Ischemia-related changes in naive and mutant forms of ubiquitin and neuroprotective effects of ubiquitin in the hippocampus following experimental transient ischemic damage

Ubiquitin binds to short-lived proteins and denatured proteins produced by various forms of injury. The loss of ubiquitin leads to an accumulation of abnormal proteins and may affect cellular structure and function. The aim of the present study is to observe the chronological changes in ubiquitin naive form and its mutant form (ubiquitin⁺¹) in the hippocampal CA1 region (CA1) after transient cerebral ischemia in gerbils. Delayed neuronal death in the CA1 was confirmed 4 days after ischemic insult with NeuN immunohistochemistry. Ubiquitin immunoreactivity and protein level in the CA1 were lowest at 12 h after ischemia/reperfusion; thereafter, they were increased with time. Ubiquitin⁺¹ immunoreactivity and protein levels in the CA1 were slightly decreased at 3 h after ischemia/reperfusion, and they were significantly increased 1 day after ischemia/reperfusion. In addition, ubiquitin and ubiquitin⁺¹ immunoreaction was expressed in astrocytes after delayed neuronal death in the ischemic CA1. To elucidate the protective effect of ubiquitin on ischemic damage, the animals were treated with ubiquitin (1.5 mg/kg body weight) intravenously via the femoral vein. Ubiquitin treatment significantly reduced ischemia-induced locomotor hyperactivity, neuronal death and reactive gliosis such as astrocytes and microglia. In addition, 5 days after ubiquitin treatment in the ischemic group, ubiquitin immunoreactivity was similar to that in the ubiquitin-treated sham group, however, ubiquitin⁺¹ immunoreactivity was higher than that in the ubiquitin-treated sham group. These findings indicate that the depletion of ubiquitin and the accumulation of ubiquitin⁺¹ in CA1 pyramidal neurons after transient cerebral ischemia may inhibit ubiquitin proteolytic pathway and this leads to delayed neuronal death of CA1 pyramidal neurons directly or indirectly after transient cerebral ischemia.     

Changes in immunoreactivity of HSP60 and its neuroprotective effects in the gerbil hippocampal CA1 region induced by transient ischemia

Heat shock proteins act as molecular chaperones and are involved in protein folding, refolding, transport, and translocation. In the present study, we observed changes in heat shock protein 60 (HSP60) immunoreactivity and protein level in the gerbil hippocampal CA1 region after 5 min of transient forebrain ischemia and its neuroprotective effect against ischemic damage. HSP60 immunoreactivity in the CA1 region began to increase in the stratum pyramidale at 30 min after ischemia/reperfusion, and peaked 24 h after ischemia/reperfusion. Thereafter, HSP60 immunoreactivity was decreased in the CA1 region with time. Seven days after ischemia/reperfusion, HSP60 immunoreactivity was increased again in the CA1 region: at this time point after ischemia/reperfusion, HSP60 immunoreactivity was expressed in glial cells in the ischemic CA1 region. HSP60 immunoreactive glial cells were astrocytes containing glial fibrillar acidic protein. In contrast, change in HSP60 immunoreactivity in the ischemic CA2/3 region was not significant compared with that in the ischemic CA1 region. In Western blot study, HSP60 protein level in the CA1 region was increased after ischemia/reperfusion and highest 24 h after ischemia/reperfusion. Animals treated with recombinant adenoviruses expressing Hsp60 (Ad-Hsp60) showed the neuroprotection of CA1 pyramidal neurons from ischemic damage. These results suggest that HSP60 may be associated with delayed neuronal death of CA1 pyramidal neurons after transient ischemia, and the induction of HSP60 protects the neurons from ischemic damage.     

Inhibition of Na/H exchange by SM-20220 attenuates free fatty acid efflux in rat cerebral cortex during ischemia–reperfusion injury

The Na⁺/H⁺ exchanger (NHE) is activated during ischemia–reperfusion in an effort to restore intracellular pH to normal levels. Inhibition of NHE with non-selective amiloride derivatives has been shown to be neuroprotective and to attenuate free fatty acid efflux during ischemia–reperfusion. We evaluated the effects of SM-20220 (20 μM), a highly selective and specific NHE inhibitor, applied topically onto rat cerebral cortex prior to and during a 20-min period of ischemia. SM-20220 application significantly reduced the ischemia-evoked efflux of myristic, palmitic, and arachidonic acids during both ischemia and reperfusion with significant decreases in linoleic and docosahexaenoic levels during reperfusion. This study confirms the importance of NHEs in eliciting free fatty acid efflux, inhibition of which may be an essential component of the neuroprotective benefits of NHE inhibitors in ischemia–reperfusion injury.     

