Dynamic Increase in Extracellular ATP Accelerates Photoreceptor Cell Apoptosis via Ligation of P2RX7 in Subretinal Hemorrhage

Photoreceptor degeneration is the most critical cause of visual impairment in age-related macular degeneration (AMD). In neovascular form of AMD, severe photoreceptor loss develops with subretinal hemorrhage due to choroidal neovascularization (CNV), growth of abnormal blood vessels from choroidal circulation. However, the detailed mechanisms of this process remain elusive. Here we demonstrate that neovascular AMD with subretinal hemorrhage accompanies a significant increase in extracellular ATP, and that extracellular ATP initiates neurodegenerative processes through specific ligation of Purinergic receptor P2X, ligand-gated ion channel, 7 (P2RX7; P2X7 receptor). Increased extracellular ATP levels were found in the vitreous samples of AMD patients with subretinal hemorrhage compared to control vitreous samples. Extravascular blood induced a massive release of ATP and photoreceptor cell apoptosis in co-culture with primary retinal cells. Photoreceptor cell apoptosis accompanied mitochondrial apoptotic pathways, namely activation of caspase-9 and translocation of apoptosis-inducing factor (AIF) from mitochondria to nuclei, as well as TUNEL-detectable DNA fragmentation. These hallmarks of photoreceptor cell apoptosis were prevented by brilliant blue G (BBG), a selective P2RX7 antagonist, which is an approved adjuvant in ocular surgery. Finally, in a mouse model of subretinal hemorrhage, photoreceptor cells degenerated through BBG-inhibitable apoptosis, suggesting that ligation of P2RX7 by extracellular ATP may accelerate photoreceptor cell apoptosis in AMD with subretinal hemorrhage. Our results indicate a novel mechanism that could involve neuronal cell death not only in AMD but also in hemorrhagic disorders in the CNS and encourage the potential application of BBG as a neuroprotective therapy.     

A chick model of retinal detachment: cone rich and novel

Background

Development of retinal detachment models in small animals can be difficult and expensive. Here we create and characterize a novel, cone-rich retinal detachment (RD) model in the chick.

Methodology/Principal Findings

Retinal detachments were created in chicks between postnatal days 7 and 21 by subretinal injections of either saline (SA) or hyaluronic acid (HA). Injections were performed through a dilated pupil with observation via surgical microscope, using the fellow eye as a control. Immunohistochemical analyses were performed at days 1, 3, 7, 10 and 14 after retinal detachment to evaluate the cellular responses of photoreceptors, Müller glia, microglia and nonastrocytic inner retinal glia (NIRG). Cell proliferation was detected with bromodeoxyuridine (BrdU)-incorporation and by the expression of proliferating cell nuclear antigen (PCNA). Cell death was detected with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). As in mammalian models of RD, there is shortening of photoreceptor outer segments and mis-trafficking of photoreceptor opsins in areas of RD. Photoreceptor cell death was maximal 1 day after RD, but continued until 14 days after RD. Müller glia up-regulated glial fibriliary acidic protein (GFAP), proliferated, showed interkinetic nuclear migration, and migrated to the subretinal space in areas of detachment. Microglia became reactive; they up-regulated CD45, acquired amoeboid morphology, and migrated toward outer retina in areas of RD. Reactive NIRG cells accumulated in detached areas.

Conclusions/Significance

Subretinal injections of SA or HA in the chick eye successfully produced retinal detachments and cellular responses similar to those seen in standard mammalian models. Given the relatively large eye size, and considering the low cost, the chick model of RD offers advantages for high-throughput studies.     

RNA Interference of GADD153 Protects Photoreceptors from Endoplasmic Reticulum Stress-Mediated Apoptosis after Retinal Detachment

Background

Apoptosis of photoreceptors plays a critical role in the vision loss caused by retinal detachment (RD). Pharmacologic inhibition of photoreceptor cell death may prevent RD. This study investigated the role of GADD153 that participates in endoplasmic reticulum (ER) stress-mediated apoptosis of photoreceptor cells after RD.

