Glucagon-like peptide-1 receptor activation reduces ischaemic brain damage following stroke in Type 2 diabetic rats

Diabetes is a strong risk factor for premature and severe stroke. The GLP-1R (glucagon-like peptide-1 receptor) agonist Ex-4 (exendin-4) is a drug for the treatment of T2D (Type 2 diabetes) that may also have neuroprotective effects. The aim of the present study was to determine the efficacy of Ex-4 against stroke in diabetes by using a diabetic animal model, a drug administration paradigm and a dose that mimics a diabetic patient on Ex-4 therapy. Furthermore, we investigated inflammation and neurogenesis as potential cellular mechanisms underlying the Ex-4 efficacy. A total of seven 9-month-old Type 2 diabetic Goto–Kakizaki rats were treated peripherally for 4 weeks with Ex-4 at 0.1, 1 or 5 μg/kg of body weight before inducing stroke by transient middle cerebral artery occlusion and for 2–4 weeks thereafter. The severity of ischaemic damage was measured by evaluation of stroke volume and by stereological counting of neurons in the striatum and cortex. We also quantitatively evaluated stroke-induced inflammation, stem cell proliferation and neurogenesis. We show a profound anti-stroke efficacy of the clinical dose of Ex-4 in diabetic rats, an arrested microglia infiltration and an increase of stroke-induced neural stem cell proliferation and neuroblast formation, while stroke-induced neurogenesis was not affected by Ex-4. The results show a pronounced anti-stroke, neuroprotective and anti-inflammatory effect of peripheral and chronic Ex-4 treatment in middle-aged diabetic animals in a preclinical setting that has the potential to mimic the clinical treatment. Our results should provide strong impetus to further investigate GLP-1R agonists for their neuroprotective action in diabetes, and for their possible use as anti-stroke medication in non-diabetic conditions

Glucagon like peptide-1 receptor agonists as neuroprotective agents for ischemic stroke: a systematic scoping review

Stroke mortality and morbidity is expected to rise. Despite considerable recent advances within acute ischemic stroke treatment, scope remains for development of widely applicable neuroprotective agents. Glucagon like peptide-1 receptor agonists (GLP-1RAs), originally licensed for the management of Type 2 Diabetes Mellitus, have demonstrated pre-clinical neuroprotective efficacy in a range of neurodegenerative conditions. This systematic scoping review reports the pre-clinical basis of GLP-1RAs as neuroprotective agents in acute ischemic stroke and their translation into clinical trials. We included 35 pre-clinical studies, 11 retrospective database studies, 7 cardiovascular outcome trials and 4 prospective clinical studies. Pre-clinical neuroprotection was demonstrated in normoglycemic models when administration was delayed by up to 24-hours following stroke induction. Outcomes included reduced infarct volume, apoptosis, oxidative stress and inflammation alongside increased neurogenesis, angiogenesis and cerebral blood flow. Improved neurological function and a trend towards increased survival were also reported. Cardiovascular outcomes trials reported a significant reduction in stroke incidence with semaglutide and dulaglutide. Retrospective database studies show a trend towards neuroprotection. Prospective interventional clinical trials are on-going, but initial indicators of safety and tolerability are favourable. Ultimately, we propose that repurposing GLP-1RAs is potentially advantageous but appropriately designed trials are needed to determine clinical efficacy and cost-effectiveness

Neuroprotective exendin-4 enhances hypothermia therapy in a model of hypoxic-ischaemic encephalopathy