Hypothyroid state does not protect but delays neuronal death in the hippocampal CA1 region following transient cerebral ischemia: Focus on oxidative stress and gliosis

We investigated protective effects of hypothyroidism on delayed neuronal death, gliosis, lipid peroxidation and Cu,Zn‐superoxide dismutase (SOD1) in the gerbil hippocampal CA1 region (CA1) after 5 min of transient cerebral ischemia. The hypothyroidism was induced by 0.025% methimazole treatment. Free triiodothyronine and thyroxine levels were markedly decreased in the hypothyroid group. Four days after ischemia/reperfusion, only a few NeuN‐immunoreactive (+) neurons were detected in the CA1 of euthyroid‐ischemia (eu‐ischemia) group; however, at this time point, the number of NeuN⁺ neurons was significantly higher in the hypothyroid‐ischemia (hypo‐ischemia) group than in the eu‐ischemia group. At 5 days postischemia, NeuN⁺ neurons were significantly decreased in the hypo‐ischemia group: The number of NeuN⁺ neurons in this group was similar to that in the eu‐ischemia group. Activations of GFAP⁺ astrocytes and Iba‐1⁺ microglia in the CA1 were higher in the eu‐ischemia group 3 and 4 days after ischemia/reperfusion. At 5 days postischemia, the activations of both the glial cells in the CA1 were similar between the two groups. 4‐Hydroxy‐2‐nonenal (HNE), a marker for lipid peroxidation, immunoreactivity in the eu‐ischemia group was higher than in the hypo‐ischemia group; at 5 days postischemia, the immunoreactivity was similar between the two groups. In contrast, SOD1 level was lower in the CA1 of the eu‐ischemia group. These results suggest that hypothyroid state does not protect against delayed neuronal death but only delays the neuronal death in the hippocampal CA1 region after transient cerebral ischemia by reducing lipid peroxidation and increasing SOD1. © 2010 Wiley‐Liss, Inc.     

Rapid decrease of high affinity ouabain binding sites in hippocampal CA1 region following short-term global cerebral ischemia in rat

High affinity [3H]ouabain binding was examined in the hippocampal CA1 region and frontal cortex of rats subjected to 5 min complete cerebral ischemia in a clinical death model, and to subsequent resuscitation. A decrease of Bmax directly after ischemia and its further gradual decrease during 120 min of reperfusion were noted in the ischemia-vulnerable CA1 region, whereas no change of Bmax was observed in frontal cortex. The apparent Kd constant showed insignificant fluctuations in either of the two brain regions. Since ouabain binds with high affinity to the neuronal (alpha +)-form of Na+/K+-ATPase, the results indicate a rapid enzyme loss in CA1 neurons. The high affinity ouabain binding test proved to be a sensitive detector of premorphological changes in nerve cell membranes in ischemia.     

Enhancement in activities of large conductance calcium-activated potassium channels in CA1 pyramidal neurons of rat hippocampus after transient forebrain ischemia

It has been reported previously that the neuronal excitability persistently suppresses and the amplitude of fast afterhyperpolarization (fAHP) increases in CA1 pyramidal cells of rat hippocampus following transient forebrain ischemia. To understand the conductance mechanisms underlying these post-ischemic electrophysiological alterations, we compared differences in activities of large conductance Ca²⁺-activated potassium (BK_Ca) channels in CA1 pyramidal cells acutely dissociated from hippocampus before and after ischemia by using inside-out configuration of patch clamp techniques. (1) The unitary conductance of BK_Ca channels in post-ischemic neurons (295 pS) was higher than that in control neurons (245 pS) in symmetrical 140/140 mM K⁺ in inside-out patch; (2) the membrane depolarization for an e-fold increase in open probability (P_o) showed no significant differences between two groups while the membrane potential required to produce one-half of the maximum P_o was more negative after ischemia, indicating no obvious changes in channel voltage dependence; (3) the [Ca²⁺]_i required to half activate BK_Ca channels was only 1 μM in post-ischemic whereas 2 μM in control neurons, indicating an increase in [Ca²⁺]_i sensitivity after ischemia; and (4) BK_Ca channels had a longer open time and a shorter closed time after ischemia without significant differences in open frequency as compared to control. The present results indicate that enhanced activity of BK_Ca channels in CA1 pyramidal neurons after ischemia may partially contribute to the post-ischemic decrease in neuronal excitability and increase in fAHP.     