Methods

Retinal detachment was created in Wistar rats by subretinal injection of hyaluronic acid. The rats were then randomly divided into four groups: normal control group, RD group, GADD153 RNAi group and vehicle group. RNA interference of GADD153 was performed using short hairpin RNA (shRNA). Expressions of GADD153 mRNA and protein were examined by RT-PCR and Western blotting analysis, respectively. GADD153 protein distribution in the retinal cells was observed using immunofluorescence confocal laser scanning microscopy. Apoptosis of retinal cells was determined by TdT-mediated fluorescein-16-dUTP nick-end labeling (TUNEL) assay.

Results

Lentivirus GADD153 shRNA with the most effective silencing effect was chosen for in vivo animal study and was successfully delivered into the retinal tissues. GADD153 mRNA and protein expressions in GADD153 RNAi group were significantly lower than those in the RD group. Silencing of GADD153 by RNAi protected photoreceptors from ER stress-induced apoptosis.

Conclusion

ER stress-mediated pathway is involved in photoreceptor cell apoptosis after RD. GADD153 is a key regulatory molecule regulating ER-stress pathways and plays a crucial role in the apoptosis of photoreceptor cells after RD.     

Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in retinopathies using non-viral and viral gene delivery to the retina and retinal pigment epithelium (RPE). Subretinal injection is the best approach to deliver viral vectors directly to RPE cells. Before the clinical trial of a gene therapy, it is inevitable to validate the efficacy of the therapy in animal models of various retinopathies. Thus, subretinal injection in mice becomes a fundamental technique for an ocular gene therapy. In this protocol, we provide the easy and replicable technique for subretinal injection of viral vectors to experimental mice. This technique is modified from the intravitreal injection, which is widely used technique in ophthalmology clinics. The representative results of RPE/choroid/scleral complex flat-mount will help to understand the efficacy of this technique and adjust the volume and titer of viral vectors for the extent of gene transduction.     

Scleral calcification and photoreceptor cell death during aging and exposure to chronic stress

Male and female Fischer 344 rats of three different ages (12, 18, and 25 months) have been examined for the presence of photoreceptor (PR) cell loss and for occurrence of scleral cartilage and bone formation. In addition, male and female rats, aged 11 months at the beginning of the experiments, were exposed to chronic stress for either 0.5, 2, 4, or 6 months. Photoreceptor cell death gradually increases during the aging process and is exacerbated by exposure to chronic stress. It is more severe in the peripheral than the central retina and exposure to stress increases this pattern of cell loss. The superior retina is more severely affected than the inferior hemisphere in aging and during stress. The incidence of scleral cartilage or bone formation increases with age in male and female rats, but with stress exposure an increase is seen in males only. Bone formations occur more frequently in male than in female animals and are almost always (97%) located in the superior hemisphere of the eye. Although there appears to be a direct relationship between photoreceptor cell death and the occurrence of scleral ossifications in group data, in individual eyes the bone formations are not always associated with severity of PR cell loss. The relationship of PR cell death and incidence of scleral ossification to gender and to exposure to stress supports a hypothesis for an endocrine basis of ocular aging.     

Trans-Corneal Subretinal Injection in Mice and Its Effect on the Function and Morphology of the Retina

Purpose

To introduce a practical method of subretinal injection in mice and evaluate injection-induced retinal detachment (RD) and damage using a dynamic imaging system, electrophysiology, and histology.

