Acute glaucoma is an ocular emergency and sight -threatening disease which is caused by a sudden increase in intraocular ocular pressure (IOP) due to blockage of aqueous humor outflow. Acute glaucoma can result in permanent loss of visual acuity and visual field (VF). Prophylactic or therapeutic medicine is rare for acute glaucoma. In animal studies, a well-established model to investigate this acute IOP spike is by fluid infusion and adjustment of the fluid level to induce high IOP within a few seconds. However, there is no blockage of aqueous outflow and the increase in intraocular pressure is unrealistically rapid. To mimic the IOP profile in human acute glaucoma attack, we propose the use of an ophthalmic viscosurgical device (OVD), Healon 5 (AMO, Santa Ana, CA, USA) which is injected intracamerally to block aqueous outflow. The IOP is allowed to increase naturally inside the globe. We found that Healon 5 can induce an acute elevation in IOP with very similar characteristics to those observed in humans. For example, the IOP profile during the attack, changes in the anterior segment and retinal nerve fibre layer (RNFL) thinning are all consistent with findings in human acute angle closure glaucoma (AACG). We believed that our new model can more accurately reflect acute glaucoma than other animal models. Based on these findings we further tested the neuroprotective effect of Lycium barbarum polysaccharides (LBP) on retinal neurons against an acute rise in IOP (attack) with the new model. L. barbarum is an herb that has been used in Chinese medicine for thousands of years. The fruit of this plant is believed to be good for the health of the eyes. In our study we found that oral administration of LBP preceding an acute glaucoma attack can preserve the visual function of the animals despite the loss of neurons in the retinal ganglion cell layer (RGCL). L. barbarum intake seems to inhibit secondary cell death and progression of the disease. In conclusion, we had successfully established a new acute glaucoma attack animal model by intracameral injection of Healon 5. This model more closely resembles the condition observed in human acute glaucoma. We also found that LBP has a prophylactic neuroprotective effect against an acute glaucoma attack in animals. It can protect the visual function and possibly inhibit secondary cell death. Oral consumption of LBP as a health supplement may provide extra benefit to people who are at high risk of developing acute glaucoma, in addition to the protective effects of LBP against other diseases. / published_or_final_version / Anatomy / Master / Master of Philosophy

Glaucoma is a neurodegenerative disease and one of the major causes of blindness in the world. Secondary degeneration is involved in glaucoma. The retinal ganglion cells (RGCs) which are vulnerable to secondary degeneration in glaucoma are the promising target population for therapeutic intervention. Partial optic nerve transection (PONT) model has been established in the last decade. Primary and secondary degeneration can be separated in different regions of retinas in this model. Therefore, PONT is a good model for the study of mechanisms of secondary degeneration and the drug screening for secondary degeneration. Lycium barbarum (L. barbarum) has been shown to be neuroprotective for cortical neurons in vitro. It has also been shown that L. barbarum could delay RGCs death in a rat ocular hypertension model. In order to further investigate the effects of L. barbarum for RGCs, two models, complete optic nerve transaction (CONT) model and PONT model, were employed in my study. My results showed that the polysaccharide extract from L. barbarum (LBP) could partly prevent RGCs from death in the inferior retinas 4 weeks after PONT whereas it could not reduce the loss of RGCs after CONT. The1,1'-dioctadecyl-3, 3, 3’, 3’-tetramethylindocarbocyanine perchlorate(DiI) labeling of RGCs whose axons were transected showed that the majority of labeled cell bodies existed in the superior retinas. The result meant that more cell bodies in the superior retinas would die from primary degeneration than in the inferior retina after PONT. Therefore my results indicated that LBP protected RGCs which would die from secondary degeneration rather than primary degeneration. The results of Terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) staining showed that RGCs underwent apoptosis 1 week after PONT. Western-blot analysis demonstrated that oxidative stress was involved in the degeneration of RGCs after PONT. Furthermore, c-Jun N-terminal kinases (JNKs) pathway was activated which was indicated by an increase ofphospho-JNK2/3(p-JNK2/3)and phospho-c-jun(p-c-jun). Our results also revealed that orally feeding of LBP could increase the expression of manganese superoxide dismutase (MnSOD) and insulin growth factor-1 (IGF-1)and decrease the expression of p-JNK2/3 and p-c-jun. The results from optic nerve (ON) study showed that glial cells, including astrocytes and microglias/macrophages, were activated after PONT. Oxidative stress and inflammation were involved in the process. Secondary degeneration of ON was not obvious and LBP exerted no protective effects on the survival of axons in the ON. The multifocal electroretinography (mfERG) study showed that both the functions of inner retinas and outer retinas were damaged after PONT. The results indicated that other cell types or the synapses between different cell types were damaged in addition to RGCs. LBP could improve the function of the whole retinas, including both inner retinas and outer retinas after PONT. In conclusion, our results indicated that LBP protected RGCs from secondary degeneration via inhibiting oxidative stress and the activation of JNK pathway.LBP could also improve the function of both inner retinas and outer retinas after PONT. / published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Retinal detachment (RD) is a leading cause of blindness and although final surgical reattachment rate has greatly improved, visual outcome in many macula-off detachments is disappointing, mainly because of photoreceptor cell death. We previously showed that both lutein and Lycium barbarum polysaccharides (LBP) are neuroprotective in a rodent model of ischemia/reperfusion injury. The objective of this study is to investigate lutein and LBP as possible pharmacological adjuncts to surgery. Lutein: 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 0.5mg/kg lutein in corn oil (treatment group) were given intraperitoneally starting 4 hours 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 experiment was repeated with treatment commencing 36 hours after RD. When lutein was given 4 hours after RD, there was significantly fewer TUNELpositive 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 hours after RD similar results were observed. 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 hours. The use of lutein in patients with RD may serve as an adjunct to surgery to improve visual outcomes. LBP: The same RD model was used for the LBP experiment. Phosphate buffered solution (PBS) or LBP in PBS was given orally through a gavage at 1mg/kg and 10mg/kg concentrations. For this experiment, animals were sacrificed 7 days after RD, and only cell counting of the ONL and TUNEL staining were performed. Both sets of results did not produce statistically significant changes with the use of LBP. Our preliminary data for the effect of LBP on retinal detachment shows no significant beneficial effect. / published_or_final_version / Medicine / Master / Master of Research in Medicine