Hypoxic-ischaemic encephalopathy remains a global health burden. Despite medical advances and treatment with therapeutic hypothermia, over 50% of cooled infants are not protected and still develop lifelong neurodisabilities, including cerebral palsy. Furthermore, hypothermia is not used in preterm cases or low resource settings. Alternatives or adjunct therapies are urgently needed. Exendin-4 is a drug used to treat type 2 diabetes mellitus that has also demonstrated neuroprotective properties, and is currently being tested in clinical trials for Alzheimer’s and Parkinson’s diseases. Therefore, we hypothesized a neuroprotective effect for exendin-4 in neonatal neurodisorders, particularly in the treatment of neonatal hypoxic-ischaemic encephalopathy. Initially, we confirmed that the glucagon like peptide 1 receptor (GLP1R) was expressed in the human neonatal brain and in murine neurons at postnatal Day 7 (human equivalent late preterm) and postnatal Day 10 (term). Using a well characterized mouse model of neonatal hypoxic-ischaemic brain injury, we investigated the potential neuroprotective effect of exendin-4 in both postnatal Day 7 and 10 mice. An optimal exendin-4 treatment dosing regimen was identified, where four high doses (0.5 µg/g) starting at 0 h, then at 12 h, 24 h and 36 h after postnatal Day 7 hypoxic-ischaemic insult resulted in significant brain neuroprotection. Furthermore, neuroprotection was sustained even when treatment using exendin-4 was delayed by 2 h post hypoxic-ischaemic brain injury. This protective effect was observed in various histopathological markers: tissue infarction, cell death, astrogliosis, microglial and endothelial activation. Blood glucose levels were not altered by high dose exendin-4 administration when compared to controls. Exendin-4 administration did not result in adverse organ histopathology (haematoxylin and eosin) or inflammation (CD68). Despite initial reduced weight gain, animals restored weight gain following end of treatment. Overall high dose exendin-4 administration was well tolerated. To mimic the clinical scenario, postnatal Day 10 mice underwent exendin-4 and therapeutic hypothermia treatment, either alone or in combination, and brain tissue loss was assessed after 1 week. Exendin-4 treatment resulted in significant neuroprotection alone, and enhanced the cerebroprotective effect of therapeutic hypothermia. In summary, the safety and tolerance of high dose exendin-4 administrations, combined with its neuroprotective effect alone or in conjunction with clinically relevant hypothermia make the repurposing of exendin-4 for the treatment of neonatal hypoxic-ischaemic encephalopathy particularly promising

Neuroprotective Effects of Diabetes Drugs for the Treatment of Neonatal Hypoxia-Ischemia Encephalopathy

The perinatal period represents a time of great vulnerability for the developing brain. A variety of injuries can result in death or devastating injury causing profound neurocognitive deficits. Hypoxic-ischemic neonatal encephalopathy (HIE) remains the leading cause of brain injury in term infants during the perinatal period with limited options available to aid in recovery. It can result in long-term devastating consequences with neurologic complications varying from mild behavioral deficits to severe seizure, intellectual disability, and/or cerebral palsy in the newborn. Despite medical advances, the only viable option is therapeutic hypothermia which is classified as the gold standard but is not used, or may not be as effective in preterm cases, infection-associated cases or low resource settings. Therefore, alternatives or adjunct therapies are urgently needed. Ongoing research continues to advance our understanding of the mechanisms contributing to perinatal brain injury and identify new targets and treatments. Drugs used for the treatment of patients with type 2 diabetes mellitus (T2DM) have demonstrated neuroprotective properties and therapeutic efficacy from neurological sequelae following HIE insults in preclinical models, both alone, or in combination with induced hypothermia. In this short review, we have focused on recent findings on the use of diabetes drugs that provide a neuroprotective effect using in vitro and in vivo models of HIE that could be considered for clinical translation as a promising treatment