Delayed hippocampal neuronal death in young gerbil following transient global cerebral ischemia is related to higher and longer-term expression of p63 in the ischemic hippocampus

The tumor suppressor p63 is one of p53 family members and plays a vital role as a regulator of neuronal apoptosis in the development of the nervous system. However, the role of p63 in mature neuronal death has not been addressed yet. In this study, we first compared ischemia-induced effects on p63 expression in the hippocampal regions (CA1–3) between the young and adult gerbils subjected to 5 minutes of transient global cerebral ischemia. Neuronal death in the hippocampal CA1 region of young gerbils was significantly slow compared with that in the adult gerbils after transient global cerebral ischemia. p63 immunoreactivity in the hippocampal CA1 pyramidal neurons in the sham-operated young group was significantly low compared with that in the sham-operated adult group. p63 immunoreactivity was apparently changed in ischemic hippocampal CA1 pyramidal neurons in both ischemia-operated young and adult groups. In the ischemia-operated adult groups, p63 immunoreactivity in the hippocampal CA1 pyramidal neurons was significantly decreased at 4 days post-ischemia; however, p63 immunoreactivity in the ischemia-operated young group was significantly higher than that in the ischemia-operated adult group. At 7 days post-ischemia, p63 immunoreactivity was decreased in the hippocampal CA1 pyramidal neurons in both ischemia-operated young and adult groups. Change patterns of p63 level in the hippocampal CA1 region of adult and young gerbils after ischemic damage were similar to those observed in the immunohistochemical results. These findings indicate that higher and longer-term expression of p63 in the hippocampal CA1 region of the young gerbils after ischemia/reperfusion may be related to more delayed neuronal death compared to that in the adults.     

Elevation of Na+-K+ ATPase immunoreactivity in GABAergic neurons in gerbil CA1 region following transient forebrain ischemia

In a previous study, we suggested that GABAergic neurons might be resistant to ischemic insult, because of the maintenance of the GABA shunt, which is one of the ATP synthetic pathways in neurons. In the present study, we identified Na(+)-K(+) ATPase immunoreactivity in the gerbil hippocampus in order to determine whether changes in Na(+)-K(+) ATPase immunoreactivity correlate with GABA shunt following ischemic insult. At 12 h after ischemia-reperfusion, Na(+)-K(+) ATPase immunoreactivity accumulated in some neurons in the CA1 region. However, the protein content of Na(+)-K(+) ATPase was not altered. Interestingly, the density of Na(+)-K(+) ATPase immunoreactivity in neurons and the protein content in the CA1 region was intensified in the 24 h post-ischemic group. As a result of double immunofluorescence study, Na(+)-K(+) ATPase immunoreactive neurons were identified with GABAergic neurons. Therefore, our findings suggest that the increase of Na(+)-K(+) ATPase in GABAergic neurons may be able to explain the resistance of these cells to ischemic insult, and support our previous hypothesis that GABA may play an important role as a metabolite in the survival of GABAergic neurons after ischemic insult.     

