transgenic pigs for two different ELOVL4 mutations: the 5 base pair deletion (5 bpdel) and the 270 stop mutation (Y270terEYFP). Retinal transgene expression, morphology and electrophysiology were examined. Results The authors obtained four lines of Y270terEYFP and one line of 5 bpdel transgenic animals. Direct fluorescence microscopy indicated that the Y270terEYFP protein is expressed in photoreceptors and mislocalised within the cell. Immunohistochemical examination of transgenic pigs showed photoreceptor loss and disorganised inner and outer segments. Electroretinography demonstrated diminished responses in both transgenic models. Conclusions These transgenic pigs provide unique animal models for examining macular degeneration and STGD3 pathogenesis.

Transplantation of full-thickness retina in the rhodopsin transgenic pig.

Purpose To establish the morphology of full-thickness neuroretinal grafts transplanted to hosts with degenerative photoreceptor disease. Methods Twenty rhodopsin transgenic pigs received a neuroretinal sheet from a neonatal normal pig in one eye. Following vitrectomy and retinotomy with bleb formation, the grafts were positioned inside the bleb between the host neuroretina and retinal pigment epithelium. After a survival time of 4 months, eye specimens were studied by light and electron microscopy as well as with immunohistochemical markers. Results One eye developed endophthalmitis in the immediate postoperative period and was terminated. Laminated grafts with correct polarity were found in 13 of the remaining 19 eyes. In most cases, these grafts had well-developed organized photoreceptors with outer segments apposed to the host retinal pigment epithelium. The inner layers of the graft contained mostly Müller cells. Both eyes of the hosts had a reduction of photoreceptor cells in most of the retina, while inner layers remained relatively intact. Conclusions Full-thickness neuroretinal grafts can be transplanted to a large animal host with photoreceptor degeneration. The transplantation procedure is relatively atraumatic to both graft and host tissue, and the grafts survive well for at least 4 months. The graft and host retina does not seem to form extensive neuronal contacts, and future work must be directed at stimulating such activity without disrupting the retinal neuronal organization.

Successful Cloning of the Yucatan Minipig Using Commercial/Occidental Breeds as Oocyte Donors and Embryo Recipients

The widespread application of porcine SCNT to biomedical research is being hampered by the large adult size (300-600 lbs) of the commercial breeds commonly used for SCNT. The Yucatan minipig, in contrast, has an adult weight of 140-150 lbs and a long history of utility in biomedical research. In order to combine the wide availability of commercial swine with the biomedical value of the Yucatan minipig, we utilized SCNT using the Yucatan as nuclear donors and commercial swine as both oocyte donors and recipients. Of six recipient gilts receiving 631 SCNT embryos, three went to term and delivered seven piglets, four of which survived to adulthood. Additionally, we obtained fetal fibroblasts from a cloned Yucatan and used them for a second round of SCNT. Of three recipients receiving 315 reconstructed embryos, one went to term and delivered three piglets, one of which survived to adulthood. Both microsatellite and D-loop sequence analysis confirmed that all of the piglets generated were nuclear-mitochondrial hybrids carrying Yucatan nuclear DNA and commercial breed mitochondrial DNA. This report shows that it is possible to produce viable Yucatan SCNT clones and opens up the possibility of developing valuable biomedical models in this porcine breed.

Effect of oviductal condition on the development of one-cell porcine embryos in mouse or rat oviducts maintained in organ culture

Abstract To determine the effect of varying oviductal conditions on porcine embryo development in mouse oviducts in organ culture, one-cell porcine embryos from each donor female were randomly assigned to the following treatments: medium alone, mouse oviducts obtained from females mated to fertile males or vasectomized males, mouse oviducts from mated fertile females flushed with culture medium, and rat oviducts. Of the embryos cultured in medium alone, 17.3% (n = 52) developed to the morula or blastocyst stage. For porcine embryos cultured in mouse oviducts, 77.1% (n = 35) developed to the morula or blastocyst stage in mouse oviducts obtained from fertile matings; 74.5% (n = 51) in mouse oviducts obtained from females mated to vasectomized males; and 58.7% (n = 46) in flushed mouse oviducts obtained from mated fertile females. Development of porcine embryos in rat oviducts from mated fertile females was limited (17.8% morula or blastocyst; n = 45). Mouse oviducts obtained from females mated to fertile or vasectomized males and not flushed supported one-cell porcine embryo development to a greater extent than flushed mouse oviducts (P 0.90). Cell numbers were similar for embryos reaching the morula or blastocyst stage across all treatments, except for embryos reaching the blastocyst stage in rat oviducts, where cell numbers were significantly lower (P

Lensectomy and vitrectomy decrease the rate of photoreceptor loss in rhodopsin P347L transgenic pigs

BackgroundPhotoreceptor degeneration in retinitis pigmentosa (RP) runs an inevitable, gradually progressive course. A wide variety of growth factors of different origins have been shown to slow the rate of degeneration in some rodent models of RP. Recently, lens-derived neurotrophic factors have been shown to rescue degenerating ganglion cells in crush models of the optic nerve. Our objective was to evaluate the potential rescue effect of lensectomy and vitrectomy (L&V) on photoreceptor degeneration in a large-animal model, the rhodopsin P347L transgenic pig.MethodsWe operated on one eye of each of 49 3-week-old pigs—15 vitrectomies and 34 L&V, 6 of which received steroids. Retinal paraffin sections were prepared for all eyes, in addition to immunohistochemistry in four eyes, 8 weeks after L&V.ResultsAt eight weeks after L&V, operated eyes showed significantly more nuclei in the outer nuclear layer (ONL) than the unoperated fellow eyes. The better preservation of the ONL persisted but was less prominent by 20 weeks after surgery. Steroid treatment did not markedly reduce the better preservation of the ONL seen at 8, 10, and 12 weeks after surgery. The significant difference in cell count between operated and unoperated eyes in the L&V group at 8 weeks was due to the difference in the number of rods, not the cones.ConclusionLensectomy and vitrectomy delay photoreceptor degeneration in rhodopsin P347L transgenic pigs. Lens-related rescue effect is a probable reason for the delayed degeneration.