Deficiency of aldose reductase attenuates inner retinal neuronal changes in a mouse model of retinopathy of prematurity.

Retinopathy of prematurity (ROP) is a leading cause of childhood blindness where vascular abnormality and retinal dysfunction are reported. We showed earlier that genetic deletion of aldose reductase (AR), the rate-limiting enzyme in the polyol pathway, reduced the neovascularization through attenuating oxidative stress induction in the mouse oxygen-induced retinopathy (OIR) modeling ROP. In this study, we further investigated the effects of AR deficiency on retinal neurons in the mouse OIR. Seven-day-old wild-type and AR-deficient mice were exposed to 75 % oxygen for 5 days and then returned to room air. Electroretinography was used to assess the neuronal function at postnatal day (P) 30. On P17 and P30, retinal cytoarchitecture was examined by morphometric analysis and immunohistochemistry for calbindin, protein kinase C alpha, calretinin, Tuj1, and glial fibrillary acidic protein. In OIR, attenuated amplitudes and delayed implicit time of a-wave, b-wave, and oscillatory potentials were observed in wild-type mice, but they were not significantly changed in AR-deficient mice. The morphological changes of horizontal, rod bipolar, and amacrine cells were shown in wild-type mice and these changes were partly preserved with AR deficiency. AR deficiency attenuated the Müller cell gliosis induced in OIR. Our observations demonstrated AR deficiency preserved retinal functions in OIR and AR deficiency could partly reduce the extent of retinal neuronal histopathology. These findings suggested a therapeutic potential of AR inhibition in ROP treatment with beneficial effects on the retinal neurons.

Neuroprotective effects of lutein in a rat model of retinal detachment.

BACKGROUND: Retinal detachment (RD) is a leading cause of blindness, and although final surgical re-attachment rate has greatly improved, visual outcome in many macula-off detachments is disappointing, mainly because of photoreceptor cell death. We previously showed that lutein is anti-apoptotic in rodent models of ischemia/reperfusion injury. The objective of this study is to investigate lutein as a possible pharmacological adjunct to surgery. METHODS: Subretinal injections of 1.4 % sodium hyaluronate were used to induce RD in Sprague-Dawley rats until their retinae were approximately 70 % detached. Daily injections of corn oil (control group) or 0.5 mg/kg lutein in corn oil (treatment group) were given intraperitoneally starting 4 h after RD induction. Animals were euthanized 3 days and 30 days after RD and their retinae were analyzed for photoreceptor apoptosis and cell survival at the outer nuclear layer (ONL) using TUNEL staining and cell counting on retinal sections. Glial fibrillary acidic protein (GFAP) and rhodopsin (RHO) expression were evaluated with immunohistochemistry. Western blotting was done with antibodies against cleaved caspase-3, cleaved caspase-8 and cleaved caspase-9 to delineate lutein's mechanism of action in the apoptotic cascade. To seek a possible therapeutic time window, the same set of experiments was repeated with treatment commencing 36 h after RD. RESULTS: When lutein was given 4 h after RD, there were significantly fewer TUNEL-positive cells in ONL 3 days after RD when compared with the vehicle group. Cell counting showed that there were significantly more nuclei in ONL in lutein-treated retinae by day 30. Treatment groups also showed significantly reduced GFAP immunoreactivity and preserved RHO expression. At day 3 after RD, Western blotting showed reduced expression of cleaved caspase-3 and cleaved caspase-8 in the treatment group. No difference was found for cleaved caspase-9. When lutein was given 36 h after RD similar results were observed. CONCLUSIONS: Our results suggest that lutein is a potent neuroprotective agent that can salvage photoreceptors in rats with RD, with a therapeutic window of at least 36 h. The use of lutein in patients with RD may serve as an adjunct to surgery to improve visual outcomes.

Hypoxia-induced oxidative stress in ischemic retinopathy.