Na+/Ca2+ exchanger 1 alters in pyramidal cells and expresses in astrocytes of the gerbil hippocampal CA1 region after ischemia

Alterations of immunoreactivity and protein contents of Na(+)/Ca(2+) exchanger 1 (NCX1) were observed in the gerbil hippocampus proper after 5 min of transient forebrain ischemia. NCX1 immunoreactivity was significantly changed in the hippocampal CA1 region, but not in the CA2/3 region after ischemia/reperfusion. In the sham-operated group, NCX1 immunoreactivity was mainly detected in CA1 pyramidal cells. However, 30 min after ischemia/reperfusion, NCX1 immunoreactivity was significantly decreased and then increased at 1 day after ischemia. At this time, NCX1 immunoreactivity in CA1 pyramidal cells was similar to that of the sham-operated group. At 3 days after ischemia, NCX1 immunoreactivity was significantly reduced in the CA1 region compared to that of the sham-operated group and NCX1 immunoreactivity was significantly increased again 4 days after ischemia. Thereafter, NCX1 immunoreactivity was decreased time-dependently in ischemia groups. Between 15 min and 6 h post-ischemia, NCX1 immunoreactivity was expressed in astrocytes in the strata oriens and radiatum of the CA1 region. From 3 days post-ischemia, NCX1 immunoreactivity was expressed in astrocytes in the strata oriens and radiatum. Ischemia-induced changes in NCX1 protein contents in the hippocampus proper concurred with immunohistochemical data post-ischemia. Our results suggest that changes in NCX1 in CA1 pyramidal cells and astrocytes after ischemia are associated with intracellular Na(+) concentrations and that NCX1 may induce an intracellular calcium overload, which may be related to neuronal death.     

Nicotinamide Prevents NAD+ Depletion and Protects Neurons Against Excitotoxicity and Cerebral Ischemia: NAD+ Consumption by SIRT1 may Endanger Energetically Compromised Neurons

Neurons require large amounts of energy to support their survival and function, and are therefore susceptible to excitotoxicity, a form of cell death involving bioenergetic stress that may occur in several neurological disorders including stroke and Alzheimer’s disease. Here we studied the roles of NAD⁺ bioenergetic state, and the NAD⁺-dependent enzymes SIRT1 and PARP-1, in excitotoxic neuronal death in cultured neurons and in a mouse model of focal ischemic stroke. Excitotoxic activation of NMDA receptors induced a rapid decrease of cellular NAD(P)H levels and mitochondrial membrane potential. Decreased NAD⁺ levels and poly (ADP-ribose) polymer (PAR) accumulation in nuclei were relatively early events (<4 h) that preceded the appearance of propidium iodide- and TUNEL-positive cells (markers of necrotic cell death and DNA strand breakage, respectively) which became evident by 6 h. Nicotinamide, an NAD⁺ precursor and an inhibitor of SIRT1 and PARP1, inhibited SIRT1 deacetylase activity without affecting SIRT1 protein levels. NAD⁺ levels were preserved and PAR accumulation and neuronal death induced by excitotoxic insults were attenuated in nicotinamide-treated cells. Treatment of neurons with the SIRT1 activator resveratrol did not protect them from glutamate/NMDA-induced NAD⁺ depletion and death. In a mouse model of focal cerebral ischemic stroke, NAD⁺ levels were decreased in both the contralateral and ipsilateral cortex 6 h after the onset of ischemia. Stroke resulted in dynamic changes of SIRT1 protein and activity levels which varied among brain regions. Administration of nicotinamide (200 mg/kg, i.p.) up to 1 h after the onset of ischemia elevated brain NAD⁺ levels and reduced ischemic infarct size. Our findings demonstrate that the NAD⁺ bioenergetic state is critical in determining whether neurons live or die in excitotoxic and ischemic conditions, and suggest a potential therapeutic benefit in stroke of agents that preserve cellular NAD⁺ levels. Our data further suggest that, SIRT1 is linked to bioenergetic state and stress responses in neurons, and that under conditions of reduced cellular energy levels SIRT1 enzyme activity may consume sufficient NAD⁺ to nullify any cell survival-promoting effects of its deacetylase action on protein substrates.     

