Galanin And Its Receptors System In Rat Diabetic Neuropathy

Diabetic neuropathy is one of the most common complications associated with diabetes. At earlier period, conspicuous clinical symptom is symmetric neurodynia, which usually develops in lower limbs. At advanced stage, it causes ulcer, diabetic foot, and leads to amputation. Pathological sections show demyelinated, atrophic nerve fibers and nerve fibers lost. Unfortunately, no satisfied treatment method is founded because of the lack of knowledge of its pathophysiology.Galanin (Gal) is a highly conserved neuron peptide, which is widespread in central and peripheral nerve system. It has been implicated that Gal take part in a number of important body functions, including nociception, neuroprotction, feeding, endocrine modulation cognition, epilepsy and cancer. The effects of Gal in nociception and neuroprotction are the research hot spot of recent years.Gal mediates its physiological effects via G protein-coupled receptor. Galanin receptor1(GalR1) and galanin receptor2(GalR2) have a broad distribution in body. The two receptors can act through Gi/o receptor subtype, inhibiting adenyl cyclase. Though linking to Gi/o, GalR2mainly shows exciting function via Gq/11to activate phospholipase C (PLC) and protein kinase C (PKC).GalRl and GalR2are extensively expressed in central nervous system and ganglia. Under normal condition, Gal is expressed only at low levels in a small number of small-sized dorsal root ganglion (DRG) neurons and in lamina II neurons in rat and a dramatically up regulation of endogenous Gal expression seems to be a compensatory response after peripheral nerve injury. Gal played a moderate inhibition of spinal nociception in normal, but it change it'expression following peripheral nerve injury and play a critical role in modulation of nociception in neuropathic states both in DRG and spinal dorsal horn (SDH) level. It is well reported that Gal elaborates its anti-pain effect through GalR1. The exact GalR1mediated Gal of antiallodynic action is unknown today. GalR2stimulates phospholipase C (PLC) and protein kinase C (PKC) activity by coupling to Gq/11thus ERK and Akt signaling pathways to take part in the neuron protect and neurite regeneration.Gal, GalR1and GalR2are thought to play an important role in many neuropathies, but whether they are responsible for diabetic neuropathic pain are still unclear. A prior research showed a delayed regeneration of Gal axons in diabetic mice after nerve injury maybe enhance neuropathic pain. A profound understanding of the mechanisms underlying Gal in diabetes is highly important for providing a new viewpoint in diabetic neuropathy and discovery a potential therapy strategy.At present study, Gal, GalR1and GalR2were studied in vivo and vitro to provide a fundamental research in diabetic neuropathy.Part I The effects of galanin on dorsal root ganglion neurons with high glucose treatment in vitroHyperglycemia in patients with diabetes eventually develops some form of diabetic neuropathy. The exposure of neurons to high glucose concentrations is considered a determinant of diabetic neuropathy. It is showed that high glucose causes DRG neurons apoptosis. Gal is not only involved in energy balance, but also has neuroprotective function. However, the effects of Gal on DRG neurons and its receptors with high glucose are not clear. The experiments designed as follows:Neurons were dissociated from embryonic day15(E15) rat DRG and cultured for48hours and randomly divided into5groups.(1) High glucose:add glucose to the neurobasal medium to acquired45mmol/L glucose at end concentration.(2) High glucose+Gal:add glucose and Gal to the neurobasal medium to acquired45mmol/L glucose and1μmol/L Gal at end concentration.(3) Gal:add Gal to the neurobasal medium to acquired1μmol/L Gal at end concentration.(4) Mannitol:add mannitol20mmol/L to create a high osmotic pressure mimicking the high glucose condition.(5) Control:normal culture without interference factors. Measure the intracellular reactive oxygen species (ROS), cell viability. Observe Hoechst33342stain and caspase-3activation to evaluate apoptosis. Analyze Gal, GalR1and GalR2expression with Western blot and Real-time RT-PCR.The results are as follows:(1) High glucose caused a rapid increasing intracellular ROS, decreases of cell viability, activating caspase-3, neuron apoptosis, and upregulation of Gal and downregulation of GalR1.(2) Administration of exogenous Gal could decrease intracellular ROS, increase cell viability, decrease caspase-3activation and neuron apoptosis caused by high glucose. Exogenous Gal decreased the upregulation of Gal caused by high glucose, but could further decrease GalR1expression.