Development of therapeutics for the treatment of diabetic brain complications

Type-2 diabetes (T2D) is characterized by hyperglycemia and hyperlipidemia, resulting in impaired insulin production and insulin resistance in peripheral tissues. Several studies have demonstrated an association between diabetes and central nervous system complications such as stroke and Alzheimer’s disease. Due to the fact that T2D is one of the fastest growing chronic illnesses, there is an urgent need to improve our knowledge on the pathogenic mechanisms to why diabetes leads to brain complications as well as to identify novel drugable targets for therapeutic use. Project 1: studies I-II Pre-clinical studies have shown that adult neurogenesis is impaired in diabetic animal models. We hypothesized that diabetes leading to neurogenesis impairment plays a role in the development of neurological complications. If so, normalizing neurogenesis in diabetes/obesity could be therapeutically useful in counteracting neurological dysfunction. The aim of studies I-II was to establish an in vitro system where to study the effect of a diabetic milieu on adult neurogenesis. Furthermore, we determined the potential role of pituitary adenylate cyclase-activating polypeptide (PACAP) and galanin to protect adult neural stem cells (NSCs) from these diabetic-like conditions. Moreover, we determined whether apoptosis and the unfolded protein response (UPR) were induced by diabetic-like conditions and whether their regulation was involved in the PACAP/galanin-mediated protective effect. Finally, we studied the potential regulation of PACAP and galanin receptors in NSCs in response to diabetic-like conditions in vitro and ex vivo. The viability of NSCs isolated from the mouse brain subventricular zone (SVZ) was assessed in presence of a diabetic milieu, as mimicked by high palmitate and glucose, which characterize diabetic glucolipotoxicity. The results show that high palmitate and glucose impair NSC viability in correlation to increased apoptosis (Bcl-2, cleaved caspase-3) and UPR signaling (CHOP, BIP, XBP1, JNK phosphorylation). We also show that PACAP and galanin counteract glucolipotoxicity via PAC1 receptor and GalR3 activation, respectively. Furthermore, we also report that PACAP and galanin receptors are regulated by diabetes in NSCs in vitro and in the SVZ ex vivo. Project 2: study III T2D is a strong risk factor for stroke and no therapy based on neuroprotection is currently available. Exendin-4 (Ex-4) is a glucagon-like peptide-1 receptor (GLP-1R) agonist in clinical use for the treatment of T2D, which has also been shown to mediate neuroprotection against stroke pre-clinically. However, the applicability of a therapy based on Ex-4 has not been investigated in a pre-clinical setting with clinical relevance. The aim of this study was to determine the potential efficacy of Ex-4 against stroke in T2D rats by using a drug administration paradigm and a dose that mimics a diabetic patient on Ex-4 therapy. Moreover, we investigated inflammation and neurogenesis as potential cellular mechanisms at the basis of Ex-4 efficacy. T2D Goto-Kakizaki (GK) rats were treated peripherally for 4 weeks with daily clinical doses of Ex-4 (0.1, 1, 5 !g/kg body weight) before inducing stroke by transient middle cerebral artery occlusion. The Ex-4 treatment was continued for 2-4 weeks thereafter. The severity of ischemic damage was measured by evaluation of stroke volume and by stereological counting of neurons in the striatum and cortex. Evaluation of stroke-induced inflammation, stem cell proliferation and neurogenesis was also quantitatively assessed by immunohistochemistry. We show that peripheral administration of Ex-4 counteracts ischemic brain damage in T2D GK rats. The results also show that Ex-4 decreased microglia infiltration and increased stroke-induced neural stem cell proliferation and neuroblast formation, while stroke-induced neurogenesis was not affected by Ex-4 treatment. Together, our data in project 1 show that we have established an in vitro assay where to study the molecular mechanism on how diabetes impact adult neurogenesis. Furthermore, our results show that this assay has the potential to be developed into a screening platform for the identification of molecules that can regulate adult neurogenesis under diabetes. In project 2, we show neuroprotective efficacy against stroke by Ex-4 in a T2D rat model, by using a pre-clinical setting with clinical relevance. Ex-4 is an anti-diabetic drug in clinical use that has been reported to show limited side effects. Thus, at least in theory stroke patients should be able to easily receive this treatment, probably with minimal risks

Glucagon-like peptide-1 (GLP-1) receptor activation dilates cerebral arterioles, increases cerebral blood flow, and mediates remote (pre)conditioning neuroprotection against ischaemic stroke.

Stroke remains one of the most common causes of death and disability worldwide. Several preclinical studies demonstrated that the brain can be effectively protected against ischaemic stroke by two seemingly distinct treatments: remote ischaemic conditioning (RIC), involving cycles of ischaemia/reperfusion applied to a peripheral organ or tissue, or by systemic administration of glucagon-like-peptide-1 (GLP-1) receptor (GLP-1R) agonists. The mechanisms underlying RIC- and GLP-1-induced neuroprotection are not completely understood. In this study, we tested the hypothesis that GLP-1 mediates neuroprotection induced by RIC and investigated the effect of GLP-1R activation on cerebral blood vessels, as a potential mechanism of GLP-1-induced protection against ischaemic stroke. A rat model of ischaemic stroke (90 min of middle cerebral artery occlusion followed by 24-h reperfusion) was used. RIC was induced by 4 cycles of 5 min left hind limb ischaemia interleaved with 5-min reperfusion periods. RIC markedly (by ~ 80%) reduced the cerebral infarct size and improved the neurological score. The neuroprotection established by RIC was abolished by systemic blockade of GLP-1R with a specific antagonist Exendin(9-39). In the cerebral cortex of GLP-1R reporter mice, ~ 70% of cortical arterioles displayed GLP-1R expression. In acute brain slices of the rat cerebral cortex, activation of GLP-1R with an agonist Exendin-4 had a strong dilatory effect on cortical arterioles and effectively reversed arteriolar constrictions induced by metabolite lactate or oxygen and glucose deprivation, as an ex vivo model of ischaemic stroke. In anaesthetised rats, Exendin-4 induced lasting increases in brain tissue PO2, indicative of increased cerebral blood flow. These results demonstrate that neuroprotection against ischaemic stroke established by remote ischaemic conditioning is mediated by a mechanism involving GLP-1R signalling. Potent dilatory effect of GLP-1R activation on cortical arterioles suggests that the neuroprotection in this model is mediated via modulation of cerebral blood flow and improved brain perfusion

