Spinal glial cell activation and cytokine secretion have been implicated in the etiology of neuropathic pain in a number of experimental models, including diabetic neuropathy. In this study, streptozotocin- (STZ-) induced diabetic rats were either untreated or treated with gabapentin (50mg/kg/day by gavage for 2 weeks, from 6 weeks after STZ). At 8 weeks after STZ, hypersensitivity was confirmed in the untreated diabetic rats as a reduced response threshold to touch, whilst mechanical thresholds in gabapentin-treated diabetic rats were no different from controls. Diabetes-associated thermal hypersensitivity was also ameliorated by gabapentin. We performed a cytokine profiling array in lumbar spinal cord samples from control and diabetic rats. This revealed an increase in L-selectin, an adhesion molecule important for neutrophil transmigration, in the spinal cord of diabetic rats but not diabetic rats treated with gabapentin. Furthermore, we found an increase in the number of neutrophils present in the parenchyma of the spinal cord, which was again ameliorated in gabapentin-treated diabetic rats. Therefore, we suggest that dysregulated spinal L-selectin and neutrophil infiltration into the spinal cord could contribute to the pathogenesis of painful diabetic neuropathy.

NIDDK, JDRF, and the Diabetic Neuropathy Study Group of EASD sponsored a meeting to explore the current status of animal models of diabetic peripheral neuropathy. The goal of the workshop was to develop a set of consensus criteria for the phenotyping of rodent models of diabetic neuropathy. The discussion was divided into five areas: (1) status of commonly used rodent models of diabetes, (2) nerve structure, (3) electrophysiological assessments of nerve function, (4) behavioral assessments of nerve function, and (5) the role of biomarkers in disease phenotyping. Participants discussed the current understanding of each area, gold standards (if applicable) for assessments of function, improvements of existing techniques, and utility of known and exploratory biomarkers. The research opportunities in each area were outlined, providing a possible roadmap for future studies. The meeting concluded with a discussion on the merits and limitations of a unified approach to phenotyping rodent models of diabetic neuropathy and a consensus formed on the definition of the minimum criteria required for establishing the presence of the disease. A neuropathy phenotype in rodents was defined as the presence of statistically different values between diabetic and control animals in 2 of 3 assessments (nocifensive behavior, nerve conduction velocities, or nerve structure). The participants propose that this framework would allow different research groups to compare and share data, with an emphasis on data targeted toward the therapeutic efficacy of drug interventions.

Benign prostatic hyperplasia (BPH)-related lower urinary tract symptoms (LUTS) and erectile dysfunction commonly coexist, and both respond to phosphodiesterase (PDE) 5 inhibitors, suggesting a shared pathophysiological mechanism. We propose that both BPH-LUTS and erectile dysfunction are caused by microvascular dysfunction within the pelvic organs, and we present an overview of preclinical and clinical studies supporting the hypothesis that, within both the penis and the lower urinary tract, a combination of endothelial and neural dysfunction leads to a vicious cycle of hypoxia, vasoconstriction, altered smooth muscle contractility, and degeneration of autonomic neurons and ganglia. This hypothesis explains much of the preclinical and clinical research relating to these two conditions, and provides a rationale for further investigation into the effects of PDE5 inhibitors on the pathophysiology and symptoms of BPH-LUTS.

Neuropathies of the peripheral and autonomic nervous systems affect up to half of all people with diabetes, and are major risk factors for foot ulceration and amputation. The aetiology is multifactorial: metabolic changes in diabetes may directly affect neural tissue, but importantly, neurodegenerative changes are precipitated by compromised nerve vascular supply. Experiments in animal models of diabetic neuropathy suggest that similar metabolic sequelae affect neurons and vasa nervorum endothelium. These include elevated polyol pathway activity, oxidative stress, the formation of advanced glycation and lipoxidation end products, and various pro-inflammatory changes such as elevated protein kinase C, nuclear factor κB and p38 mitogen activated protein kinase signalling. These mechanisms do not work in isolation but strongly interact in a mutually facilitatory fashion. Nitrosative stress and the induction of the enzyme poly (ADP-ribose) polymerase form one important link between physiological stressors such as reactive oxygen species and the pro-inflammatory mechanisms. Recently, evidence points to endoplasmic stress and the unfolded protein response as forming another crucial link. This review focuses on the aetiopathogenesis of neurovascular changes in diabetic neuropathy, elucidated in animal studies, and on putative therapeutic targets the majority of which have yet to be tested for efficacy in clinical trials.