Methods

After full dilation of a 2-month-old C57BL/6J mouse pupil, the cornea near the limbus was punctured with a 30 ½-gague disposable beveled needle. A 33 ½-gauge blunt needle was inserted through the corneal perforation into the anterior chamber, avoiding the lens before going deeper into the vitreous cavity, and penetrating the inner retina to reach the subretinal space. The mice were divided into four groups: in group 1, about 80–100% of the retina was filled with subretinally injected solution; in group 2, approximately 50–70% of the retina was filled with injected solution; in group 3, the procedures were stopped before solution injection; and non-injected eyes were used as the negative control in group 4. An optical coherence tomography (OCT) imaging system was used to monitor retinal reattachment during the first three days following the injections. Histological and functional changes were examined by light microscopy and electroretinography (ERG) at five weeks post-injection.

Results

After a short-term training, a 70% success rate with 50% or more coverage (i.e., retinal blebs occupied 50% or more retinal area and filled with the injected solution) with minimal injection-related damages can be achieved. Bleb formation was associated with retinal detachment (RD) between the neuroretina and the retinal pigment epithelium (RPE) layer. Partial RD could be observed at post-injection day 1, and by day 2 most of the retina had reattached. At 5 weeks post-injection, compared to uninjected control group 4, the b-wave amplitudes of ERG decreased 22% in group 1, 16% in group 2, and 7% in group 3; the b-wave amplitudes were statistically different between the uninjected group and the groups with either 50–70% or 80–100% coverage. The subretinal injection-induced RD reattached and became stable at five weeks post-injection, although some photoreceptor damage could still be observed in and around the injection sites, especially in 80–100% coverage group.

Conclusions

Trans-corneal subretinal injection is effective and practical, although subretinal injection-related damages can cause some morphological and functional loss.     

Heat shock protein 70 induction by valproic acid delays photoreceptor cell death by N‐methyl‐N‐nitrosourea in mice

Retinal degenerative diseases (RDs) are a group of inherited diseases characterized by the loss of photoreceptor cells. Selective photoreceptor loss can be induced in mice by an intraperitoneal injection of N‐methyl‐N‐nitrosourea (MNU) and, because of its selectivity, this model is widely used to study the mechanism of RDs. Although it is known that calcium‐calpain activation and lipid peroxidation are involved in the initiation of cell death, the precise mechanisms of this process remain unknown. Heat shock protein 70 (HSP70) has been shown to function as a chaperone molecule to protect cells against environmental and physiological stresses. In this study, we investigated the role of HSP70 on photoreceptor cell death in mice. HSP70 induction by valproic acid, a histone deacetylase inhibitor, attenuated the photoreceptor cell death by MNU through inhibition of apoptotic caspase signals. Furthermore, HSP70 itself was rapidly and calpain‐dependently cleaved after MNU treatment. Therefore, HSP70 induction by valproic acid was dually effective against MNU‐induced photoreceptor cell loss as a result of its anti‐apoptotic actions and its ability to prevent HSP70 degradation. These findings might help lead us to a better understanding of the pathogenic mechanism of RDs.

     

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.     

食蟹猴脑出血模型的建立及评价

目的建立稳定的食蟹猴自体股动脉抗凝血脑出血模型。方法健康成年食蟹猴8只,采用新鲜抗凝自体股动脉血1．5mL,立体定向一次性缓慢匀速注入右侧基底节区屏状核内侧,利用MRI、PET、神经功能缺损评分和组织病理学对模型进行评价。结果动物在麻醉苏醒后出现了与血肿部位相对应的神经系统定位体征,影像学检查血肿腔位于右侧基底节,注血部位大片代谢缺损区,中线向左侧移位。组织病理学检查损伤局部周围可见多量泡沫细胞、淋巴细胞及单核巨噬细胞浸润,部分损伤脑组织已被肉芽组织机化。结论自体股动脉抗凝血定向脑内注射法可建立稳定、可靠的食蟹猴脑出血模型,注血量、注血部位和速度是模型成功与否的关键。     

