The Therapeutic Potential And Mechanisms Of Mitiglinide On Rodent Models Of Ischemic Stroke

Stroke is a common disease of the central nervous system, which seriously harms to human health and life because of its high incidence, mortality and morbidity. A thrombosis, an embolism or systemic hypo-perfusion, all of which lead to a sharp reduction of blood flow to the brain and insufficient oxygen and glucose delivery to support cellular homeostasis, can cause an ischemic stroke and result in focal or global tissue necrosis, softening and neurological deficit ultimately. Ischemic stroke accounts for approximately 85% of all cases. To date, recombinant tissue plasminogen activator (rtPA) is still the only effective drug for acute ischemic stroke which was approved by the United States food and drug administration (FDA). However, the extensive exclusion criteria, together with the short therapeutic window and narrow age range, severely restrict its benefits on promoting reperfusion and improving the long-term clinical outcome. In company with unremitting efforts on researches of the ischemic injury cascade of biochemical reactions in acute phase after stroke, masses of drugs have been proposed as neuroprotective agents. Whereas, all neuroprotective therapies in present have not been established in clinical routine for various reasons. The effective treatment is still in development, which calls for more investigation to the underlying pathological mechanisms and therapeutic strategies of stroke in the future.Mitiglinide (MGN), a aderivative of benzylsuccinic acid, is generally referred to as non-sulfonylurea insulin secretagogues for the treatment of type 2 diabetic patients. MGN promotes insulin release from pancreatic β-cells by specifically binding to sulfonylurea receptors 1 (SUR1) and inhibiting ATP-sensitive potassium (KATp, Kir6.2/SUR1) channels, which results in membrane depolarization and Ca2+ influx through voltage-gated calcium channels. These events lead to an increase of intracellular calcium and subsequent exocytosis of insulin-containing granules.It has been recently reported that MGN prevents postprandial increases in oxidative stress and markers of inflammation in patients with diabetes mellitus. Also, MGN preserves the cardioprotective effect of ischemic preconditioning in isolated perfused rat hearts. Given to Diabetes has been identified to be an independent risk factor for ischemic stroke, and increases the risk by 2 to 5 times, several antidiabetic drugs have been reported to be against stroke. As a Rapid-onset and short-acting insulinotropic agent, MGN is more effective and safer than other insulin secretagogues, and has a lower risk because of selective action on Kir6.2/SUR1, which means the affinity of MGN binding to the KATp channel (Kir6.2/SUR1) on pancreatic β-cells is higher than that of other drugs. Both KATp and NCca2+ -ATP channels are regulated by SUR1,which expressed all cells of the neurovascular unit, and involved in many forms of central nervous system (CNS) injury, including cerebral ischemia, traumatic brain injury (TBI), spinal cord injury (SCI), and subarachnoid hemorrhage (SAH). As a promising target, SUR1 is potently and selectively blocked by MGN.Whether MGN exerts the preventive or protective roles on ischemic stroke is still unclear so far.Based on these findings, we first applied MGN in a model of transient focal cerebral ischemia in mice to investigate its roles on the injury induced by stroke. Furthermore, we undertook preclinical investigations of MGN, providing experimental evidences for the new pharmacological application of MGN.Secondly, we analyzed mechanisms and molecular targets underlying MGN-afforded neuroprotection by using WT, Kir6.1+/- and Kir6.2-/- mice. Finally, we observed the effects of MGN on neurorestorations in tMCAo mice. Our study suggests that MGN is a novel and potent candidate engaging pleiotropic mechanisms in multiple cell types of the neuro vascular unit for further development as a stroke treatment.Part Ⅰ The efficacy evaluation of therapeutic potentials of mitiglinide on acute injuries in rodent models of ischemic strokeAIM:To the investigate effects of MGN on tMCAo mice and to undertake preclinical evaluation of its efficacy with different rodent models of ischemic stroke.METHODS:Ischemia