INTRODUCTION:The vasa nervorum comprises a network of small diameter blood vessels that provide blood supply to nerves and ganglia. The cell bodies of autonomic nerves innervating the urogenital organs are housed in the major pelvic ganglia (MPG) in rats. The vasa nervorum of rat MPG have not been characterized previously, and it is not known whether these blood vessels are innervated by neuronal nitric oxide synthase (nNOS) containing nitrergic nerves.AIM:To characterize the blood vessels in and around the rat MPG and to assess their nitrergic innervation.MAIN OUTCOME MEASURES:Characterization of small blood vessels in and around the rat MPG and expression of nNOS in nerve fibers around those blood vessels.METHODS:MPG were obtained from healthy Sprague Dawley rats, fixed in paraformaldehyde, frozen and sectioned using a cryostat. The blood vessels and their nitrergic innervation were assessed with immunohistochemistry using antibodies against alpha-smooth muscle actin (smooth muscle marker), CD31 (endothelial marker), collagen IV (basal membrane marker) and nNOS. The immunofluorescence was imaged using a laser scanning confocal microscope.RESULTS:The neuronal cell bodies were contained within a capsule in the MPG. Blood vessels were observed within the capsule of the MPG as well as outside the capsule. The blood vessels inside the capsule were CD31-positive capillaries with no smooth muscle staining. Outside the capsule capillaries, arterioles and venules were observed. The extra-capsular arterioles and venules, but not the capillaries were innervated by nNOS-positive nerve fibers.CONCLUSIONS:This study, to our knowledge, is the first to demonstrate the blood vessel distribution pattern and their nitrergic innervation in the rat MPG. While similar studies in human pelvic plexus are warranted, these results suggest that the blood flow in the MPG may be regulated by nitrergic nerve fibers and reveal a reciprocal relationship between nerves and blood vessels.

Erectile dysfunction (ED) due to diabetes mellitus remains difficult to treat medically despite advances in pharmacotherapeutic approaches in the field. This unmet need has resulted in a recent re-focus on the pathophysiology, in order to understand the cellular and molecular mechanisms leading to ED in diabetes. Diabetes-induced ED is often resistant to PDE5 inhibitor treatment, thus there is a need to discover targets that may lead to novel approaches for a successful treatment. The aim of this brief review is to update the reader in some of the latest development on that front, with a particular focus on the role of impaired neuronal blood flow and the formation of advanced glycation endproducts.

We investigated the phenotype of cells involved in leukostasis in the early stages of streptozotocin-induced diabetes in mice by direct observation and by adoptive transfer of calcein-AM-labeled bone marrow-derived leukocytes from syngeneic mice. Retinal whole mounts, confocal microscopy, and flow cytometry ex vivo and scanning laser ophthalmoscopy in vivo were used. Leukostasis in vivo and ex vivo in retinal capillaries was increased after 2 weeks of diabetes (Hb A(1c), 14.2 ± 1.2) when either donor or recipient mice were diabetic. Maximum leukostasis occurred when both donor and recipient were diabetic. CD11b(+), but not Gr1(+), cells were preferentially entrapped in retinal vessels (fivefold increase compared with nondiabetic mice). In diabetic mice, circulating CD11b(+) cells expressed high levels of CCR5 (P = 0.04), whereas spleen (P = 0.0001) and retinal (P = 0.05) cells expressed increased levels of the fractalkine chemokine receptor. Rosuvastatin treatment prevented leukostasis when both recipient and donor were treated but not when donor mice only were treated. This effect was blocked by treatment with mevalonate. We conclude that leukostasis in early diabetic retinopathy involves activated CCR5(+)CD11b(+) myeloid cells (presumed monocytes). However, leukostasis also requires diabetes-induced changes in the endothelium, because statin therapy prevented leukostasis only when recipient mice were treated. The up-regulation of the HMG-CoA reductase pathway in the endothelium is the major metabolic dysregulation promoting leukostasis.