A2E,a component of lipofuscin,is pro‐angiogenic in vivo

A recent study in vitro demonstrated that a major lipofuscin component, A2E, serves as a retinoic acid receptor ligand. The current study investigated the effects of A2E on retinal pigment epithelial (RPE) cells in vivo and was performed to extend the understanding of the effects of A2E. Firstly, subretinal injection of A2E was performed and 3 weeks after the injection, and it was demonstrated that subretinal injection of A2E induced RPE cell death, and concomitant upregulation of vascular endothelial growth factor (VEGF) in the RPE and choroid. The upregulation of VEGF was attenuated by an RARα antagonist. Next we performed laser photocoagulation in mice that accumulated A2E either after subretinal injection, by Ccl2 gene knockout or by aging demonstrated that mice that accumulated A2E in the RPE, which showed higher rates of choroidal neobascularization (CNV) formation after weak laser injury than the controls and the formation of CNV was inhibited by an RARα antagonist in all models tested. The data suggest that A2E accumulation induces RPE cell death, and concomitant increase of VEGF. Accumulation of A2E alone is not sufficient to induce CNV in vivo, but induces the expression of VEGF in RPE and choroid. The mice that accumulated A2E in RPE cells are vulnerable to CNV development via RAR activation, at least in part. J. Cell. Physiol. 220: 469–475, 2009. © 2009 Wiley‐Liss, Inc.     

Distinct effects of inflammation on gliosis, osmohomeostasis, and vascular integrity during amyloid beta-induced retinal degeneration

In normal retinas, amyloid-β (Aβ) accumulates in the subretinal space, at the interface of the retinal pigment epithelium, and the photoreceptor outer segments. However, the molecular and cellular effects of subretinal Aβ remain inadequately elucidated. We previously showed that subretinal injection of Aβ(1-42) induces retinal inflammation, followed by photoreceptor cell death. The retinal Müller glial (RMG) cells, which are the principal retinal glial cells, are metabolically coupled to photoreceptors. Their role in the maintenance of retinal water/potassium and glutamate homeostasis makes them important players in photoreceptor survival. This study investigated the effects of subretinal Aβ(1-42) on RMG cells and of Aβ(1-42)-induced inflammation on retinal homeostasis. RMG cell gliosis (upregulation of GFAP, vimentin, and nestin) on day 1 postinjection and a proinflammatory phenotype were the first signs of retinal alteration induced by Aβ(1-42). On day 3, we detected modifications in the protein expression patterns of cyclooxygenase 2 (COX-2), glutamine synthetase (GS), Kir4.1 [the inwardly rectifying potassium (Kir) channel], and aquaporin (AQP)-4 water channels in RMG cells and of the photoreceptor-associated AQP-1. The integrity of the blood-retina barrier was compromised and retinal edema developed. Aβ(1-42) induced endoplasmic reticulum stress associated with sustained upregulation of the proapoptotic factors of the unfolded protein response and persistent photoreceptor apoptosis. Indomethacin treatment decreased inflammation and reversed the Aβ(1-42)-induced gliosis and modifications in the expression patterns of COX-2, Kir4.1, and AQP-1, but not of AQP-4 or GS. Nor did it improve edema. Our study pinpoints the adaptive response to Aβ of specific RMG cell functions.     

Intraocular iron injection induces oxidative stress followed by elements of geographic atrophy and sympathetic ophthalmia