Oxidative stress plays a crucial role in the pathogenesis of retinal ischemia/hypoxia, a complication of ocular diseases such as diabetic retinopathy (DR) and retinopathy of prematurity (ROP). Oxidative stress refers to the imbalance between the production of reactive oxygen species (ROS) and the ability to scavenge these ROS by endogenous antioxidative systems. Free radicals and ROS are implicated in the irreversible damage to cell membrane, DNA, and other cellular structures by oxidizing lipids, proteins, and nucleic acids. Anti-oxidants that can inhibit the oxidative processes can protect retinal cells from ischemic/hypoxic insults. In particular, treatment using anti-oxidants such as vitamin E and lutein, inhibition of nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) or related signaling pathways, and administration of catalase and superoxide dismutase (SOD) are possible therapeutic regimens for DR, ROP, and other retinal ischemic diseases. The role of oxidative stress in the pathogenesis of DR and ROP as well as the underlying mechanisms involved in the hypoxia/ischemia-induced oxidative damage is discussed. The information provided will be beneficial in understanding the underlying mechanisms involved in the pathogenesis of the diseases as well as in developing effective therapeutic interventions to treat oxidative stress-induced damages.

Anti-inflammatory effects of lutein in retinal ischemic/hypoxic injury: in vivo and in vitro studies.

PURPOSE: Lutein protects retinal neurons by its anti-oxidative and anti-apoptotic properties in ischemia/reperfusion (I/R) injury while its anti-inflammatory effects remain unknown. As Müller cells play a critical role in retinal inflammation, the effect of lutein on Müller cells was investigated in a murine model of I/R injury and a culture model of hypoxic damage. METHODS: Unilateral retinal I/R was induced by a blockade of internal carotid artery using the intraluminal method in mice. Ischemia was maintained for 2 hours followed by 22 hours of reperfusion, during which either lutein (0.2 mg/kg) or vehicle was administered. Flash electroretinogram (flash ERG) and glial fibrillary acidic protein (GFAP) activation were assessed. Lutein's effect on Müller cells was further evaluated in immortalized rat Müller cells (rMC-1) challenged with cobalt chloride-induced hypoxia. Levels of IL-1ß, cyclooxygenase-2 (Cox-2), TNFα, and nuclear factor-NF-kappa-B (NF-κB) were examined by Western blot analysis. RESULTS: Lutein treatment minimized deterioration of b-wave/a-wave ratio and oscillatory potentials as well as inhibited up-regulation of GFAP in retinal I/R injury. In cultured Müller cells, lutein treatment increased cell viability and reduced level of nuclear NF-κB, IL-1ß, and Cox-2, but not TNFα after hypoxic injury. CONCLUSIONS: Reduced gliosis in I/R retina was observed with lutein treatment, which may contribute to preserved retinal function. Less production of pro-inflammatory factors from Müller cells suggested an anti-inflammatory role of lutein in retinal ischemic/hypoxic injury. Together with our previous studies, our results suggest that lutein protected the retina from ischemic/hypoxic damage by its anti-oxidative, anti-apoptotic, and anti-inflammatory properties.

Aldose reductase deficiency reduced vascular changes in neonatal mouse retina in oxygen-induced retinopathy.

PURPOSE: Retinal neovascularization is the major pathologic process in many ocular diseases and is associated with oxidative stress. Deficiency of aldose reductase (AR), the first enzyme in the polyol pathway for glucose metabolism, has been shown to reduce oxidative stress and blood vessel leakage. The present study aimed to investigate the effect of AR deficiency on retinal neovascularization in a murine oxygen-induced retinopathy (OIR) model. METHODS: Seven-day-old wild-type (WT) and AR-deficient (AR(-/-)) mice were exposed to 75% oxygen for 5 days and then returned to room air. Vascular obliteration, neovascularization, and blood vessel leakage were analyzed and compared. Immunohistochemistry for AR, nitrotyrosine (NT), poly(ADP-ribose) (PAR), glial fibrillary acidic protein (GFAP), and Iba-1, as well as Western blots for vascular endothelial growth factor (VEGF), phospho-Erk (p-Erk), phospho-Akt (p-Akt), and phospho-IκB (p-IκB) were performed. RESULTS: Compared with WT OIR retinae, AR(-/-) OIR retinae displayed significantly smaller central retinal vaso-obliterated area, less neovascularization, and reduced blood vessel leakage. Significantly reduced oxidative stress and glial responses were also observed in AR(-/-) OIR retinae. Moreover, reduced microglial response in the avascular area but increased microglial responses in the neovascular area were found with AR deficiency. Furthermore, expression levels of VEGF, p-Erk, p-Akt, and p-IκB were significantly reduced in AR(-/-) OIR retinae. CONCLUSIONS: Our observations indicated that AR deficiency reduced retinal vascular changes in the mouse model of OIR, indicating that AR can be a potential therapeutic target in ischemia-induced retinopathy.