It has been known that light input can affect the emotions of a person. The depressive syndrome Seasonal Affective Disorder (SAD) is an effective example of the power of light in changing the mood of a person. Patients with SAD have recurring depressive episodes that follow seasonal changes, which is due to the changing daylight hours. This phenomenon suggests that there would be receptors in the retina that would not simply be responsible for vision, but also for the regulation of non-visual signals such as emotion. In many animals, projections have been found from the retina to the dorsal raphe nucleus (DRN). This brain region is a serotonergic area and has been found to be involved in the occurrence of depression. As such, the cells in the retina which were found to have projections to the DRN have a high possibility to be involved in emotion regulation. Retinal Ganglion Cells (RGCs) are classified into many types. A specific type known as an alpha cell is suspected to be the DRN-projecting subtype. This study uses Lycium Barbarum Polysaccharide (LBP) as a treatment in protecting the large RGCs from corticosterone (CORT) -induced damage. The aim is to observe if LBP will provide neuroprotection to large sized RGCs damaged by 40mg/kg or 50mg/kg CORT, and hence if LBP can be further investigated as a possible anti-depressant drug. This study observed that although LBP did not reduce large cell deaths, it reduced cell atrophy of the RGCs under high dosage of CORT (50mg/kg). For the same number of cells counted, treatment groups with a high dose CORT injection found more cells over 300μm2 in area than cells under 300μm2. Also, it was found that the temporal quadrants were more sensitive to cell size change than the nasal quadrants, paving way for more in-depth research of the spatial sensitivity to CORT or to LBP. The findings in this study indicate that LBP does indeed have a neuroprotective effect on large RGCs, although this effect is limited and as of yet seems conditional, as this study ignores the effect of CORT and LBP on other large cell properties such as the dendritic field size and the amount of synapses. Further studies are needed to determine the mechanism of the neuroprotective effect of LBP and to determine the exact site of action LBP works on. / published_or_final_version / Anatomy / Master / Master of Medical Sciences

published_or_final_version / abstract / toc / Botany / Master / Master of Philosophy

published_or_final_version / abstract / Obstetrics and Gynaecology / Master / Master of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