Iron has been implicated in the pathogenesis of age‐related retinal diseases, including age‐related macular degeneration (AMD). Previous work showed that intravitreal (IVT) injection of iron induces acute photoreceptor death, lipid peroxidation, and autofluorescence (AF). Herein, we extend this work, finding surprising chronic features of the model: geographic atrophy and sympathetic ophthalmia. We provide new mechanistic insights derived from focal AF in the photoreceptors, quantification of bisretinoids, and localization of carboxyethyl pyrrole, an oxidized adduct of docosahexaenoic acid associated with AMD. In mice given IVT ferric ammonium citrate (FAC), RPE died in patches that slowly expanded at their borders, like human geographic atrophy. There was green AF in the photoreceptor ellipsoid, a mitochondria‐rich region, 4 h after injection, followed later by gold AF in rod outer segments, RPE and subretinal myeloid cells. The green AF signature is consistent with flavin adenine dinucleotide, while measured increases in the bisretinoid all‐trans‐retinal dimer are consistent with the gold AF. FAC induced formation carboxyethyl pyrrole accumulation first in photoreceptors, then in RPE and myeloid cells. Quantitative PCR on neural retina and RPE indicated antioxidant upregulation and inflammation. Unexpectedly, reminiscent of sympathetic ophthalmia, autofluorescent myeloid cells containing abundant iron infiltrated the saline‐injected fellow eyes only if the contralateral eye had received IVT FAC. These findings provide mechanistic insights into the potential toxicity caused by AMD‐associated retinal iron accumulation. The mouse model will be useful for testing antioxidants, iron chelators, ferroptosis inhibitors, anti‐inflammatory medications, and choroidal neovascularization inhibitors.     

Nanoceria inhibit the development and promote the regression of pathologic retinal neovascularization in the Vldlr knockout mouse

Many neurodegenerative diseases are known to occur and progress because of oxidative stress, the presence of reactive oxygen species (ROS) in excess of the cellular defensive capabilities. Age related macular degeneration (AMD), diabetic retinopathy (DR) and inherited retinal degeneration share oxidative stress as a common node upstream of the blinding effects of these diseases. Knockout of the Vldlr gene results in a mouse that develops intraretinal and subretinal neovascular lesions within the first month of age and is an excellent model for a form of AMD called retinal angiomatous proliferation (RAP). Cerium oxide nanoparticles (nanoceria) catalytically scavenge ROS by mimicking the activities of superoxide dismutase and catalase. A single intravitreal injection of nanoceria into the Vldlr-/- eye was shown to inhibit: the rise in ROS in the Vldlr-/- retina, increases in vascular endothelial growth factor (VEGF) in the photoreceptor layer, and the formation of intraretinal and subretinal neovascular lesions. Of more therapeutic interest, injection of nanoceria into older mice (postnatal day 28) resulted in the regression of existing vascular lesions indicating that the pathologic neovessels require the continual production of excessive ROS. Our data demonstrate the unique ability of nanoceria to prevent downstream effects of oxidative stress in vivo and support their therapeutic potential for treatment of neurodegenerative diseases such as AMD and DR.     

Oxygen-induced retinopathy in mice with retinal photoreceptor cell degeneration

Aims

It is reported that retinal neovascularization seems to rarely co-exist with retinitis pigmentosa in patients and in some mouse models; however, it is not widely acknowledged as a universal phenomenon in all strains of all animal species. We aimed to further explore this phenomenon with an oxygen-induced retinopathy model in mice with retinal photoreceptor cell degeneration.

Main methods

Oxygen-induced retinopathy of colored and albino mice with rapid retinal degeneration were compared to homologous wild-type mice. The retinas were analyzed using high-molecular-weight FITC-dextran stained flat-mount preparation, hematoxylin and eosin (H&E) stained cross-sections, an immunohistochemical test for vascular endothelial growth factor (VEGF) distribution and Western blotting for VEGF expression after exposure to hyperoxia between postnatal days 17 (P17) and 21.

Key findings

Leakage and areas of non-perfusion of the retinal blood vessels were alleviated in the retinal degeneration mice. The number of preretinal vascular endothelial cell nuclei in the retinal degeneration mice was smaller than that in the homologous wild-type mice after exposure to hyperoxia (P < 0.01). The degree of oxygen-induced retinopathy was positively correlated with the VEGF expression level. However, the VEGF expression level was lower in the retinal degeneration mice.

Significance

Proliferative retinopathy occurred in mice with rapid retinal degeneration, but retinal photoreceptor cell degeneration could partially restrain the retinal neovascularization in this rapid retinal degeneration mouse model.     

