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Despite intensive effort and resulting gains in understanding the mechanisms underlying neuropathic pain, limited success in therapeutic approaches have been attained. A recently identified, nonchannel, nonneurotransmitter therapeutic target for pain is the enzyme soluble epoxide hydrolase (sEH). The sEH degrades natural analgesic lipid mediators, epoxy fatty acids (EpFAs), therefore its inhibition stabilizes these bioactive mediators. Here we demonstrate the effects of EpFAs on diabetes induced neuropathic pain and define a previously unknown mechanism of pain, regulated by endoplasmic reticulum (ER) stress. The activation of ER stress is first quantified in the peripheral nervous system of type I diabetic rats. We demonstrate that both pain and markers of ER stress are reversed by a chemical chaperone. Next, we identify the EpFAs as upstream modulators of ER stress pathways. Chemical inducers of ER stress invariably lead to pain behavior that is reversed by a chemical chaperone and an inhibitor of sEH. The rapid occurrence of pain behavior with inducers, equally rapid reversal by blockers and natural incidence of ER stress in diabetic peripheral nervous system (PNS) argue for a major role of the ER stress pathways in regulating the excitability of the nociceptive system. Understanding the role of ER stress in generation and maintenance of pain opens routes to exploit this system for therapeutic purposes. Here we define the causative role of endoplasmic reticulum (ER) stress on selective modulation of pain signaling. High levels of ER stress and neuropathic pain in diabetic animals are reduced using ER stress blockers. In healthy animals, turning on the ER stress signal transduction cascade generates an immediate but lasting and site restricted painful phenotype, which is reversible by ER stress blockers. This previously unnoticed mechanism explains the broad lack of efficacy of available analgesics and should ignite the discovery of a new generation of therapeutics that do not directly quell ion channel or neurotransmitter activity.

Diabetic patients and streptozotocin (STZ)-induced diabetes mellitus (DM) models exhibit signals of brain dysfunction, evidenced by neuronal damage and memory impairment. Astrocytes surrounding capillaries and synapses modulate many brain activities that are connected to neuronal function, such as nutrient flux and glutamatergic neurotransmission. As such, cognitive changes observed in diabetic patients and experimental models could be related to astroglial alterations. Herein, we investigate specific astrocyte changes in the rat hippocampus in a model of DM induced by STZ, particularly looking at glial fibrillary acidic protein (GFAP), S100B protein and glutamate uptake, as well as the content of advanced glycated end products (AGEs) in serum and cerebrospinal fluid (CSF), as a consequence of elevated hyperglycemia and the content of receptor for AGEs in the hippocampus. We found clear peripheral alterations, including hyperglycemia, low levels of proinsulin C-peptide, elevated levels of AGEs in serum and CSF, as well as an increase in RAGE in hippocampal tissue. We found specific astroglial abnormalities in this brain region, such as reduced S100B content, reduced glutamate uptake and increased S100B secretion, which were not accompanied by changes in GFAP. We also observed an increase in the glucose transporter, GLUT-1. All these changes may result from RAGE-induced inflammation; these astroglial alterations together with the reduced content of GluN1, a subunit of the NMDA receptor, in the hippocampus may be associated with the impairment of glutamatergic communication in diabetic rats. These findings contribute to understanding the cognitive deficits in diabetic patients and experimental models.

Diabetes mellitus (DM) is a metabolic disorder associated with micro- and macrovascular alterations that contribute to the cognitive impairment observed in diabetic patients. Signs of breakdown of the blood–brain barrier (BBB) and the blood–cerebrospinal fluid barrier (BCSFB) have been found in patients and animal models of DM. Breakdown of the BBB and BCSFB can lead to disruptions in cerebral homeostasis and eventually neural dysfunction and degeneration. However, our understanding of the biochemistry underlying barrier protein modifications is incomplete. Herein, we evaluated changes in the levels of specific proteins in the BBB (occludin, claudin-5, ZO-1, and aquaporin-4) and BCSFB (claudin-2 and aquaporin-1) in the hippocampus of diabetic rats, and we also investigated the functional alterations in these barriers. In addition, we evaluated the ability of exendin-4 (EX-4), a glucagon-like peptide-1 agonist that can cross the BBB to reverse the functional and biochemical modifications observed in these animals. We observed a decrease in BBB proteins (except ZO-1) in diabetic rats, whereas the EX-4 treatment recovered the occludin and aquaporin-4 levels. Similarly, we observed a decrease in BCSFB proteins in diabetic rats, whereas EX-4 reversed such changes. EX-4 also reversed alterations in the permeability of the BBB and BCSFB in diabetic rats. Additionally, altered cognitive parameters in diabetic rats were improved by EX-4. These data further our understanding of the alterations in the central nervous system caused by DM, particularly changes in the proteins and permeability of the brain barriers, as well as cognitive dysfunction. Furthermore, these data suggest a role for EX-4 in therapeutic strategies for cognitive dysfunction in DM.

