Aims/hypothesis The impact of AGEs and advanced lipoxidation end-products (ALEs) on neuronal and Müller glial dysfunction in the diabetic retina is not well understood. We therefore sought to identify dysfunction of the retinal Müller glia during diabetes and to determine whether inhibition of AGEs/ALEs can prevent it. Methods Sprague-Dawley rats were divided into three groups: (1) non-diabetic; (2) untreated streptozotocininduced diabetic; and (3) diabetic treated with the AGE/ ALE inhibitor pyridoxamine for the duration of diabetes. Rats were killed and their retinas were evaluated for neuroglial pathology. Results AGEs and ALEs accumulated at higher levels in diabetic retinas than in controls (p<0.001). AGE/ALE immunoreactivity was significantly diminished by pyridoxamine treatment of diabetic rats. Diabetes was also associated with the up-regulation of the oxidative stress marker haemoxygenase-1 and the induction of glial fibrillary acidic protein production in Müller glia (p<0.001). Pyridoxamine treatment of diabetic rats had a significant beneficial effect on both variables (p < 0.001). Diabetes also significantly altered the normal localisation of the potassium inwardly rectifying channel Kir4.1 and the water channel aquaporin 4 to the Müller glia end-feet interacting with retinal capillaries. These abnormalities were prevented by pyridoxamine treatment. Conclusions/interpretation While it is established that AGE/ALE formation in the retina during diabetes is linked to microvascular dysfunction, this study suggests that these pathogenic adducts also play a role in Müller glial dysfunction.

OBJECTIVEErythropoietin (EPO) may be protective for early stage diabetic retinopathy, although there are concerns that it could exacerbate retinal angiogenesis and thrombosis. A peptide based on the EPO helix-B domain (helix B-surface peptide [pHBSP]) is nonerythrogenic but retains tissue-protective properties, and this study evaluates its therapeutic potential in diabetic retinopathy.RESEARCH DESIGN AND METHODSAfter 6 months of streptozotocin-induced diabetes, rats (n = 12) and age-matched nondiabetic controls (n = 12) were evenly split into pHBSP and scrambled peptide groups and injected daily (10 μg/kg per day) for 1 month. The retina was investigated for glial dysfunction, microglial activation, and neuronal DNA damage. The vasculature was dual stained with isolectin and collagen IV. Retinal cytokine expression was quantified using real-time RT-PCR. In parallel, oxygen-induced retinopathy (OIR) was used to evaluate the effects of pHBSP on retinal ischemia and neovascularization (1–30 μg/kg pHBSP or control peptide).RESULTSpHBSP or scrambled peptide treatment did not alter hematocrit. In the diabetic retina, Müller glial expression of glial fibrillary acidic protein was increased when compared with nondiabetic controls, but pHBSP significantly reduced this stress-related response (P < 0.001). CD11b+ microglia and proinflammatory cytokines were elevated in diabetic retina responses, and some of these responses were attenuated by pHBSP (P < 0.01–0.001). pHBSP significantly reduced diabetes-linked DNA damage as determined by 8-hydroxydeoxyguanosine and transferase-mediated dUTP nick-end labeling positivity and also prevented acellular capillary formation (P < 0.05). In OIR, pHBSP had no effect on preretinal neovascularization at any dose.CONCLUSIONSTreatment with an EPO-derived peptide after diabetes is fully established can significantly protect against neuroglial and vascular degenerative pathology without altering hematocrit or exacerbating neovascularization. These findings have therapeutic implications for disorders such as diabetic retinopathy.