(3) Different experimental conditions didn't impact GalR2expression. The results of the present study indicate for the first time that Gal and its receptor system are involved in high glucose-induced DRG neuronal injury. Gal and GalR1, but not GalR2, display plasticity in high glucose and/or exogenous Gal treated DRG neurons. Exogenous Gal can rescue DRG neurons from apoptosis induced by high glucose. The contribution of exogenous Gal on neuroprotection appears to be quite significant. The data strengthen the rationale of the in vivo study. These results provide rationale and experimental evidence for development and further studies of Gal on therapeutic strategy for improving diabetic neuropathy. Part Ⅱ The effects and mechanisms of galanin on diabetic neuropathy in diabetic ratsDiabetic neuropathy can cause hyperalgia and allodynia, but the mechanism is still unclear. DRG is involved in diabetic neuropathy. DRG has been identified as the target tissue in diabetic somatosensory neuropathy. SDH is involved in nociceptive transmission in painful diabetic neuropathy. It has been showed that Gal and its receptors are involved in transmission and regulation of neuropathic pain. This experiment researches the effects of Gal and its receptor on neuropathic pain in streptozotocin (STZ) induced diabetic rats.The experiments designed as follows:Diabetic rats were induced by intraperitoneal injection of STZ (55mg/kg). The expression of Gal, GalRl, and GalR2in DRG and SDH were detected by Western blot and Real-time RT-PCR. The mechanical stimulation test (von Frey filaments test) was done after intrathecal injection of Gal, Gal antagonist M35, selective GalRl agonist M617, or selective GalR2agonist AR-M1896.The results are as follows:(1) Diabetic rats induced by STZ produce a stable mechanical allodynia after four weeks.(2) GalR1expression decreased in the DRG of diabetic rats.(3) GalR2expression decreased in DRG and SDH of diabetic rats.(4) Exogenous Gal could reduce allodynia. M35, a receptor blocker of Gal could block this effect.(5) Intrathecal injection of M617, selective GalR1agonist, could reduce allodynia, while AR-M1896, selective GalR2agonist, could not reduce allodynia.The results of the present study demonstrated that Gal and its receptor signaling system is involved in diabetic neuropathic pain sensitization processing. GalRl, but not GalR2, is involved in diabetic neuropathic pain sensitization or transmission and is one of the potential therapeutic targets on diabetic neuropathic pain. These findings provide rationale and experimental evidence for development and further studies of Gal therapeutic strategy to alleviate diabetic neuropathic pain symptoms.Part Ⅲ The protection effect of galanin in sciatic nerve injury diabetic ratsDiabetic neuropathy lead pain and sciatic nerve damage, regeneration deficits. However, the mechanism causing these neurological diseases is unclear. It has been proved that Gal and its receptors are involved in nerve transmission and regulation of pain in our reseach (part Ⅱ). This research further studied Gal and its receptors in neuropathic pain and nerve regeneration in STZ-induced diabetic rats.The experiments designed as follows:STZ-induced diabetic rats12weeks and the same age normal rats randomly divided into six groups:(1) diabetic pinch+Gal:diabetic rats after sciatic nerve pinched, daily intrathecal injection of3μg of exogenous Gal.(2) diabetic pinch:diabetic rat sciatic nerve pinched.(3) diabetic sham:sham-operated diabetic rats, did not pinch the sciatic nerve.(4) normal pinch+Gal:normal rats after sciatic nerve pinched, daily intrathecal injection of exogenous3μg Gal.(2) Normal pinch:normal rat sciatic nerve pinched.(3) control group:sham-operated normal rats, did not pinch the sciatic nerve. The mechanical and thermal stimulation were tested in all groups. The toe width, nerve conduction velocity (NCV) and morphological regeneration of the sciatic nerve were measured in each group. The expression of Gal, GalRl and GalR2of DRG and SDH were measured in each group.The results are as follows:(1) Pinch injury in diabetic rats sciatic nerve increased neuropathic pain.(2) Exogenous Gal significantly reduced neuropathic pain in diabetic and normal sciatic nerve pinch rat.(3) Exogenous Gal significantly improved toe width, sciatic NCV and nerve regeneration in diabetic and normal sciatic nerve pinch rat.(4) The expression of Gal, GalR1and GalR2showed a great plasticity in DRG and SDH in a variety of situations.Gal and its receptor system take part in the pathophysiological process of diabetic neuropathic pain and nerve regeneration deficits. The expressions of Gal and its receptor1and2are changed after sciatic nerve pinch diabetic rats, which show a certain relationship with pathological processes of diabetic neuropathy. Exogenous Gal can improve diabetes-induced neuropathic pain, NCV and nerve regeneration deficits. These findings provide rationale and experimental evidence for development and further studies of Gal therapeutic strategy to alleviate diabetic neuropathy.