Prehospital exenatide in hyperglycemic stroke-A randomized trial

Objectives Hyperglycemia is a predictor for poor stroke outcome. Hyperglycemic stroke patients treated with thrombolysis have an increased risk of intracranial hemorrhage. Insulin is the gold standard for treating hyperglycemia but comes with a risk of hypoglycemia. Glucagon-like peptide-1 receptor agonists (GLP-1RA) are drugs used in type 2 diabetes that have a low risk of hypoglycemia and have been shown to exert neuroprotective effects. The primary objective was to determine whether prehospital administration of the GLP-1RA exenatide could lower plasma glucose in stroke patients. Secondary objective was to study tolerability and safety. Materials & Methods Randomized controlled trial comparing exenatide administrated prehospitally with a control group receiving standard care for hyperglycemia. Patients with Face Arm Speech Test >= 1 and glucose >= 8 mmol/L were randomized. Glucose was monitored for 24 hours. All adverse events were recorded. Results Nineteen patients were randomized, eight received exenatide. An interim recruitment failure analysis with subsequent changes of the protocol was made. The study was stopped prematurely due to slow inclusion. No difference was observed in the main outcome of plasma glucose at 4 hours, control vs exenatide (mean, SD); 7.0 +/- 1.9 vs 7.6 +/- 1.6; P = .56). No major adverse events were reported. Conclusions We found no evidence that prehospital exenatide had effect on hyperglycemia. However, it was given without adverse events in this study with limited sample size that was prematurely stopped due to slow inclusion.Peer reviewe

Save the brain! ; studies on GLP-1-mediated neuroprotection

Background: Patients with type 2 diabetes (T2D) suffer stroke more often and have worse recovery. Moreover, hyperglycemic stroke patients treated with thrombolytic therapy have more intracranial bleedings. Insulin is the gold standard for treatment of hyperglycemia, but has a significant risk of hypoglycemia, especially if rapid normalization of glucose is the goal. Glucagon like peptide-1 based drugs, such as receptor agonists (GLP-1RA) and dipeptidylpeptidase-4 inhibitors (DPP-4i) that raise the endogenous GLP-1 levels are used to treat T2D. Interestingly these drugs have been shown to reduce stroke incidence; and, importantly for this thesis, to exert acute neuroprotective effects in animal models if given before a stroke. Aim: To determine if GLP-1RA and DPP-4i are neuroprotective in animal models when given after stroke and to identify some of the underlying mechanisms. Furthermore, the aim was to determine the GLP-1 levels in humans after stroke and their relationship to outcome. Finally, to test treating hyperglycemic stroke patients with GLP-1RA early after stroke: in the ambulance. Study 1: An experimental animal study determining the effect of giving the GLP-1RA exendin-4 (Ex-4) after the onset of stroke. We did this in both young and healthy animals as well as aged and obese/diabetic animals. Experimental stroke was induced by middle cerebral artery occlusion (MCAO). The main finding was that exendin-4 is neuroprotective if given after MCAO, both in young and old animals. The effect was time-sensitive with effect at 1.5 and 3 h after MCAO, but lost at the 4.5 h time point. Study 2: An experimental animal study where we determined if DPP-4i treatment had neuroprotective properties when started after the onset of stroke. Additionally, we studied if the effect was dependent of the GLP-1 receptor by using a GLP-1R -/- knockout model mouse. The main finding was that DPP-4 inhibitors require chronic pre-treatment to be effective. Furthermore, we showed that the effect is not dependent on the GLP-1 receptor. Study 3: In rats with diabetes (GK-rats) or without (Wistars) we studied the impact of aging in diabetes on regulatory GABA-ergic interneurons (key cells involved in stroke recovery) and whether treatment with the GLP-1RA exendin-4 could revert the observed changes. Main findings: 1: The number of GABAeric neurons decreased in aged diabetic animals. 2: Treatment with Ex-4 had effect on the subpopulation of GABAeric neurons positive for calbindin. Study 4: We determined the endogenous GLP-1 levels in patients treated with thrombolytic therapy for ischemic stroke. A group of 59 patients underwent a OGTT at day 2-4 during the hospital stay. A repeat OGTT was performed 3 months later. At the three-month follow-up functional stroke outcome was measured with mRS. 27 healthy controls underwent one OGTT. The main findings were that the GLP-1 level was higher in stroke patients and remained unchanged 3 months later. The GLP-1 level was not associated with functional outcome. Study 5: A randomized clinical trial examining the feasibility of prehospital treatment with exenatide in hyperglycemic patients (8-15 mmol/L) with suspected stroke. Patients were followed for 24 h. 19 patients were randomized, 8 received exenatide. There was no evidence of a difference in the main outcome of glucose at 4 hours. No adverse events were reported. Conclusion: Both GLP-1RA and DPP-4i are neuroprotective against stroke. The effect is, however, time sensitive with the need for early initiation after stroke (GLP-1RA) or chronic prestroke treatment (DPP-4i). Additionally, the effect of DPP-4i is GLP-1 receptor independent. The findings encourage the use of these drugs for the treatment of diabetes