stroke was induced in two to three months old male C57BL/6J mice or Sprague Dawley (SD) rats using the advanced intraluminal transient/permanent middle cerebral artery occlusion (tMCAo/pMCAo) model. Glinides (RGN, NGN, or MGN) was intragastric administered (i.g.) to mice 60 min after the withdrawal of the monofilament. The brain water content and neurological deficits were measured in each group after cerebral ischemia. Infarct volume was measured by TTC staining and morphologic changes were observed by Nissl staining. Fasting blood glucose (FBG) was examined by cutting the tail of mice. Blockade and subsequent restoration of cerebral blood flow was confirmed by laser Doppler and two dimensional laser speckle imaging techniques.RESULTS:1) Cerebral blood flow was continuously assessed by recording laser Doppler flow to ensure appropriate ischemia and reperfusion. Animals that showed CBF reduction of equal to or greater than 80%of baselines were randomly assigned to vehicle, or drugs treatment groups. When given intragastrically 1 h after reperfusion, MGN (0.05～1 mg/kg) yielded significant reduction of infarct volumes, brain water content and neurological deficits, although no decreases in FBG among these dosages was observed.2) Differences with respect to brain injury by both NGN and RGN was observed in terms of a marked reduction of brain infarct, edema, and neurological deficits, confirming the neuroprotective effects of glinides at low doses without hypoglycemia.3) MGN improved of histological outcomes in mice transient focal ischemia; nevertheless, did not induce any significant change in regional cerebral blood flow (rCBF).4) Protective effect of MGN was significant until mice were treated in 6 hours after Ischemia/reperfusion.5) MGN was cerebroprotective in both transient and permanent models of SD rats by improvement of histological and functional outcomes.CONCLUSIONS:1. MGN is robust neuroprotective against cerebral ischemia at low doses without hypoglycemia.2. MGN improved of histological outcomes in mice transient focal ischemia, whereas did not influence rCBF at all.3. MGN has a strong beneficial effect with long clinically relevant therapeutic time windows over 6 hours in tMCAo mouse model.4. MGN exhibits cerebroprotection in both transient and permanent ischemia models of rats.Part II The neuroprotective mechanisms involved in mitiglinide-mediated neuroprotection on the cerebral ischemia/reperfusion-induced acute injury in miceAIM:The present study was designed to evaluate the neuroprotective effects of MGN on NVU dysfunction in tMCAo mice and further investigate the mechanisms underlying MGN-afforded neuroprotection.METHODS:Transient focal cerebral ischemia was induced in two to three-month-old male mouse by intraluminal occlusion of the right MCA for 60 min as described previously. They were all treated with MGN (0.4 mg/kg, i.g.) atl h after reperfusion and sacrificed at 24 h or 72 h. The mortality rate and neurological deficits were measured in each group after cerebral ischemia. Immunohistochemistry and immunofluorescence were performed to detect the morphological changes of neurons, astrocytes, microglia and other neurovascular components in cortex and striatum with quantifications by stereological counts. Western blotting assay and Image J software were applied to analyze levels of matrix metalloproteinases 9 (MMP-9), occluding, apoptosis (including Bcl-2, Bax and caspase 3), endoplasmic reticulum stress (ERS, including GRP78, Chop and caspase 12), and inflammation reaction(NF-κB signal p65 in nuclei and IL-1β production and maturation in cytoplasm). The level of oxidative stress was evaluated via measuring expressions of Malondialdehyde (MDA) and Superoxide Dismutase (SOD) by commercial assay kits. Moreover, Wild type (WT), Kir6.2-/- and Kir6.1+/- mice were subjected to further determine which the beneficial effects of MGN was attributable to.RESULTS:1) MGN (0.4 mg/kg) reduced neuronal death and loss, and inhibited the gliosis in cortex and striatum of tMCAo mice.2) MGN attenuated occludin and collagen Ⅳ degradation through decreased MMP9 expression.3) The mechanisms for neuroprotection afforded by MGN were involved in suppressing apoptosis, ERS, and oxidative stress induced by cerebral ischemia/reperfusion.4) MGN ameliorated post-ischemic