Experimental Differentiation of Intraocular Masses Using Ultrahigh-Field Magnetic Resonance Imaging – A Case Series

Purpose

The case reports presented here were compiled to demonstrate the potential for improved diagnosis and monitoring of disease progress of intraocular lesions using ultrahigh-field magnetic resonance microscopy (MRM) at 7.1 Tesla.

Methods

High-resolution ex vivo ocular magnetic resonance (MR) images were acquired on an ultrahigh-field MR system (7.1 Tesla, ClinScan, Bruker BioScan, Germany) using a 2-channel coil with 4 coil elements and T2-weighted turbo spin echo (TSE) sequences of human eyes enucleated because of different intraocular lesions. Imaging parameters were: 40×40 mm field of view, 512×512 matrix, and 700 µm slice thickness. The results were correlated with in vivo ultrasound and histology of the enucleated eyes.

Results

Imaging was performed in enucleated eyes with choroidal melanoma, malignant melanoma of iris and ciliary body with scleral perforation, ciliary body melanoma, intraocular metastasis of esophageal cancer, subretinal bleeding in the presence of perforated corneal ulcer, hemorrhagic choroidal detachment, and premature retinopathy with phthisis and ossification of bulbar structures. MR imaging allowed differentiation between solid and cystic tumor components. In case of hemorrhage, fluid-fluid levels were identified. Melanin and calcifications caused significant hypointensity. Microstructural features of eye lesions identified by MRM were confirmed by histology.

Conclusion

This study demonstrates the potential of MRM for the visualization and differential diagnosis of intraocular lesions. At present, the narrow bore of the magnet still limits the use of this technology in humans in vivo. Further advances in ultrahigh-field MR imaging will permit visualization of tumor extent and evaluation of nonclassified intraocular structures in the near future.     

Dysregulation in retinal para-inflammation and age-related retinal degeneration in CCL2 or CCR2 deficient mice

We have shown previously that a para-inflammatory response exists at the retinal/choroidal interface in the aging eye; and this response plays an important role in maintaining retinal homeostasis under chronic stress conditions. We hypothesized that dysregulation of the para-inflammatory response may result in an overt pro-inflammatory response inducing retinal degeneration. In this study, we examined this hypothesis in mice deficient in chemokine CCL2 or its cognate receptor CCR2. CCL2- or CCR2-deficient mice developed retinal degenerative changes with age, characterized as retinal pigment epithelial (RPE) cell and photoreceptor cell death. Retinal cell death was associated with significantly more subretinal microglial accumulation and increased complement activation. In addition, monocytes from CCL2- or CCR2-deficient mice had reduced capacity for phagocytosis and chemotaxis, expressed less IL-10 but more iNOS, IL-12 and TNF-α when compared to monocytes from WT mice. Complement activation at the site of RPE cell death resulted in C3b/C3d but not C5b-9 deposition, indicating only partial activation of the complement pathway. Our results suggest that altered monocyte functions may convert the protective para-inflammatory response into an overtly harmful inflammation at the retina/choroidal interface in CCL2- or CCR2-deficient mice, leading to RPE and photoreceptor degeneration. These data support a concept whereby a protective para-inflammatory response relies upon a normally functioning innate immune system. If the innate immune system is deficient chronic stress may tip the balance towards an overt inflammatory response causing cell/tissue damage.     

Resin duct size and density as ecophysiological traits in fire scars of Pseudotsuga menziesii and Larix occidentalis

Background and Aims

Resin ducts (RDs) are features present in most conifer species as defence structures against pests and pathogens; however, little is known about RD expression in trees following fire injury. This study investigates changes in RD size and density in fire scars of Douglas fir (Pseudotsuga menziesii) and western larch (Larix occidentalis) as a means to evaluate the ecophysiological significance of traumatic resinosis for tree defence and survival.