Lycium barbarum extracts protect the brain from blood-brain barrier disruption and cerebral edema in experimental stroke.

BACKGROUND AND PURPOSE: Ischemic stroke is a destructive cerebrovascular disease and a leading cause of death. Yet, no ideal neuroprotective agents are available, leaving prevention an attractive alternative. The extracts from the fruits of Lycium barbarum (LBP), a Chinese anti-aging medicine and food supplement, showed neuroprotective function in the retina when given prophylactically. We aim to evaluate the protective effects of LBP pre-treatment in an experimental stroke model. METHODS: C57BL/6N male mice were first fed with either vehicle (PBS) or LBP (1 or 10 mg/kg) daily for 7 days. Mice were then subjected to 2-hour transient middle cerebral artery occlusion (MCAO) by the intraluminal method followed by 22-hour reperfusion upon filament removal. Mice were evaluated for neurological deficits just before sacrifice. Brains were harvested for infarct size estimation, water content measurement, immunohistochemical analysis, and Western blot experiments. Evans blue (EB) extravasation was determined to assess blood-brain barrier (BBB) disruption after MCAO. RESULTS: LBP pre-treatment significantly improved neurological deficits as well as decreased infarct size, hemispheric swelling, and water content. Fewer apoptotic cells were identified in LBP-treated brains by TUNEL assay. Reduced EB extravasation, fewer IgG-leaky vessels, and up-regulation of occludin expression were also observed in LBP-treated brains. Moreover, immunoreactivity for aquaporin-4 and glial fibrillary acidic protein were significantly decreased in LBP-treated brains. CONCLUSIONS: Seven-day oral LBP pre-treatment effectively improved neurological deficits, decreased infarct size and cerebral edema as well as protected the brain from BBB disruption, aquaporin-4 up-regulation, and glial activation. The present study suggests that LBP may be used as a prophylactic neuroprotectant in patients at high risk for ischemic stroke.

Lutein enhances survival and reduces neuronal damage in a mouse model of ischemic stroke.

INTRODUCTION: Stroke is one of the leading causes of death worldwide. Protective agents that could diminish the injuries induced by cerebral ischemia/reperfusion (I/R) are crucial to alleviate the detrimental outcome of stroke. The aim of this study is to investigate the protective roles of lutein in cerebral I/R injury. METHODS: Two-hour cerebral ischemia was induced by unilateral middle cerebral artery occlusion (MCAo) in mice. Either lutein (0.2 mg/kg) or vehicle was given to mice intraperitoneally 1h after MCAo and 1h after reperfusion. Neurological deficits were evaluated at 22 h after reperfusion while survival rate was assessed daily until 7 days after reperfusion. Brains were cut into 2mm-thick coronal slices and stained with 2% 2,3,5-triphenyltetrazolium chloride to determine the infarct size after MCAo. Paraffin-embedded brain sections were prepared for TUNEL assay and immunohistochemistry. Protein lysate was collected for Western blotting experiments. RESULTS: Higher survival rate, better neurological scores, smaller infarct area and smaller infarct volume were noted in the lutein-treated group. Immunohistochemistry data showed a decrease of immunoreactivity of nitrotyrosine, poly(ADP-ribose) and NFκB in the lutein-treated brains. Western blotting data showed decreased levels of Cox-2, pERK, and pIκB, but increased levels of Bcl-2, heat shock protein 70 and pAkt in the lutein-treated brains. CONCLUSIONS: Post-treatment of lutein protected the brain from I/R injury, probably by its anti-apoptotic, anti-oxidative and anti-inflammatory properties. These suggest that lutein could diminish the deleterious outcomes of cerebral I/R and may be used as a potential treatment for stroke patients.

Lycium barbarum polysaccharides reduce neuronal damage, blood-retinal barrier disruption and oxidative stress in retinal ischemia/reperfusion injury.