inflammation in the peri-infarct area of brain.5) The protective effect of treatment with MGN was abolished by Kir6.1 knockdown, which was preserved in Kir6.2 knockout mice on the contrary.CONCLUSIONS:1. MGN alleviates the neurovascular disruption in tMCAo mouse model.2. The underlying mechanism is attributed to that MGN interferes multiple deleterious processes, including apoptosis, ERS, oxidative stress, inflammation and so on.3. The neuroprotective effect of MGN is dependent on Kir6.1 but not Kir6.2 containing KATp channels in tMCAo mouse model.Part Ⅲ The effects of mitiglinide on functional recovery after cerebral ischemia/reperfusion in miceAIM:To evaluate the effects of MGN treatment on brain repair and its correlation with functional outcomes after cerebral ischemia/reperfusion.METHODS:Survival rate was measured in animals for 20 days after induction of stroke using the intraluminal MCAo mouse model as described previously. Mice received continuous daily injections of MGN (0.4 mg/kg/d, i.g.) beginning at 60 min after reperfusion on Day 1 until the end of the experiment. Sensory and motor coordination deficits were analyzed using the foot fault test, rotarod test, cylinder test and the corner turn test on Days 3,7,14,21 and 28. Deficits in learning and memory were assessed by means of the Morris water-maze task. Mice received injections of 5-bromo-2-deoxyuridine (BrdU,50 mg/kg) four times on Day 29 after stroke, and were transcardially perfused with 4% paraformaldehyde at Days 30 and 58. Brain frozen sections were stained with anti-BrdU antibody alone or in combination with with anti-NeuN or anti-GFAP antibodys for analyze MGN-mediated effects on post-stroke neurogenesis.RESULTS:1) Long-term treatment of MGN improved survival of tMCAo. mice.2) MGN showed a faster recovery of behavioral deficits in the forelimb use asymmetry, corner turn test, foot fault test and rotarod test after ischemia/reperfusion. 3) MGN enhanced the ability to acquire task and learn platform location (escape latency) and ability to recall position of platform on probe trial as indicated by time spent the goal quadrant where the platform had been previously located.4) Application of MGN is associated with increasement in numbers of BrdU positive cells in both and SGZ on Day 30 or 58 after ischemia/reperfusion.5) MGN pushed BrdU positive cells to migrate toward the ischemic lesion and preferentially differentiate to mature neurons(BrdU/NeuN).CONCLUSIONS:1. MGN exhibits the improvements in the quality of life and living after transient focal cerebral ischemia.2. MGN facilitates brain repair through enhancing neurogenesis after cerebral ischemia/reperfusion.In summary, the innovations of the present study lie in:1. MGN plays the neuroprotective effects and has a long therapeutic time window in rodent models of ischemic stroke. In three different rodent models of ischemic stroke, MGN exhibits the significant cerebroprotection against histological and functional damage, with wide therapeutic and clinically relevant time windows. This is academically reported for the first time that hypoglycemic drugs MGN might serve as a neuroprotective agent, which not only expanding the pharmacological applications of MGN, but also providing a new candidate drug for stroke treatment.2. MGN is a novel and potent neuroprotective agent targeting multiple deleterious dysregulations and neurovascular disruption after stroke. MGN is a novel and potent therapy targeting neurovascular disruption and post-stroke dysregulation in multiple cellular pathways (apoptosis, ERS, inflammation, oxidative stress, etc). The therapeutic potential of MGN is abolished by Kir6.1 knockdown but preserved when Kir6.2 gene is deficient, suggesting Kir6.1 may be the molecular target for MGN. The findings here illustrate the mechanism and target underlying of neuroprotection exerted by MGN, which might open a new therapeutic windows in stroke therapy.3. MGN improves functional recovery by enhancing neurogenesis after stroke. Long-term treatment of MGN enhances neurogenesis and neurorepair around the injury at delayed time-points after cerebral ischemia, thus leading to an improved functional outcome. Our findings provide a strong foundation for a translational research program to study MGN in patients with ischemic stroke.