Background Diabetes accelerates memory dysfunction in a continuous, slowly pathological process. Studies suggest that the time course of certain biomarkers can characterize the pathological course of the disease to provide information for early intervention. Thus, there is an urgent need for validated biomarkers to characterize the cognitive impairment induced by DM. We aimed to detect changes in cerebrospinal fluid biomarkers such as amyloid β42, phosphorylated tau protein, interleukin 6, and acetylcholine in diabetic rats over time, and to analyse the relationship between diabetes and cognitive impairment. Methods Rats were injected once intraperitoneally with 1% of streptozotocin to establish a diabetic model. Index changes were investigated longitudinally and all were measured at the end of the experiment at day 75. Aβ42, P-tau, IL-6, and ACh levels in CSF, insulin levels in plasma, and Aβ42 levels in plasma and brain tissue were measured by ELISA. Results Compared with control, the diabetic model showed ACh in CSF to be decreased by day 15, continuing lower out to day 75. Aβ42 changes in brain and blood showed the same trends but exhibited minima at different time points: day 30 in CSF and day 15 in plasma. After the minimum, Aβ42 in cerebrospinal fluid rose and levelled off lower than in the control group, whereas Aβ42 in plasma rose and went above the controls at day 30, slowly trending upwards for the remainder of the experiment. P-tau protein in CSF in diabetic rats showed an increasing trend, becoming significantly different from the controls at day 60 and day 75. Aβ42 in CSF was strongly negatively correlated with blood glucose at day 15 and was negatively correlated with insulin in serum, particularly at day 45. Conclusion Our longitudinal research model suggest that changes in the measured biomarkers appear before learning and memory impairments do. Aβ42 and ACh in the diabetes model group clearly changed from day 0 to day 45, and then P-tau and IL-6 varied significantly from day 45 to day 75. The reduced ACh levels observed possibly correlated with the factors common to changes in Aβ42, P-tau, and IL-6.

Differential Effects of Diabetes on Rat Choroid Plexus Ion Transporter Expression Richard D. Egleton , Christopher C. Campos , Jason D. Huber , Rachel C. Brown and Thomas P. Davis From the Department of Pharmacology, College of Medicine, The University of Arizona, Tucson, Arizona Abstract Though diabetes is a disease with vascular complications, little is known about its effects on the blood-brain barrier or the blood-cerebrospinal fluid barrier (BCSFB). The BCSFB is situated at choroid plexuses located in the lateral, third, and fourth ventricles. Choroid plexuses are the primary site of cerebrospinal fluid (CSF) production and express numerous ion transporters. Previous studies have shown a perturbation of ion transport in the periphery and brain during diabetes. In this study, we investigated the effect of diabetes on ion transporters in the choroid plexuses of streptozotocin (STZ)-induced diabetic rats. Diabetes was induced in male Sprague-Dawley rats by intraperitoneal injection of STZ (60 mg/kg in citrate buffer, confirmed by glucose analysis: 601 ± 22 mg/dl diabetic rats, 181 ± 46 mg/dl age-matched controls); and at 28 days, rats were killed, choroid plexuses harvested, and protein extracted. Western blot analyses were carried out using antibodies for ion transporters, including Na + -K + -2Cl − cotransporter and the Na + -K + -ATPase α1-subunit. The efflux of the K + analog 86 Rb + from choroid plexus was also studied. Diabetic rats showed an increase in expression of the Na + -K + -2Cl − cotransporter and the Na + -K + -ATPase α1-subunit, as compared with age-matched controls, a decrease in Na + -H + exchanger expression, and no change in Na + -K + -ATPase β1- or β2-subunit. The net effect of these changes was a 66% increase in 86 Rb + efflux from diabetic choroid plexus compared with controls. These changes in expression may affect choroid plexus ion balance and thus significantly affect CSF production in diabetic rats. Footnotes Address correspondence and reprint requests to Dr. Thomas P. Davis, Department of Pharmacology, P.O. Box 245050, The University of Arizona, Tucson, AZ 85724. E-mail: davistp{at}u.arizona.edu . Received for publication 14 July 2002 and accepted in revised form 24 February 2003. aCSF, artificial cerebrospinal fluid; BBB, blood-brain barrier; BCSFB, blood-cerebrospinal fluid barrier; CSF, cerebrospinal fluid; PKC, protein kinase C; STZ, streptozotocin. DIABETES