A review of stress-induced hyperglycaemia in the context of acute ischaemic stroke: Definition, underlying mechanisms, and the status of insulin therapy

The transient elevation of blood glucose produced following acute ischaemic stroke (AIS) has been described as stress-induced hyperglycaemia (SIH). SIH is common even in patients with AIS who have no previous diagnosis of diabetes mellitus. Elevated blood glucose levels during admission and hospitalization are strongly associated with enlarged infarct size and adverse prognosis in AIS patients. However, insulin-intensive glucose control therapy defined by admission blood glucose for SIH has not achieved the desired results, and new treatment ideas are urgently required. First, we explore the various definitions of SIH in the context of AIS and their predictive value in adverse outcomes. Then, we briefly discuss the mechanisms by which SIH arises, describing the dual effects of elevated glucose levels on the central nervous system. Finally, although preclinical studies support lowering blood glucose levels using insulin, the clinical outcomes of intensive glucose control are not promising. We discuss the reasons for this phenomenon

Incretin mimetics as pharmacological tools to elucidate and as a new drug strategy to treat traumatic brain injury

Traumatic brain injury (TBI), either as an isolated injury or in conjunction with other injuries, is an increasingly common occurring event. An estimated 1.7 million injuries occur within the US each year and 10 million people are affected annually worldwide. Indeed, some one-third (30.5%) of all injury-related deaths in the U.S. are associated with TBI, which will soon outstrip many common diseases as the major cause of death and disability. Associated with a high morbidity and mortality, and no specific therapeutic treatment, TBI has become a pressing public health and medical problem. The highest incidence of TBI occurs among young adults (15 to 24 years age) as well as in the elderly (75 years and older) who are particularly vulnerable as injury, often associated with falls, carries an increased mortality and worse functional outcome following lower initial injury severity. Added to this, a new and growing form of TBI, blast injury, associated with the detonation of improvised explosive devices in the war theaters of Iraq and Afghanistan, are inflicting a wave of unique casualties of immediate impact to both military personnel and civilians, for which long-term consequences remain unknown and may potentially be catastrophic. The neuropathology underpinning head injury is becoming increasingly better understood. Depending on severity, TBI induces immediate neuropathological effects that for the mildest form may be transient but with increasing severity cause cumulative neural damage and degeneration. Even with mild TBI, which represents the majority of cases, a broad spectrum of neurological deficits, including cognitive impairments, can manifest that may significantly influence quality of life. In addition, TBI can act as a conduit to longer-term neurodegenerative disorders. Prior studies of glucagon-like peptide-1 (GLP-1) and long-acting GLP-1 receptor agonists have demonstrated neurotrophic/neuroprotective activities across a broad spectrum of cellular and animal models of chronic neurodegenerative (Alzheimer's and Parkinson's diseases) and acute cerebrovascular (stroke) disorders. In line with the commonality in mechanisms underpinning these disorders as well as TBI, the current article reviews this literature and recent studies assessing GLP-1 receptor agonists as a potential treatment strategy for mild to moderate TBI