Introduction.Activation of the DNA repair enzyme, poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP), in response to hyperglycemia-driven oxidative/nitrosative stress, may be an important mechanism in the development of vascular and neural complications in diabetes mellitus. However, a role for PARP in diabetic erectile dysfunction (ED) has not been demonstrated.Aim.To assess whether treatment with a novel PARP-1 inhibitor, GPI 15427, could improve neurovascular dysfunction in corpus cavernosum (CC) from diabetic mice.Methods.Diabetes was induced by streptozotocin in male MF1 mice; duration was 6 weeks. Intervention GPI 15427 treatment (20 mg/kg/day intraperitoneal [i.p.]) was given for 2 weeks following 4 weeks of untreated diabetes. CC strips were mounted in aerated organ baths for measurement of pharmacological or electrical stimulation-evoked changes in smooth muscle tension.Main Outcome Measures.Contractile responses to noradrenergic stimulation and to pharmacological agents stimulating endothelium-dependent and -independent relaxation, and nerve-mediated relaxations against a background precontraction.Results.Contractions in response to phenylephrine or activation of noradrenergic nerves were not significantly altered by diabetes. In contrast, maximum nitrergic nerve-mediated relaxation of phenylephrine-precontracted CC was approximately 28% reduced by diabetes: GPI 15427 treatment completely corrected this diabetic deficit. Similarly, maximal nitric oxide (NO)-mediated endothelium-dependent and -independent relaxations to acetylcholine and sodium nitroprusside, against phenylephrine precontraction, were attenuated approximately 37% and 23% by diabetes, respectively. These deficits were completely reversed by PARP-1 inhibition. Furthermore, GPI 15427 corrected a modest diabetic deficit in sensitivity to nitroprusside (EC50 reduced by 0.14 log units); a similar trend was observed for acetylcholine-induced relaxation.Conclusions.GPI 15427 treatment provides marked benefits for NO-dependent neurovascular function in diabetic mouse CC. Therefore, PARP-1 inhibition may be worthy of further investigation for diabetes-associated ED. Nangle MR, Cotter MA, and Cameron NE. Poly(ADP-ribose) polymerase inhibition reverses nitrergic neurovascular dysfunctions in penile erectile tissue from streptozotocin-diabetic mice. J Sex Med 2010;7:3396-3403.

Erythropoiesis-stimulating agents (ESAs) are used to treat anemia associated with renal failure. It is now known that these agents also show a broad range of cell- and tissue-protective effects. In the current study, we explored whether an ESA, epoetin delta, affects vascular pathology linked to diabetes mellitus (DM). In a rat model of streptozotocin-induced DM, we investigated, by pre-embedding electron-immunocytochemistry, whether epoetin delta affects DM-induced structural changes in cerebrovascular endothelium of the rat basilar artery and influences the subcellular distribution of endothelial nitric oxide synthase (eNOS). Epoetin delta treatment influenced DM-induced changes to the distribution of eNOS in, and the structure of, the endothelial cell. This may indicate potential beneficial effects of epoetin delta on cerebrovascular endothelium and suggests eNOS as a possible target molecule of epoetin delta in DM.

In diabetes mellitus (DM) reduced motor and sensory properties of peripheral nerves are linked with the dysfunction of neural vasculature. We investigated C-fibers and microvessels of sciatic nerve of normal, DM, and DM + epoetin delta-treated rats. C-fibers immunoreactive for calcitonin gene-related peptide (CGRP), tyrosine hydroxylase (TH), epoetin receptor (EpoR), and common β receptor subunit of the interleukin 3 receptor (IL-3Rβ) were present in all rats, whereas in DM and epoetin-treated rats C-fibers also showed neuronal (nNOS) and inducible (iNOS) nitric oxide synthases. The cross-sectional area of CGRPpositive C-fibers was decreased in DM, but it recovered after epoetin treatment. In all conditions, vascular endothelium showed scarce immunolabeling for endothelial nitric oxide synthase (eNOS); the profound immunoreactivity for eNOS, EpoR, and IL-3Rβ was in pericytes. Some perivascular autonomic nerves were damaged and IL-3Rβ positive. Findings are discussed in terms of declined sensory conduction velocity in DM, its improvement after epoetin treatment, and the possible vascular contribution to these phenomena.

