Role Of Fyn-mediated NMDA Receptor Function In Prediabetic Neuropathy In Mice

Diabetes mellitus(DM)is a metabolic syndrome characterized by hyperglycemia caused by the interaction of genetic and environmental factors.With the aging of the population and the change of people’s life style,the incidence of diabetes in China increases year by year,which brings heavy burden to families and society.One of most common complications of diabetes mellitus is diabetic neuropathy,which affects nerve fibers of the peripheral nervous system.Diabetic neuropathy occurs in more than 50%of adult diabetic patients.Patients with diabetic neuropathy often experience hyperalgesia or allodynia,which are accompanied by excessive sensitivity to nociceptive stimuli,or may perceive normal stimuli as painful stimuli.However,the current neuropathological mechanisms of diabetic neuropathy have not yet been fully elucidated.It is still a challenge to cure diabetic neuropathy.Previous studies have illustrated that the spinal cord dorsal horn is critical in the perception of nociceptive signals in diabetic neuropathy.It has been proposed that central sensitization can occur in the dorsal horn of the spinal cord after inflammation or nerve injury,which may result in increased primary afferents,neuroplasticity in the spinal cord,and hyperactivity of dorsal horn neurons in the spinal cord in diabetic neuropathic pain.One of the putative mechanisms underlying the hyperactivity of dorsal horn neurons in the spinal cord may involve N-methyl-D-aspartate(NMDA)receptors.In support of this,administration of NMDA induces a greater increase in calcium influx in spinal lamina Ⅱ neurons in nerve-ligated rats relative to control rats.Consistent with this,NMDAR antagonism remarkably reduces evoked responses of dorsal horn neurons in spinal nerve-ligated rats.Such an effect might involve NR2B subunit-containing NMDARs,since extensive evidence has shown that spinal NR2B subunit-containing NMDARs play a critical role in nociceptive processing and pathological pain.Most animal studies of diabetic neuropathy have been carried out in diabetic rodents prepared by injections of streptozotocin(STZ),a pancreatic-cell cytotoxin.These rodents develop a syndrome that resembles type 1 diabetes.With this model of diabetes,it has been shown that STZ-induced diabetic rats exhibit mechanical allodynia and hyperalgesia.In addition,type 1 diabetic neuropathy is associated with increased glutamate release from primary afferent terminals in the spinal cord.Furthermore,the tactile allodynia in STZ-induced diabetic rats is associated with increased expression of NR2B subunits of NMDARs in the spinal cord.However,it remains unclear whether NMDARs are involved in diabetic neuropathy in type 2 diabetic animals.Given that type 2 diabetes accounts for 90-95%of all diagnosed cases of diabetes in adult humans and animal studies have demonstrated diabetic neuropathy in obesity-associated prediabetic animals,it will be important to examine the role of NMDARs in prediabetic neuropathy.NMDA receptor function is critically regulated by Src-family tyrosine kinases.In the spinal cord,NR2A and NR2B are the predominant subunits.NR2B subunits are highly expressed in the superficial dorsal horn of the spinal cord.Importantly,both NR2A and NR2B subunits can be tyrosine phosphorylated in the central nervous system.A previous study has demonstrated that NR2B-containing NMDARs in the spinal cord are responsible for the maintenance of chemically induced neuropathic pain and that NR2B phosphorylation at Tyr1472 by Fyn kinase is essential for the maintenance of neuropathic pain.However,few studies have been conducted to examine the role of Fyn kinase in prediabetic neuropathy.Therefore,the present study was designed to evaluate the role of Fyn kinase and NMDARs in the spinal cord in prediabetic neuropathy with an animal model of high-fat diet-induced prediabetes.PART I ROLE OF NR2B SUBUNIT OF NMDARs FUNCTION IN PREDIABETIC NEUROPATHY IN MICEObjectives:To evaluate the