Histochemical study of Ca2+-ATPase activity in ischemic CA1 pyramidal neurons in the gerbil hippocampus

Although cytosolic Ca²⁺ accumulation plays a pivotal role in delayed neuronal death, there have been no investigations on the role of the cellular Ca²⁺ export system in this novel phenomenon. To clarify the function of the Ca²⁺-pump in delayed neuronal death, the plasma membrane Ca²⁺-ATPase activity of CA1 pyramidal neurons was investigated ultracytochemically in normal and ischemic gerbil hippocampus. To correlate enzyme activity with delayed neuronal death, histochemical detection was performed at various recirculation times after 5 min of ischemia produced by occlusion of the bilateral carotid arteries. At 10 min after ischemia, CA1 pyramidal neurons showed weak Ca²⁺-ATPase activity. Although enzyme activity had almost fully recovered 2 h after ischemia, it was reduced again 6 h after ischemia. Thereafter, Ca²⁺-ATPase activity on the plasma membrance of CA1 pyramidal neurons decreased progressively, losing its localization on day 3. On day 4 following ischemia, reaction products were diffusely scattered throughout the whole cell body. Our results indicate that, after once having recovered from ischemic damage, severe disturbance of the membrane Ca²⁺ export system proceeds from the early stage of delayed neuronal death and disturbs the re-export of accumulated cytosolic Ca²⁺, which might contribute to delayed neuronal death. Occult disruption of Ca²⁺ homeostasis seems to occur from an extremely early stage of delayed neuronal death in CA1 pyramidal cells.     

Folic acid deficiency increases delayed neuronal death, DNA damage, platelet endothelial cell adhesion molecule-1 immunoreactivity, and gliosis in the hippocampus after transient cerebral ischemia

Folic acid deficiency increases stroke risk. In the present study, we examined whether folic acid deficiency enhances neuronal damage and gliosis via oxidative stress in the gerbil hippocampus after transient forebrain ischemia. Animals were exposed to a folic acid-deficient diet (FAD) for 3 months and then subjected to occlusion of both common carotid arteries for 5 min. Exposure to an FAD increased plasma homocysteine levels by five- to eightfold compared with those of animals fed with a control diet (CD). In CD-treated animals, most neurons were dead in the hippocampal CA1 region 4 days after ischemia/reperfusion, whereas, in FAD-treated animals, this occurred 3 days after ischemia/reperfusion. Immunostaining for 8-hydroxy-2'-deoxyguanosine (8-OHdG) was performed to examine DNA damage in CA1 neurons in both groups after ischemia, and it was found that 8-OHdG immunoreactivity in both FAD and CD groups peaked at 12 hr after reperfusion, although the immunoreactivity in the FAD group was much greater than that in the CD group. Platelet endothelial cell adhesion molecule-1 (PECAM-1; a final mediator of neutrophil transendothelial migration) immunoreactivity in both groups increased with time after ischemia/reperfusion: Its immunoreactivity in the FAD group was much higher than that in the CD group 3 days after ischemia/reperfusion. In addition, reactive gliosis in the ischemic CA1 region increased with time after ischemia in both groups, but astrocytosis and microgliosis in the FAD group were more severe than in the CD group at all times after ischemia. Our results suggest that folic acid deficiency enhances neuronal damage induced by ischemia.     

Activation of mitogen-activated protein kinases after transient forebrain ischemia in gerbil hippocampus.

We investigated the expression, activation, and distribution of c-Jun N-terminal kinases (JNKs), p38 mitogen-activated protein kinases (p38s) and extracellular signal-regulated kinases (ERKs) using Western blotting and immunohistochemistry in gerbil hippocampus after transient forebrain ischemia to clarify the role of these kinases in delayed neuronal death (DND) in the CA1 subfield. Immunoblot analysis demonstrated that activities of JNK, p38, and ERK in whole hippocampus were increased after 5 min of global ischemia. We used an immunohistochemical study to elucidate the temporal and spatial expression of these kinases after transient global ischemia. The immunohistochemical study showed that active JNK and p38 immunoreactivities were enhanced at 15 min of reperfusion and then gradually reduced and disappeared in the hippocampal CA1 region. On the other hand, in CA3 neurons, active JNK and p38 immunoreactivities were enhanced at 15 min of reperfusion and peaked at 6 hr of reperfusion and then gradually reduced but was continuously detected 72 hr after ischemia. Active ERK immunoreactivity was observed transiently in CA3 fibers and dentate gyrus. Pretreatment with SB203580, a p38 inhibitor, but not with PD98059, an ERK kinase 1/2 inhibitor, reduced ischemic cell death in the CA1 region after transient global ischemia by inhibiting the activity of p38. These findings indicate that the p38 pathway may play an important role in DND during brain ischemia in gerbil. Components of the pathway are important target molecules for clarifying the mechanism of neuronal death.     