Aim: Interleukin‐6 (IL‐6), a member of the neuropoietic cytokine family, participates in neural development and has neurotrophic activity. Recent research has also indicated actions to improve vasa nervorum function in diabetes. Both these facets are potentially relevant for treatment of diabetic neuropathy. The aim of this study was to determine whether IL‐6 treatment corrected changes in neurovascular function in streptozotocin‐induced diabetic rats. Methods: After 1 month of diabetes, rats were given IL‐6 for 1 month. The rats were subjected to sensory testing and measurements of nerve conduction velocities and nerve blood flow by hydrogen clearance microelectrode polarography. Further groups were used to study responses of the isolated gastric fundus and renal artery. Results were statistically analysed using ANOVA and post hoc tests. Results: Diabetic rats showed mechanical hyperalgesia, thermal hyperalgesia, and tactile allodynia. The former was unaffected by IL‐6 treatment, whereas the latter two measures were corrected. Immunohistochemical staining of dorsal root ganglia for IL‐6 did not reveal any changes with diabetes or treatment. The results showed that 22 and 17.4% slowing of sciatic motor and saphenous sensory nerve conduction velocities, respectively, with diabetes were improved by IL‐6. Sciatic endoneurial perfusion was halved by diabetes and corrected by IL‐6. A 40.6% diabetic deficit in maximal non‐adrenergic, non‐cholinergic relaxation of gastric fundus to nerve stimulation was unaffected by IL‐6. Renal artery endothelium‐dependent relaxation was halved by diabetes, the endothelium‐derived hyperpolarizing factor (EDHF) component being severely attenuated. IL‐6 did not affect nitric oxide‐mediated vasorelaxation, but markedly improved EDHF responses. Conclusions: IL‐6 improved aspects of small and large nerve fibre and vascular endothelium dysfunction in diabetic rats. The functional benefits related to increased nerve blood flow via an EDHF mechanism, and IL‐6 could have therapeutic potential in diabetic neuropathy and vasculopathy, which should be further evaluated.

Erectile dysfunction is an early manifestation of autonomic neuropathy in diabetes mellitus, and it also acts as an early marker of vascular disease. The nitric oxide system that controls erection, both neurally and via the endothelium, is particularly vulnerable to diabetes [1]. In addition to haematopoietic functions, erythropoietin has neuroprotective/neurotrophic actions. Erythropoietin receptors are found in the innervation and sinusoidal endothelium of corpus cavernosum [2]. Treatment with erythropoietin accelerated the recovery of erectile function following cavernous nerve injury in rats [3]. Impaired vascular perfusion could contribute to diabetic autonomic neuropathy [1]. Blood flow to the major pelvic ganglion, (MPG) which houses the cell bodies of nerves innervating the penis, has never been investigated in diabetes or any other disease state. The aims were to assess the effects of erythropoietin-delta treatment on erectile function and MPG blood flow in the streptozotocin-induced diabetic rat model.

Neuropathy is a common complication of diabetes mellitus, which reduces the quality of life and may be life-threatening. The etiology is complex and multifactorial: hyperglycemia and dyslipidemia give rise to oxidative stress and formation of advanced glycation and lipoxidation end products. These stimulate inflammatory processes, nuclear factor B (NF kappa B) activation being of central importance. Many of the drugs that have been developed for treatment of diabetic complication at least in part work through suppressing either NF kappa B activation itself, or the production of cytokines that stimulate NF kappa B, such as tumor necrosis factor (TNF) alpha. To date there have been few tests of drugs that are specific inhibitors of the NF kappa B/TNF alpha axis. However preliminary results in animal models are encouraging and go some way in establishing the NF kappa B cascade as an important therapeutic target for diabetic vascular complications in general, and neuropathy in particular.