role of NR2B subunit of NMDARs function in prediabetic neuropathy in mice.Methods:1.Mice experiment group:Adult male C57BL6/SV129 mice were randomly divided into normal control group and diabetic group,40 in each group.Animal model of high-fat diet-induced prediabetes:The diabetic mice were fed with high-fat diet and the control mice were fed with normal mouse chow,both of which were kept for 16 weeks or 24 weeks.Body weight and blood glucose data for both groups were collected during the first week after the end of the 16 and 24 weeks of feeding,respectively.2.Behavioral tests:All behavioral testing was conducted at 16 wk or 24 wk on normal diet or high-fat diet treatment in wild-type mice or Fyn-/-mice.Each mouse received two behavioral tests.To test mouse tactile response,a von Frey assay was conducted.Mice were placed in a clear plastic cage on top of a wire mesh grid that allowed access to their hind paws for the duration of the analysis.Mechanical withdrawal thresholds with von Frey monofilaments were measured to track the progression of neuropathy and evaluate the effects of acute intrathecal administration of Ro 25-6981 on neuropathy,using the up-down method to determine 50%withdrawal thresholds.The next day after Von Frey assay,a hot plate test was also performed to track progression of thermal sensitivity.3.Detection of the expression of NR2B subunit in each group:Spinal cord tissue of mice after 16 weeks or 24 weeks of diet was collected,and the expression of NR2B subunit in the spinal cord was analyzed by Western blotting assay and the phosphorylation of NR2B subunit at Tyr1472.The discussion of NR2B activation below refers to normalized pNR2B/total NR2B levels.4.Behavioral tests after inhibition of NR2B subunit:Intrathecal administrations of vehicle(i.e.,1%DMSO in phosphate-buffered saline)or Ro 25-6981,a selective NR2B subunit-containing NMDAR antagonist,were conducted through the intervertebral space in unanesthetized mice between L5 and L6 of the spinal cord Five minutes after the injections,mice behavioral tests were conducted.Results:1.Effect of high-fat diet treatment on body weight,blood glucose level and food intake in mice:After the first high-fat diet for the first week,the mice showed a slightly increased body weight and food intake compared to the normal-diet mice,But blood glucose levels did not increase.After 16 or 24 weeks of high-fat diet,body weight,food intake and blood glucose levels in mice relative to the normal diet control mice were significantly increased.2.Effect of a high-fat diet on tactile allodynia and thermal hyperalgesia in mice:High-fat diets at 16 and 24 weeks resulted in tactile allodynia and thermal hyperalgesia in mice compared to the normal diet control mice.In addition,tactile allodynia and thermal analgesia were more severe in mice fed a high-fat diet for 24 weeks compared to a 16-week high-fat diet.3.Effect of high-fat diet on expression of NR2B subunit in mouse spinal cord:Compared with normal diet control mice,NR2B subunit expression in spinal cord of high-fat diet mice increased at 16 and 24 weeks.In addition,the expression of NR2B subunit in spinal cord at high-fat diet at 24 weeks was stronger than that at 16 weeks of high-fat diet.Phosphorylation of the NR2B subunit at Tyr1472 also showed similar changes,ie an increase in NR2B activation(the ratio of p-NR2B to NR2B).4.Intrathecal injection of NR2B NMDA receptor antagonist on the behavior of mice:Intrathecal injection of Ro 25-6981 can alleviate high-fat diet-induced tactile allodynia,but does not affect the thermal pain subsided.When the amount of intrathecal Ro 25-6981 was increased from 5 μg to 20 μg,the paw withdrawal threshold of mice increased significantly.However,any dose of intrathecal injection of Ro 25-6981 did not change the licking latency of mice.Conclusion:The increased expression/function of NR2B subunit-containing NMDARs contribute to the progression of neuropathy in type 2 diabetes.PART II ROLE OF FYN-MEDIATED NR2B SIGNALING IN PREDIABETIC NEUROPATHY