Diabetes mellitus (DM) patients experience memory and cognitive deficits. The mechanisms underlying this dysfunction in the brain of DM patients are not fully understood, and therefore, no optimized therapeutic strategy has been established so far. The aim of the present study was to assess whether irisin was able to improve memory and cognitive performance in a streptozotocin-induced diabetic mouse model. A diabetic mouse model was established and behavioral tests were performed. We also set up primary cultures for mechanism studies. Western blots and EMSA were used for molecular studies. Significant impairment of cognition and memory was observed in these DM mice, which could be effectively prevented by irisin cotreatment. We also found upregulated levels of GFAP protein, reduced synaptic protein expression, and increased levels of interleukin-1β (IL-1β) and interleukin-6 (IL-6) in the brains; however, irisin significantly attenuated these cellular responses. Meanwhile, our results demonstrated that irisin inhibited the activation of P38, STAT3, and NFκB proteins of DM mice. Furthermore, our results suggested that irisin might regulate the function of P38, STAT3, and NFκB in hippocampal tissues of DM mice. Collectively, irisin inhibited neuroinflammation in STZ-induced DM mice by inhibiting cytokine release and improving their cognitive function. Our findings revealed the mechanism of irisin’s anti-inflammatory effect in the CNS.

Microglia, the resident phagocytes of the retina, are believed to influence the development of retinopathy, but their exact contributions to vascular integrity and neuronal loss are unknown. Therefore, utilizing two models of microglia depletion, we aimed to deplete and repopulate microglia to clarify the contribution of microglia to neuronal loss and vascular damage in the diabetic retina in an STZ-induced model of hyperglycemia. Here, we report that 2 weeks exposure to diphtheria toxin (DTx) in diabetic CX3CR1 CreER :R26 iDTR transgenic mice induced a 62% increase in Iba1 + microglia associated with an increase in TUJ1 + axonal density and prevention of NeuN + RBPMS + neuronal loss. Conversely, diabetic PBS controls exhibited robust TUJ1 + axonal and NeuN + RBPMS + neuronal loss compared to non-diabetic controls. A 2-week recovery period from DTx was associated with a 40% reduction in angiogenesis and an 85% reduction in fibrinogen deposition into the diabetic retina in comparison to diabetic PBS-treated controls. Analysis of microglia morphology and marker expression revealed that following a 2-week recovery period microglia displayed a P2RY12 + Ly6C – phenotype and high transformation index (TI) values complimented by a ramified-surveillant morphology closely resembling non-diabetic controls. In contrast, diabetic PBS-treated control mice displayed P2RY12 + Ly6C + microglia, with a 50% reduction in TI values with an amoeboid morphology. To validate these observations were due to microglia depletion, we used PLX-5622 to assess vascular and neuronal damage in the retinas of diabetic mice. Confocal microscopy revealed that PLX-5622 also induced an increase in TUJ1 + axonal density and prevented fibrinogen extravasation into the diabetic retina. mRNAseq gene expression analysis in retinal isolates revealed that PLX-5622-induced microglia depletion and repopulation induced a downregulation in genes associated with microglial activation and phagocytosis, B2m , Cx3cr1, and Trem2 , and complement-associated synaptic pruning, C1qa , C1qb , and C1qc . Although the levels of microglia depletion induced with DTx in the CX3CR1 CreER :R26 iDTR model and those induced with the CSF-1R antagonists are distinct, our results suggest that microglia depletion and replenishment is neuroprotective by inducing the proliferation of a homeostatic microglia pool that supports neuronal and vascular integrity.