Methods

Transverse and tangential microsections were prepared for light microscopy and image analysis in order to analyse axial and radial RDs, respectively. Epithelial cells of RDs and fusiform rays associated with radial RDs were also examined. RDs were compared between normal xylem and wound xylem at four different section heights along the fire-injured stem.

Key Results

Following fire injury, P. menziesii axial RDs narrowed by 38–43 % in the first year after injury, and the magnitude of this change increased with stem height. Larix occidentalis axial RDs widened by 46–50 % in the second year after injury. Radial RDs were of equivalent size in P. menziesii, but widened by 162–214 % in L. occidentalis. Fusiform rays were larger following fire injury, by 4–14 % in P. menziesii and by 23–38 % in L. occidentalis. Furthermore, axial RD density increased in both species due to the formation of tangential rows of traumatic RDs, especially in the first and second years after injury. However, radial RD density did not change significantly.

Conclusions

These results highlight traumatic resinosis as a species-specific response. Pseudotsuga menziesii produce RDs of equivalent or reduced size, whereas L. occidentalis produce wider RDs in both the axial and radial duct system, thereby increasing resin biosynthesis and accumulation within the whole tree. Larix occidentalis thus appears to allocate more energy to defence than P. menziesii.     

Hyperplastic neuroretinopathy and disorder of pigment epithelial cells precede accelerated retinal degeneration in the SJL/N mouse

We have found a complex eye disease in the SJL/N mouse. This animal is closely related to the SJL/J mouse, which is homozygous for retinal degeneration (rd) and which also suffers from extraocular reticulum cell sarcomas at around 200 days of age. In the SJL/N animal, a high incidence of subretinal tumor is present at 9 days after birth. Furthermore, we have observed an extensive neuroretinal hyperplasia, a phenomenon that is termed hyperplastic neuroretinopathy, and that is probably the consequence of elevated levels of cytokines in the animals. In addition to these anomalies, the SJL/N mouse shows progressive dystrophy of the retinal pigment epithelium (RPE) from day 4 onwards, and accelerated photoreceptor cell degeneration is completed by day 16. The early RPE dystrophy appears to be a secondary autoimmune disease, since cells in this structure and in the choroid develop MHC class II antigens, whereas we suspect that the accelerated photoreceptor cell loss is induced by a soluble toxic agent. The F₁ progeny derived from cross-breeding the SJL/N and Balb/c +/+ strains also shows a high incidence of subretinal tumor and hyperplastic neuroretinopathy, but neither the RPE dystrophy nor retinal degeneration.     

In Vitro and In Vivo Proteomic Comparison of Human Neural Progenitor Cell‐Induced Photoreceptor Survival

Retinal degenerative diseases lead to blindness with few treatments. Various cell‐based therapies are aimed to slow the progression of vision loss by preserving light‐sensing photoreceptor cells. A subretinal injection of human neural progenitor cells (hNPCs) into the Royal College of Surgeons (RCS) rat model of retinal degeneration has aided in photoreceptor survival, though the mechanisms are mainly unknown. Identifying the retinal proteomic changes that occur following hNPC treatment leads to better understanding of neuroprotection. To mimic the retinal environment following hNPC injection, a co‐culture system of retinas and hNPCs is developed. Less cell death occurs in RCS retinal tissue co‐cultured with hNPCs than in retinas cultured alone, suggesting that hNPCs provide retinal protection in vitro. Comparison of ex vivo and in vivo retinas identifies nuclear factor (erythroid‐derived 2)‐like 2 (NRF2) mediated oxidative response signaling as an hNPC‐induced pathway. This is the first study to compare proteomic changes following treatment with hNPCs in both an ex vivo and in vivo environment, further allowing the use of ex vivo modeling for mechanisms of retinal preservation. Elucidation of the protein changes in the retina following hNPC treatment may lead to the discovery of mechanisms of photoreceptor survival and its therapeutic for clinical applications.