The use of paramagnetic microbeads for the occlusion of the iridocorneal angle produces a sustained elevation of IOP with fewer injections and avoids the risk of visual axis occlusion. It represents a simple and effective method for the induction of experimental glaucoma.

Tuberous sclerosis (TSC) is an inherited syndrome in which tumours in multiple organs are characterised by activation of mammalian target of rapamycin complex 1 (mTORC1). Previous work suggests that mTORC1 activation is associated with feedback inhibition of Akt, a substrate of mTORC2. This could limit TSC-associated tumour growth but lead to paradoxical promotion of tumour cell survival upon treatment with mTOR inhibitors. However, Akt/mTOR signalling has not been fully investigated in TSC-associated tumours and it has been uncertain whether mTOR inhibition can prevent TSC-associated renal tumourigenesis. In this study, we investigated Akt/mTOR signalling in renal tumours using a Tsc2(+/-) mouse model and tested whether mTOR inhibition could prevent renal tumourigenesis. We found that all renal lesions including cysts, adenomas and carcinomas exhibited activation of both Akt and mTORC1 as evidenced by increased protein expression and phosphorylation of Akt and mTOR and their downstream targets. Protein kinase Cα was also highly expressed and phosphorylated in these lesions, consistent with activation of mTORC2. Surprisingly, IRS proteins were highly expressed, in contrast to a striking decrease seen in cultured Tsc2(-/-) mouse embryonic fibroblasts, suggesting one mechanism through which loss of feedback inhibition of Akt may occur in mTORC1 hyperactivated Tsc-associated tumours. Long-term treatment with rapamycin reduced both Akt and mTORC1 activity in normal kidney tissues and blocked the development of all types of renal lesions. In conclusion, in contrast to previous studies, we found that Akt signalling is not inhibited in Tsc-associated renal lesions and that by partially inhibiting the Akt/mTOR pathway, rapamycin is highly effective in preventing Tsc-associated tumours.

Tuberous sclerosis (TSC) is an inherited tumour syndrome caused by mutations in TSC1 or TSC2 that lead to aberrant activation of mTOR. Tumour responses in TSC patients to rapamycin, an allosteric inhibitor of mTOR, or its analogs are partial and reversible probably due to feedback activation of Akt. In this study, we examined the efficacy of GSK2126458, an ATP-competitive dual inhibitor of PI3K/mTOR, in comparison to rapamycin for treatment of renal tumours in genetically engineered Tsc2+/- mice. We found that both GSK2126458 and rapamycin caused significant reduction in number and size of solid renal tumours. GSK2126458 also significantly reduced the number and size of all lesions (cystic, papillary and solid) although to a lesser extent compared to rapamycin. GSK2126458 inhibited both PI3K and mTOR while rapamycin exerted stronger inhibitory effect on mTORC1 in renal tumours. Furthermore, GSK2126458 and rapamycin suppressed proliferation of tumour cells. Importantly, GSK2126458 increased apoptosis of solid tumours but rapamycin did not. Further investigations are therefore needed to test whether rapamycin in combination with GSK2126458 could promote apoptosis and thus improve therapy of TSC-associated renal tumours.

Tuberous sclerosis (TSC) is an inherited tumor syndrome caused by mutations in TSC1 or TSC2 that lead to aberrant activation of mTOR and development of tumors in multiple organs including the kidneys. The mTOR inhibitors rapamycin and everolimus (rapalogs) have demonstrated clinical efficacy in treating TSC-associated tumors including renal angiomyolipomas. However, tumor responses are usually only partial, and regrowth occurs after drug withdrawal. TSC-associated tumors are highly vascular, and TSC patients with renal angiomyolipomas have elevated levels of circulating vascular endothelial growth factor (VEGF) A and VEGFD. Sorafenib inhibits multiple kinases including VEGF receptors and has been used to treat metastatic epithelioid angiomyolipoma in one case, but formal trials have not been undertaken. In this study, we investigated tumor angiogenesis and the therapeutic efficacy of everolimus in combination with sorafenib for renal tumors in Tsc2+/− mice. We found that these tumors exhibited remarkably variable angiogenesis despite consistent aberrant activation of mTOR and increased expression of HIF1α and VEGFA. Treatment of 11-month-old Tsc2+/− mice for 2 months with a combination of everolimus and sorafenib significantly reduced the number and size of solid renal tumors, whereas everolimus or sorafenib alone did not. These results suggest that inhibition of mTOR and multiple kinases including VEGF receptors using combination therapy could hold promise for the treatment of TSC-associated tumors that have responded inadequately to a rapalog alone.

Retinal ganglion cell (RGC) dendritic atrophy is an early feature of many forms of retinal degeneration, providing a challenge to RGC classifi cation. The characterization of these changes is complicated by the possibility that selective labeling of any particular class can confound the estimation of dendritic remodeling. To address this issue we have developed a novel, robust, and quantitative RGC classifi cation based on proximal dendritic features which are resistant to early degeneration. RGCs were labeled through the ballistic delivery of DiO and DiI coated tungsten particles to whole retinal explants of 20 adult Brown Norway rats. RGCs were grouped according to the Sun classifi cation system. A comprehensive set of primary and secondary dendrite features were quantifi ed and a new classifi cation model derived using principal component (PCA) and discriminant analyses, to estimate the likelihood that a cell belonged to any given class. One-hundred and thirty one imaged RGCs were analyzed; according to the Sun classifi cation, 24% ( n = 31) were RGC A , 29% ( n = 38) RGC B , 32% ( n = 42) RGC C , and 15% ( n = 20) RGC D . PCA gave a 3 component solution, separating RGCs based on descriptors of soma size and primary dendrite thickness, proximal dendritic fi eld size and dendritic tree asymmetry. The new variables correctly classifi ed 73.3% ( n = 74) of RGCs from a training sample and 63.3% ( n = 19) from a hold out sample indicating an effective model. Soma and proximal dendritic tree morphological features provide a useful surrogate measurement for the classifi cation of RGCs in disease. While a defi nitive classifi cation is not possible in every case, the technique provides a useful safeguard against sample bias where the normal criteria for cell classifi cation may not be reliable.