Background: This study investigated the effects of Laurocerasus officinalis Roem (cherry laurel; CL), a traditionally consumed fruit, on cognitive performance and selected neurobiochemical and metabolic pathways in a nontransgenic streptozotocin (STZ)-induced Alzheimer’s disease (i.c.v. STZ) model and an STZ-induced type 2 diabetes mellitus (T2DM; i.p. STZ) model. Method: Fifty-seven adult male Sprague–Dawley rats were allocated to control, T2DM, and Alzheimer (ALZ) model groups, with subgroup interventions including CL supplementation and, in the T2DM model, metformin as a comparator. Spatial learning and memory were assessed using the Morris Water Maze. Serum and brain tissue levels of GSK3-β, glutathione (GSH), interleukin-1 (IL-1), GLUT4, GLP-1, β-amyloid (Aβ), and acetylcholinesterase (AChE) were quantified. Results: Serum GSK3-β levels did not differ significantly between groups, whereas brain tissue GSK3-β showed significant between-group differences. CL increased GSH levels in both models, with significant elevations in serum and brain tissue GSH in the ALZ model following CL administration; in the T2DM model, GSH increased after both CL and metformin. In the ALZ model, CL was associated with decreased serum Aβ and AChE levels and improved Morris Water Maze performance, reflected by reduced escape latencies. Conclusions: CL supplementation was associated with antioxidant enhancement and modulation of amyloid- and cholinergic-related measures, alongside improved spatial learning performance in the STZ-induced nontransgenic ALZ model. In addition, CL reduced blood glucose in the T2DM model. Given the likely contribution of fruit phytochemicals (including total phenolics), further studies are warranted to better define the bioactive composition and mechanisms underlying these effects.

BackgroundImpaired insulin signaling pathway in the brain in type 2 diabetes (T2D) is a risk factor for Alzheimer disease (AD). Glucagon-like peptide-1 (GLP-1) and its receptor agonist are widely used for treatment of T2D. Here we studied whether the effects of exendin-4 (EX-4), a long-lasting GLP-1 receptor agonist, could reduce the risk of AD in T2D.Materials and MethodsType 2 diabetes rats were injected with EX-4 for 28 consecutive days. Blood glucose and insulin levels, as well as GLP-1 and insulin in cerebrospinal fluid, were determined during the experiment. The phosphorylation level of tau at individual phosphorylation sites, the activities of phosphatidylinositol 3 kinase/protein kinase B (PI3K/AKT), and glycogen synthase kinase-3β (GSK-3β) were analyzed with Western blots.ResultsThe levels of phosphorylated tau protein at site Ser199/202 and Thr217 level in the hippocampus of T2D rats were found to be raised notably and evidently decreased after EX-4 intervention. In addition, brain insulin signaling pathway was ameliorated after EX-4 treatment, and this result was reflected by a decreased activity of PI3K/AKT and an increased activity of GSK-3β in the hippocampus of T2D rats as well as a rise in PI3K/AKT activity and a decline in GSK-3β activity after 4 weeks intervention of EX-4.ConclusionsThese results demonstrate that multiple days with EX-4 appears to prevent the hyperphosphorylation of AD-associated tau protein due to increased insulin signaling pathway in the brain. These findings support the potential use of GLP-1 for the prevention and treatment of AD in individuals with T2D.

Painful Diabetic Neuropathy (PDN) is a severe complication of diabetes, featured by intricate aetiology and multiple side effects of current therapeutic approaches. In recent years, the glymphatic system has attracted increasing attention for its role in PDN. This study investigated the regulatory effects and underlying mechanisms of Oxymatrine (OMT) on the spinal glymphatic system in PDN rat models, aiming to provide novel therapeutic insights for PDN. The PDN rat model was established by high-fat and high-sugar diet combined with streptozotocin (STZ) induction. The 50% paw withdrawal threshold (50% PWT) was measured by Von Frey filaments to evaluate neuropathic pain. Spinal glymphatic system function was observed via Magnetic Resonance Imaging (MRI). Western blotting was used to detect the expression of Aquaporin-4 (AQP-4), Metalloproteinase-9 (MMP-9), NF-κB p65, p-p65, Nrf2 and HO-1. Immunofluorescence was performed to assess AQP4 polarization and nuclear expression of p65. In addition, the levels of oxidative stress indicators (GSH, SOD, MDA) and inflammatory factors (IL-1β, IL-6, TNF-α) were determined. OMT treatment significantly alleviated PDN-related symptoms and improved the detected indicators. It effectively reduced oxidative stress and inflammatory levels, upregulated the expression of Nrf2 and HO-1, downregulated MMP-9 expression, repaired AQP-4 polarisation, and restored the function of the spinal glymphatic system in PDN rats. This study provides a theoretical foundation for the potential application of OMT as a therapeutic agent for PDN, and its multi-target regulatory mechanism offers new directions for PDN treatment.