Neuronal cell death, glial cell activation, retinal swelling and oxidative injury are complications in retinal ischemia/reperfusion (I/R) injuries. Lycium barbarum polysaccharides (LBP), extracts from the wolfberries, are good for "eye health" according to Chinese medicine. The aim of our present study is to explore the use of LBP in retinal I/R injury. Retinal I/R injury was induced by surgical occlusion of the internal carotid artery. Prior to induction of ischemia, mice were treated orally with either vehicle (PBS) or LBP (1 mg/kg) once a day for 1 week. Paraffin-embedded retinal sections were prepared. Viable cells were counted; apoptosis was assessed using TUNEL assay. Expression levels of glial fibrillary acidic protein (GFAP), aquaporin-4 (AQP4), poly(ADP-ribose) (PAR) and nitrotyrosine (NT) were investigated by immunohistochemistry. The integrity of blood-retinal barrier (BRB) was examined by IgG extravasations. Apoptosis and decreased viable cell count were found in the ganglion cell layer (GCL) and the inner nuclear layer (INL) of the vehicle-treated I/R retina. Additionally, increased retinal thickness, GFAP activation, AQP4 up-regulation, IgG extravasations and PAR expression levels were observed in the vehicle-treated I/R retina. Many of these changes were diminished or abolished in the LBP-treated I/R retina. Pre-treatment with LBP for 1 week effectively protected the retina from neuronal death, apoptosis, glial cell activation, aquaporin water channel up-regulation, disruption of BRB and oxidative stress. The present study suggests that LBP may have a neuroprotective role to play in ocular diseases for which I/R is a feature.

Roles of paroxetine and corticosterone on adult mammalian ciliary body cell proliferation.

BACKGROUND: The neurogenesis in retina of adult mammals is generally abolished, and this renders the retina lack of regenerative capacity. Despite this, there is a small population of nestin-positive cells in the ciliary epithelium which retains neurogenic potential. The present study aimed at investigating the effect of two drugs, corticosterone and paroxetine, on the cell proliferation of the ciliary body. METHODS: Adult Sprague-Dawley rats were given vehicle, corticosterone, paroxetine, or both corticosterone and paroxetine treatment for 14 days. Cell proliferation in the ciliary body was quantified using 5-bromo-2-deoxyuridine (BrdU) immunohistochemistry. Co-labelling of BrdU and stem cell marker was used to phenotype the BrdU immunoreactive cells. RESULTS: Corticosterone treatment suppressed while paroxetine treatment increased the cell proliferation of the ciliary body. Co-labelling with cell markers revealed that the BrdU positive cells also showed nestin expression but not glial fibrillary acidic protein (GFAP). CONCLUSIONS: The results illustrate that proliferation of retinal progenitor cells situated in ciliary body are subjected to regulation by selective serotonin reuptake inhibitors (SSRI) and corticosteroid, which is similar to our previous findings in neurogenic regions in central nervous system (CNS). Paroxetine treatment could reverse the suppressive effect of corticosterone on ciliary body cell proliferation. This provides information for future investigation of retinal stem cell biology and potential treatment of retinal degenerative diseases.

Lutein protects RGC-5 cells against hypoxia and oxidative stress.

Retinal ischemia and oxidative stress lead to neuronal death in many ocular pathologies. Recently, we found that lutein, an oxy-carotenoid, protected the inner retina from ischemia/reperfusion injury. However, it is uncertain whether lutein directly protects retinal ganglion cells (RGCs). Here, an in vitro model of hypoxia and oxidative stress was used to further investigate the neuroprotective role of lutein in RGCs. Cobalt chloride (CoCl(2)) and hydrogen peroxide (H(2)O(2)) were added to a transformed RGC cell line, RGC-5, to induce chemical hypoxia and oxidative stress, respectively. Either lutein or vehicle was added to cultured cells. A higher cell count was observed in the lutein-treated cells compared with the vehicle-treated cells. Our data from this in vitro model revealed that lutein might protect RGC-5 cells from damage when exposed to either CoCl(2)-induced chemical hypoxia or H(2)O(2)-induced oxidative stress. These results suggest that lutein may play a role as a neuroprotectant.

