BRB disruption, glial cell activation, oxidative stress and neuronal death are major concerns in retinal I/R injury. Neuroprotective agents that can retard or prevent these damages are beneficial in treating many ocular diseases in which retinal I/R is a complication. In our present study, we showed that pre-treating mice with LBP for 1 week could protect the animals from I/R injury by reducing neuronal cell death, retinal swelling, glial activation, BRB disruption and oxidative stress.
In the present study, unilateral retinal ischemia was induced by occluding the ICA which supplies blood to the ophthalmic artery 
. This model is one of the commonly used animal retinal I/R models 
. Different from other retinal I/R model, the unilateral ICA occlusion model is a purely vascular model and does not involve any mechanical injury to the eye during the experimental operation 
. The ophthalmic artery occlusion and blood resupply by ECA and ICA can be easily monitored by the duration of filament kept inside the vessel lumen. This model induces complete but reversible retinal ischemia injury 
and leads to a considerable cell loss in inner retina by the blockade of ophthalmic artery 
. It mimics the clinical situation of transient monocular amaurosis fugax 
and other ocular diseases in which retinal I/R is a complication. Therefore, this artery occlusion model is a good tool for exploring neuroprotective agents against retinal I/R injury.
is a dried fruit that is used as a food or a medicine according to Chinese tradition 
. It has been claimed that LBP can exhibit anti-aging, anti-tumor, cytoprotective, neuro-modulation, and immune modulation effects 
. LBP can attenuate breakage of DNA by oxidation in the testicle cells in mice 
. It has also been shown to protect DNA damage of peripheral blood lymphocytes against oxidative stress 
. However, researches on the protective effects of LBP against ocular diseases are still ongoing. In the present study, we aim to look at the protective effects of LBP against retinal I/R injury. We focus on four aspects that are closely related to retinal I/R injury: anti-apoptosis, preservation of BRB integrity, prevention of retinal swelling, and anti-oxidation.
Retinal I/R induces neuronal death in the inner retina, especially RGC. An extensive loss of cells in the GCL has been seen after retinal I/R injury 
. A majority of these neurons die by apoptosis during retinal I/R injury 
. Our data show an increased number of apoptotic nuclei in inner retinal layers, especially in the GCL, of the ischemic retina. The result is highly reproducible and comparable to our previously published data 
. In previous studies, only a few TUNEL-positive apoptotic cells are found in inner retina with 1 hr ischemia injury although similar animal model is used 
. The discrepancy is probably due to the shorter ischemia period when compared with that in our study. Retinal function, assessed by the a-wave and b-wave of electroretinography, is deteriorated in the ischemic eye 
, suggesting that the function of retinal neurons is weakened in retinal I/R injury. Similar to a previous study 
, we also show that retinal I/R caused detrimental injury to amacrine cells, as indicated by the great reduction in calretinin expression and the dis-organization of IPL stratification. Apart from calretinin-expressing amacrine cells, fewer nNOS-expressing amacrine cells were observed in I/R retina 
. Comparable phenomenon was noted in bipolar cell immunoreactivity in the present study. Apart from neuronal damage, I/R injury also induces impairment in synaptic connections of retinal neurons 
. Vesl-1L/Homer 1c (V-1L) is a marker for used as a marker for the assessment of changes in synaptic connectivity preceding apoptosis and during early stages of apoptosis of RGCs 
. A reduction of V-1L immunoreactivity indicates that the synaptic connections of RGCs and other inner retinal neurons, including amacrine cells and bipolar cells, are attenuated after I/R. In this study, our first aim to investigate the anti-apoptotic and neuroprotective effects of LBP in retinal I/R injury. Significantly fewer apoptotic cells were seen in GCL and INL of LBP-treated ischemic retina. The detrimental effects on amacrine cells and bipolar cells due to I/R injury was minimized. In outer retina, LBP has been shown to decrease apoptosis in photoreceptors of rd1 mice with photoreceptor degeneration 
. Its beneficial protection is as good as other neuroprotective agents such as lutein and zeaxanthin 
. Our data elucidated that LBP is anti-apoptotic not only in outer retina, but also inner retina. Moreover, our earlier studies showed that LBP protects against beta-amyloid peptide neurotoxicity by inhibiting pro-apoptotic signaling pathways such as JNK, PKR and caspase-3 
. It is therefore likely that LBP may exert its neuroprotective effects via inhibition of JNK, PKR and caspase-3 activity in retinal I/R injury.
BRB is a protective barrier which consists of the outer and inner BRB. The role of BRB is to maintain the homeostatic condition of retinal microenvironment and exclude harmful substance getting into the retina 
. The outer barrier is formed by the retinal pigment epithelium, separating the outer retina from the choroid 
. The inner BRB is formed by the tight junctions of the vascular endothelial cells and sheathed by the Muller cell processes 
. In many ocular disease including ischemic retinal vein/artery occlusion and diabetic retinopathy, breakdown of the inner BRB increases retinal vascular permeability, resulting in retinal edema and cell death 
. In the present study, we examined BRB integrity by assessing blood vessel leakage using IgG immunohistochemistry. The majority of IgG was confined in the lumen of blood vessels in normal retina. However, IgG extravasations were present outside the vessel lumen after I/R injury, indicating blood vessel leakage and BRB breakdown. IgG extravasations was minimized in ischemic retina treated with LBP. This suggests that LBP could prevent or minimize the disruption of BRB in retinal I/R injury, which has not been studied previously.