transmitter(s) underlying nitric oxide synthase (NOS)-independent neural inhibition in the internal anal sphincter (IAS) is still uncertain. The present study investigated the role of purinergic transmission. Contractile and electrical responses to electrical field stimulation of nerves (0.1-5 Hz for 10 -60 s) were recorded in strips of mouse IAS. A single stimulus generated a 28-mV fast inhibitory junction potential (IJP) and relaxation. The NOS inhibitor N -nitro-L-arginine (L-NNA) reduced the fast IJP duration by 20%. Repetitive stimulation at 2.5-5 Hz caused a more sustained IJP and sustained relaxation. L-NNA reduced relaxation at 1 Hz and the sustained IJP at 2.5-5 Hz. All other experiments were carried out in the presence of NOS blockade. IJPs and relaxation were significantly reduced by the P2 receptor antagonists 4-[[4-formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-2-pyridinyl]azo]-1,3-benzenedisulfonic acid (PPADS) (100 M), by desensitization of P2Y receptors with adenosine 5Ј-[␤-thio]diphosphate (ADP-␤S) (10 M), and by the selective P2Y1 receptor blocker 2Ј-deoxy-N 6 -methyl adenosine 3Ј,5Ј-diphosphate (MRS2179) (10 M). Relaxation and IJPs were also significantly reduced by the K ϩ channel blocker apamin (1 M). Removal of extracellular potassium (Ko) increased IJP amplitude to 205% of control, whereas return of Ko 30 min later hyperpolarized cells by 19 mV and reduced IJP amplitude to 50% of control. Exogenous ATP (3 mM) relaxed muscles in the presence of TTX (1 M) and hyperpolarized cells by 15 mV. In conclusion, these data suggest that purinergic transmission significantly contributes to NOS-independent neural inhibition in the mouse IAS. P2Y1 receptors, as well as at least one other P2 receptor subtype, contribute to this pathway. Purinergic receptors activate apaminsensitive K ϩ channels as well as other apamin-insensitive conductances leading to hyperpolarization and relaxation.gastrointestinal; enteric; motor inhibition; NANC transmission THE INTERNAL ANAL SPHINCTER (IAS) aids in maintaining fecal continence and permits evacuation of fecal contents during the rectoanal inhibitory reflex (RAIR). To fulfill these functions, the IAS usually exists in a state of contraction that can be relaxed by inhibitory nerves during the defecation reflex. In the present study we examined inhibitory motor innervation to the mouse IAS. The predominant neurotransmitters underlying inhibitory neural responses in the gastrointestinal tract include nitric oxide (NO), VIP, pituitary adenylate cyclase-activating polypeptide, and ATP (or a related compound). There is still controversy regarding the contribution of these neurotransmitters to neural inhibition in the IAS. Whereas some studies suggest that neural inhibition is almost exclusively due to NO in combination with VIP (40), earlier studies of the guinea pig (39), rat (14), and rabbit (32) IAS provided evidence that a purinergic neural pathway contributes as well.Membrane hyperpolarization is an important mechanism contributing to inhibitory motor responses. ...

Excitatory motor innervation to the internal anal sphincter (IAS) of the monkey, the rabbit and mouse were compared. Contractile responses to electrical field stimulation of nerves (EFS, atropine 1 micromol L(-1) and N(omega)-nitro-L-arginine 100 micromol L(-1) present throughout) were examined in isolated strips of IAS. In the monkey IAS, EFS caused frequency dependent (1-30 Hz) contractions which were abolished by guanethidine (10 micromol L(-1)) or phentolamine (3 micromol L(-1)). The sympathetic neurotransmitter noradrenaline (NA) also caused concentration-dependent (10 nmol L(-1)-100 micromol L(-1)) contractions which were abolished by phentolamine revealing a small relaxation that was abolished by propranolol (3 micromol L(-1)). In contrast, EFS caused only relaxation of the mouse and rabbit IAS which was not affected by guanethidine. Furthermore, NA relaxed these muscles and relaxation was nearly abolished by combined addition of phentolamine and propranolol. In conclusion, the monkey IAS is functionally innervated by sympathetic nerves that contract the muscle via excitatory alpha-adrenergic receptors. In contrast, no significant motor function could be identified for sympathetic nerves in the rabbit or mouse IAS although adrenergic receptors linked to muscle inhibition are present. These data reveal species dependent differences in sympathetic motor innervation and suggest that some species are more appropriate than others as models for motor innervation to the human IAS.