Effect of lutein on retinal neurons and oxidative stress in a model of acute retinal ischemia/reperfusion.

PURPOSE: Retinal ischemia/reperfusion (I/R) occurs in many ocular diseases and leads to neuronal death. Lutein, a potent antioxidant, is used to prevent severe visual loss in patients with early age-related macular degeneration (AMD), but its effect on I/R insult is unclear. The objective of the present study is to investigate the neuroprotective effect of lutein on retinal neurons after acute I/R injury. METHODS: Unilateral retinal I/R was induced by the blockade of internal carotid artery using intraluminal method in mice. Ischemia was maintained for 2 hours followed by 22 hours of reperfusion, during which either lutein or vehicle was administered. The number of viable retinal ganglion cells (RGC) was quantified. Apoptosis was investigated using TUNEL assay. Oxidative stress was elucidated using markers such as nitrotyrosine (NT) and poly(ADP-ribose) (PAR). RESULTS: In vehicle-treated I/R retina, severe cell loss in ganglion cell layer, increased apoptosis as well as increased NT and nuclear PAR immunoreactivity were observed. In lutein-treated I/R retina, significantly less cell loss, decreased number of apoptotic cells, and decreased NT and nuclear PAR immunoreactivity were seen. CONCLUSIONS: The neuroprotective effect of lutein was associated with reduced oxidative stress. Lutein has been hitherto used principally for protection of outer retinal elements in AMD. Our study suggests that it may also be relevant for the protection of inner retina from acute ischemic damage.

Implantation of neurotrophic factor-treated sensory nerve graft enhances survival and axonal regeneration of motoneurons after spinal root avulsion.

We previously showed that motor nerves are superior to sensory nerves in promoting axon regeneration after spinal root avulsion. It is, however, impractical to use motor nerves as grafts. One potential approach to enhancing axonal regeneration using sensory nerves is to deliver trophic factors to the graft. Here, we examined the regulation of receptors for brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, ciliary neurotrophic factor, and pleiotrophin after root avulsion in adult rats. We then tested their survival-promoting and neuroregenerative effects on spinal motoneurons. The results showed that receptors for brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor were upregulated and that these trophic factors promoted survival and axonal regeneration of motoneurons when they were injected into the sensory nerve graft before implantation. In contrast, receptors for ciliary neurotrophic factor and pleiotrophin were downregulated after avulsion. Ciliary neurotrophic factor did not promote survival and axonal regeneration, whereas pleiotrophin promoted axonal regeneration but not survival of injured spinal motoneurons. Our results suggest that infusion of trophic factors into sensory nerve grafts promote motoneuron survival and axonal regeneration. The technique is technically easy and is, therefore, potentially clinically applicable.

Enhanced survival of melanopsin-expressing retinal ganglion cells after injury is associated with the PI3 K/Akt pathway.

In the present study, we studied the factors that contribute to the injury-resistant property of melanopsin-expressing retinal ganglion cells (mRGCs). Since phosphatidylinositol-3 kinase (PI3 K)/Akt signaling pathway is one of the well-known pathways for neuronal cell survival, we investigated the survival of mRGCs by applying the PI3 K/Akt specific inhibitors after injury. Two injury models, unilateral optic nerve transection and ocular hypertension, were adopted using Sprague-Dawley rats. Inhibitors of PI3 K/Akt were injected intravitreally following injuries to inhibit the PI3 K/Akt signaling pathway. Retinas were dissected after designated survival time, immunohistochemistry was carried out to visualize the mRGCs using melanopsin antibody and the number of mRGCs was counted. Co-expression of melanopsin and phospho-Akt (pAkt) was also examined. Compared to the survival of non-melanopsin-expressing RGCs, mRGCs showed a marked resistance to injury and co-expressed pAkt. Application of PI3 K/Akt inhibitors decreased the survival of mRGCs after injury. Our previous study has shown that mRGC are less susceptible to injury following the induction of ocular hypertension. In this study, we report that mRGCs were injury-resistant to a more severe type of injury, the optic nerve transection. More importantly, the PI3 K/Akt pathway was found to play a role in maintaining the survival of mRGCs after injury.