IN MICEObjectives:To evaluate the role of Fyn-mediated NR2B signaling in prediabetic neuropathy in mice.Methods:1.Mice experiment group:Adult male Fyn wild-type C57BL6/SV129 mice and Fyn knockout(Fyn-/-)mice(C57BL6/SV129 background),40 in each.Normal male wild-type and Fyn-/-mice were fed high-fat diet for 16 or 24 weeks.Body weight and blood glucose data were collected during the first week after 16 and 24 weeks of high fat diet treatment.2.Behavioral tests:All behavioral testing was conducted at 16 wk or 24 wk on normal diet or high-fat diet treatment in wild-type mice or Fyn-/-mice.Each mouse received two behavioral tests.To test mouse tactile response,a von Frey assay was conducted.Mice were placed in a clear plastic cage on top of a wire mesh grid that allowed access to their hind paws for the duration of the analysis.Mechanical withdrawal thresholds with von Frey monofilaments were measured to track the progression of neuropathy and evaluate the effects of acute intrathecal administration of Ro 25-6981 on neuropathy,using the up-down method to determine 50%withdrawal thresholds.The next day after Von Frey assay,a hot plate test was also performed to track progression of thermal sensitivity and measure the effects of acute intrathecal administration of Ro 25-6981 on thermal sensitivity.3.Detection of the expression of Fyn kinase and NR2B subunit in each group:The spinal cord tissues of mice treated with diet for 16 weeks or 24 weeks were collected and the expression of Fyn kinase,NR2B subunit and NR2B phosphorylation in the spinal cord were analyzed by Western blotting.4.Detection of physical interaction between Fyn kinase and NR2B subunit:Coimmunoprecipitation assays were conducted by immunoprecipitation of the spinal cord samples with mouse monoclonal anti-Fyn antibody followed by probe of the blots with the antibody against NR2B in separate groups of wild-type mice.Results:1.Changes in Body Weight,Blood Glucose,and Food Intake in High-Fat Diet:Fyn-/-mice and wild-type mice showed similar changes in body weight,blood glucose levels and food intake after high-fat diet.After the first high-fat diet,Fyn-/-mice showed slightly increased body weight and food intake compared to the normal diet,but did not increase their blood glucose levels.After 16 or 24 weeks of high-fat diet,Fyn-/-mice showed more pronounced weight gain.In addition,there was a significant increase in food intake in the high fat diet fed group as compared to the normal diet control mice.In addition,blood glucose levels in Fyn-/-mice increased relative to normal diet control mice after a 16 or 24 week high-fat diet.2.Effect of high-fat diet on tactile allodynia and thermal hyperalgesia in mice:A high-fat diet resulted in thermal pain relief in Fyn-/-mice but did not cause tactile allodynia.Compared to normal diet Fyn-/-mice,mice with prediabetic Fyn-/-mice showed reduced thermal hyperalgesia but no tactile allodynia 16 and 24 weeks after treatment with a high-fat diet.In addition,the thermal pain relief in mice was more pronounced at 24 weeks in the high-fat diet compared to 16 weeks in the high-fat diet.3.Effect of high-fat diet on Fyn kinase expression in the spinal cord of mice:High-fat diet failed to change the expression of Fyn kinase in the spinal cord of wild-type mice.However,the high-fat diet promoted the interaction of Fyn kinase and NR2B subunits in the spinal cord of wild-type mice compared to the normal diet-fed mice,and this interaction was greater in the 24-week high-fat diet than at 16 weeks Enhanced.4.Effect of high-fat diet on expression of NR2B subunit in mouse spinal cord:High-fat diets at 16 and 24 weeks increased expression of NR2B subunit in spinal cord of Fyn-/-mice compared with normal diet control mice,But the expression of NR2B subunit increased more at 24 weeks.However,the high-fat diet did not alter the level of phosphorylation of Tyr1472 at NR2B in the spinal cord of Fyn-/-mice.Conclusion:Fyn-mediated NR2B signaling plays a critical role in regulation of prediabetic neuropathy.