In order to understand the possible mechanisms in the glaucoma-related retinopathy, the role of the vasoconstrictor, endothelin-1 (ET-1), receptor for advanced glycation end-products (RAGE) as well as its ligand, Aβ in the degeneration of retinal ganglion cells (RGCs) were studied in experimental models. In addition, the relationship of ET-1, RAGE and Aβ for the RGC protective mechanism of Lycium Barbarum (LB) was also investigated. In the first part, ET-1 together with its receptors, ETA and ETB, were studied to understand their possible roles in chronic ocular hypertension (COH). The neuronal protective mechanism of LB was also determined by using a well established COH rat model. In normal rats, ET-1 and its receptors, ETA and ETB, were distributed in the retina, vasculature and optic nerve. Interestingly, ET-1 expression was up-regulated after COH. LB could decrease the expression of ET-1 and regulate its receptors (up-regulation of ETB and down-regulation of ETA in vasculature; up-regulation of ETA and down-regulation of ETB in RGCs) under the condition of COH. These data suggested that the RGC protective mechanism of LB might be related to its ability to regulate the biological effects of ET-1. To investigate the pathogenic effect of ET-1 in glaucoma, in the second part, we used transgenic mice with over-expression of ET-1 on endothelial cells (TET-1 mice). We found that beginning at 10-12 months, TET-1 mice showed a progressive retinal degeneration (loss of RGCs associated with neurons in the inner nuclear layer and outer nuclear layer of the retina) without elevation of the intraocular pressure (IOP). The data demonstrated that TET-1 mice may serve as a potential model to investigate the role of endothelial ET-1 in the pathogenesis of normal tension glaucoma and other degenerative retinopathy. To investigate whether LB plays a role on neuronal protection other than in COH, in the third part, we used an acute ocular hypertension (AOH)-induced ischemia mouse model. We found that LB could rescue RGCs under AOH insult, associating with blood vessel protection (decreasing the damage of blood-retinal-barriers and rescuing the survival of endothelial cells and pericytes) and inhibiting retinal gliosis. We also found the protective mechanism of LB was closely correlated with down-regulation of the expression of RAGE, ET-1, APP (amyloid precursor protein), AGE (advanced glycation end-product) as well as Aβ; therefore to reduce the damage effects of these RAGE-mediated reactions to the retinal neurons, blood vessels and glial cells involved in the ischemic insult. Taken together, the present study demonstrated that TET-1 mice may be a potential model for investigating the role of ET-1 in degenerative retinopathies, such as normal tension glaucoma. We also showed the neuronal protective mechanism of LB in vivo was associated with inhibiting the biological effect of ET-1 and down-regulating the damage signaling pathways mediated by the activation of RAGE and its ligands (AGE and Aβ). These results provided further understandings in the mechanism of the glaucoma-related retinopathy. In addition, LB could be a neuroprotective agent to the retina following both chronic and acute injuries. / published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Ischemic stroke is a devastating cerebrovascular disease resulting in high mortality rate and distressing sequelae such as hemiplegia, ataxia and even visual impairment. Retinal ischemia refers to a common pathological feature shared by many blinding diseases including retinal vascular occlusions, diabetic retinopathy, glaucoma, and retinopathy of prematurity. Ischemia/reperfusion injury is implicated in both of these pathological conditions, which greatly impact on one’s daily life. The eventual consequence of the insult is irreversible neuronal cell death and functional deterioration. Apart from current symptomatic treatment for these diseases, researchers and clinicians are dedicated to look for ideal neuroprotectant to meet the clinical needs. Traditional Chinese medicine has been received accumulating attention in recent years, and Lycium barbarum is one of them. The polysaccharides (LBP) utilized in the present study are the rich extracts of the fruit of Lycium barbarum that has been shown to exert many biological effects. This study aims to evaluate its protective effects in cerebral and retinal ischemia, which has not yet been fully investigated. A well-established rodent model, middle cerebral artery occlusion, was utilized in the present study to mimic cerebral and retinal ischemia/reperfusion injury. In the study of cerebral ischemia, both pre-treatment and post-treatment of LBP were explored. Seven-day LBP pre-treatment revealed significant protection against neurological deficits and cerebral infarction. Besides, it attenuated cerebral edema and glial activation, as well as preserved blood-brain barrier integrity. Further study showed that these beneficial effects of LBP pre-treatment might act via anti-apoptosis, antioxidation and anti-inflammation. However, similar findings were not noted in LBP post-treatment experiments, possibly due to the timing of intervention. In the investigation of retinal ischemia, the observation time was prolonged to 7 days after the insult. Electroretinogram was used to evaluate the functional alternation of retinal neurons. Sustained retinal dysfunction was induced by two-hour ischemia. LBP pre-treatment with continuous daily supplementation effectively alleviated visual dysfunction and protected the retina from morphological impairment including neuronal death, glial activation and blood-retinal barrier disruption. Similarly, these protective effects might be associated with the involvement of attenuation of apoptosis and oxidative stress. In conclusion, LBP pre-treatment with continuous daily supplementation protected the brain and retina, both functionally and morphologically, from ischemia/reperfusion injury. This dosing regimen hold great promise in serving as a prophylactic neuroprotectant in patients at high risk for ischemic stroke, as well as preserving normal visual function and reducing irreversible neuronal death in ischemic retinopathies. Further studies on the active ingredients and underlying mechanisms would be informative for better application of LBP in clinical situation. / published_or_final_version / Ophthalmology / Doctoral / Doctor of Philosophy