Disruption of BRB leads to swelling of astrocytes and Muller cells processes associated with the activation of GFAP and AQP4 under ischemic conditions 
. These causes further retinal edema and tissue damage. In normal conditions, water and ion homeostasis in retina is controlled by Muller cells via the transmembrane aquaporin water channels 
. Muller cells do not express GFAP under normal physiological situations. The end feet of retinal astrocytes and Muller cells wrap around the blood vessels of the superficial retina and express AQP4 
. GFAP up-regulation is a hallmark of astrocyte and Muller cell activation and the resulting reactive gliosis 
. Expression of GFAP in Muller cells and astrocytes occurs under pathological conditions such as I/R injury 
. In addition, over-expression of AQP4 is associated with water influx into the retina during injury 
. Water influx is further exaggerated during reperfusion. Swelling and hypertrophy of Muller cells occur consequently upon overloading of intracellular K+
and the movement of water inside cells 
. It has been shown that the deletion of AQP4 gene can protect retina against swelling in a mouse model of ischemia, thus emphasizing the importance of AQP4 in water transport 
. In the present study, activation of GFAP and AQP4 was observed in the vehicle-treated retina, indicating the role of Muller cells and AQP4 controlling water transport in retina. Moreover, retinal swelling was noted in ischemic retina. These results indicate a close association among the BRB integrity, the control of water flux and retinal swelling. Here, we show that LBP pre-treatment could diminish the activation of GFAP and AQP4, as well as retinal swelling in retinal I/R injury, implying the inhibitory effects of LBP in retinal swelling by minimizing the activation of Muller cell and AQP4.
Oxidative stress plays a role in retinal I/R injury due to the high content of polyunsaturated fatty acid in retina. Reactive oxygen species (ROS) and free radical formation during I/R facilitates lipid peroxidation of membrane, denature of protein and DNA damage 
. The breakdown of DNA strands activates the nuclear enzyme poly(ADP-ribose) polymerase (PARP) to produce PAR 
. PARP cleaves nicotinamide adenine dinucleotide, and subsequently leads to energy failure and cell death 
. Another pathway of inducing oxidative stress is the nitrosative injury. Free radical formation facilitates nitric oxide (NO) production, which reacts with superoxide to from peroxynitrite, a strong oxidant 
. Peroxynitrite leads to nitration of tyrosine residues of cells to form NT 
. Therefore, NT is an indicator for oxidative-nitrosative stress. Increased immunoreactivity of PAR and NT was observed in the vehicle-treated I/R retina, indicating increased oxidative stress associated with retinal I/R. In addition, oxidative stress has been shown to have an injurious role in BRB integrity by disruption of tight junctions in retina 
. ROS generated in ischemia up-regulates vascular endothelial growth factor (VEGF) gene expression 
. Tight junction damage and increased VEGF expression attribute to an increase in vascular permeability and subsequent BRB breakdown 
. Treatment with anti-oxidants, including Lycium barbarum
, increases glutathione peroxidase activity and glutathione levels, and decreases cystine concentrations in retina of rd1/rd1
mice, indicating a decreased level of oxidative stress in retinal generation 
. In the present data, LBP-treated I/R retina had a less extent of oxidative stress, indicated by a decreased level of PAR immunoreactivity. These findings suggest that one of the possible mechanisms for LBP to exhibit its cytoprotective effects is via attenuation of oxidative stress. The decrease in oxidative stress may attribute to prevent or diminish BRB breakdown induced by I/R injury. However, LBP treatment did not lower NT immunoreactivity after I/R injury. This may imply that LBP does not have anti-oxidative effect against nitrosative stress. Further investigation is required to confirm this observation.
Increasing lines of evidence have demonstrated that modulation of immune responses can affect the degenerative processes of neurons in the CNS 
. In fact, previous studies have shown that LBP can enhance immune function 
, suggesting that the beneficial effects of LBP may also be mediated by immuno-modulations. Our recent study also demonstrated that LBP protect RGC by modulating the activation of microglia in an animal model of chronic ocular hypertension 
. We speculate that neuroprotection conferred by LBP may be a combination of many different mechanisms including anti-apoptosis, anti-oxidation and immuno-modulation in addition to our current findings i.e. protection of the BRB and maintenance of the water homeostasis via the down-regulation of AQP4.
Our data indicated that pre-treatment with LBP for 1 week could effectively protect the mouse from retinal I/R injury. The present study hence suggests that LBP may be used as a preventive TCM for diseases associated with I/R such as amaurosis fugax and acute glaucoma. Further studies are warranted to evaluate the therapeutic effects of LBP and other potential neuroprotective mechanisms.