Polyethylene glycol-based homologated ligands for nicotinic acetylcholine receptors.

A homologous series of polyethylene glycol (PEG) monomethyl ethers were conjugated with three ligand series for nicotinic acetylcholine receptors. Conjugates of acetylaminocholine, the cyclic analog 1-acetyl-4,4-dimethylpiperazinium, and pyridyl ether A-84543 were prepared. Each series was found to retain significant affinity at nicotinic receptors in rat cerebral cortex with tethers of up to six PEG units. Such compounds are hydrophilic ligands which may serve as models for fluorescent/affinity probes and multivalent ligands for nAChR.

Cervical sympathectomy regulates expression of key angiogenic factors in the rat choroid.

Age-related macular degeneration is the leading cause of blindness in people over the age of 55. In addition to an increased risk of vision loss due to macular degeneration, aging results in a substantial loss of sympathetic nerve activity. We have previously shown that loss of sympathetic nerve activity to the eye causes significant remodeling of the choroidal vasculature. The hypothesis of the present study was that the choroidal remodeling noted after sympathectomy was due to alterations in key angiogenic growth factors. To test this hypothesis, female Sprague-Dawley rats underwent superior cervical ganglionectomy, which eliminates all sympathetic innervation to the eye. Six weeks after surgery, eyes were removed, and the choroidal tissue was processed for real-time PCR to measure gene expression and western blot analysis to assess protein expression. Gene and protein expression were significantly increased for vascular endothelial growth factor (VEGF) and pigment epithelial-derived growth factor (PEDF) in the sympathectomized eye, as compared to the contralateral eye (P < 0.05). Protein expression was increased 4-fold for angiopoietin1, with no change in steady-state gene expression. For both p53 and placental growth factor, steady-state mRNA levels were significantly decreased, while protein expression was significantly increased. Protein expression for Flt-1 was decreased significantly, with reduced gene expression. These results suggest that the vascular remodeling noted in the choroidal blood vessels after sympathectomy is a complex process involving numerous growth factor families. Therefore, modulation of sympathetic nerve activity may be a suitable mechanism to prevent the vascular growth associated with macular degeneration.

Cervical sympathectomy causes photoreceptor-specific cell death in the rat retina.

Changes in the regulation of the vasculature of the eye may be related to some age-related ocular diseases. We have previously shown that loss of sympathetic innervation, as can normally occur with age, resulted in substantial vascular growth of the choroid. The current study was designed to determine whether changes induced by sympathetic denervation causes significant loss of photoreceptors and increased glial cell reactivity in the retina. Sympathetic denervation was performed followed by immunohistochemistry, TUNEL staining, and protein expression analysis to investigate photoreceptor loss. There was a significant reduction (30%) in photoreceptor numbers in the sympathectomized eye. This loss was due to apoptosis, as there was over a doubling in apoptotic cell numbers after sympathectomy. This loss of photoreceptors in the sympathectomized eye resulted in a significantly reduced width of the outer nuclear layer of the retina when compared to the contralateral eye. Increased glial fibrillary acidic protein (GFAP) staining was also noted after sympathectomy in the ganglion cell layer with streaking toward the bipolar cell layer. These results suggest that loss of sympathetic innervation may cause significant changes to the physiology of the choroid.

Beta-adrenergic receptor regulation of pigment epithelial-derived factor expression in rat retina.

In the present study, we have examined a potential mechanism by which sympathetic nerves regulate PEDF and whether its down regulation may be responsible for increased capillary density observed after sympathectomy. Six weeks post-sympathectomy, eyes were removed from female Sprague-Dawley rats for Western blot analysis, RNA isolation, real-time PCR, and immunohistochemistry for measurement of PEDF expression. The contralateral or left eye was used as an intra-animal control. In addition, retinal pigment epithelial cells were grown in culture and treated with norepinephrine and propranolol. An ELISA assay was used to determine the amount of PEDF secreted into the RPE media. Quantitative results of Western blot analysis and real-time PCR confirm that both steady-state gene expression and protein levels of PEDF are significantly decreased in the sympathectomized retina (P<0.05) when compared to the contralateral retina. Qualitative results of immunohistochemistry verify that PEDF is located predominantly in the RPE cell layer of the retina, and levels are decreased in the sympathectomized retina. ELISA results illustrate that norepinephrine significantly increases PEDF secretion by RPE cells and propranolol slightly decreases PEDF secretion into RPE cell medium. In conclusion, down regulation of PEDF may contribute to the increased capillary density of the outer plexiform layer in the retina noted after sympathectomy. Furthermore, expression of PEDF was significantly increased after treatment of norepinephrine in RPE medium demonstrating a role of beta-adrenergic regulation of PEDF. Since sympathetic nerves are damaged in diabetes and PEDF appears to be regulated by beta-adrenergic receptors, these results suggest a role for sympathetic nerves in diabetic retinopathy. This knowledge, in turn, may be used for future treatment and prevention of diabetic retinopathy and other ocular diseases.