SM-20220, a Na(+)/H(+) exchanger inhibitor: effects on ischemic brain damage through edema and neutrophil accumulation in a rat middle cerebral artery occlusion model

The Na(+)/H(+) exchanger (NHE) is activated during ischemia-reperfusion in an effort to restore intracellular pH to normal levels. The NHE is recognized to exist as a distinct protein in the plasma membranes of a variety of cells. We investigated the pharmacological effects of a Na(+)/H(+) exchanger inhibitor, SM-20220 (N-(aminoiminomethyl)-1-methyl-1-H-indole-2-carboxamide methanesulfonate), on ischemic brain damage, edema and neutrophil accumulation at 72 h after middle cerebral artery (MCA) occlusion in a rat MCA occlusion model. SM-20220 was intravenously administered as a bolus injection immediately after occlusion, followed by a continuous infusion over 2.5 h. At 72 h after occlusion, the infract area was measured using hematoxylin-eosin staining and, using the same slices, neutrophils in the brain were immuno-stained with anti-myeloperoxidase (n=11). In a separate study, rat behavior was scored and scaled, and brains removed for the determination of water content by the dry-weight method. SM-20220 significantly (P<0.05) attenuated cerebral infarct volume, water content, and the neutrophil accumulation at 72 h after the MCA occlusion, and ameliorated neurological deficits. SM-20220, an NHE inhibitor prevented the progress of cerebral ischemic damage and edema following MCA occlusion in rats though a possible mechanism that may be due to the inhibition of neutrophil accumulation. The NHE in neutrophils may enhance the progress of cerebral damage following cerebral ischemia-reperfusion.     

The immunoreactivity and activity of adenylate cyclase type I are changed in the hippocampal CA1 region after transient forebrain ischemia in gerbils

Adenylate cyclase (AC) has a specific sensitivity to Ca²⁺/calmodulin. AC-I, one of the mediator of learning and memory, plays an important role in signal transduction underlying learning and memory function. In the present study, we found ischemia-related changes of AC-I in the hippocampal CA1 region, but not in the CA2/3 region, after 5 min of transient forebrain ischemia in gerbils. In the sham-operated group, AC-I immunoreactive neurons were detected in pyramidal and non-pyramidal cells in the hippocampus proper. AC-I immunoreactivity was significantly increased at 3 h in the CA1 region after ischemic insult. Thereafter, AC-I immunoreactivity was gradually decreased. Four days after ischemic insult, AC-I-immunoreactive CA1 pyramidal cells in the stratum pyramidale were very few due to delayed neuronal death. The results of Western blot analysis showed that changes of AC-I protein contents were similar to immunohistochemical data after ischemic insult. Gpp(NH)p-dependent AC-I activity in hippocampal CA1 region was not changed in all groups, while Ca²⁺/calmodulin-dependent AC-I activity in hippocampal CA1 region was significantly decreased 24 h after ischemia–reperfusion. These results suggest that the decrease of AC-I activity may be associated with impairment of neurodevelopment and neuroplasticity including learning and memory although the AC-I immunoreactivity was maintained 24 h postischemic group compared to that of the sham-operated group.     

Astrocyte targeted overexpression of Hsp72 or SOD2 reduces neuronal vulnerability to forebrain ischemia

Brief forebrain ischemia is a model of the delayed hippocampal neuronal loss seen in patients following cardiac arrest and resuscitation. Previous studies demonstrated that selective dysfunction of hippocampal CA1 subregion astrocytes occurs hours to days before delayed neuronal death. In this study we tested the strategy of directing protection to astrocytes to protect neighboring neurons from forebrain ischemia. Two well‐studied protective proteins, heat shock protein 72 (Hsp72) or superoxide dismutase 2 (SOD2), were genetically targeted for expression in astrocytes using the astrocyte‐specific human glial fibrillary acidic protein (GFAP) promoter. The expression constructs were injected stereotacticly immediately above the hippocampal CA1 region on one side of the rat brain two days prior to forebrain ischemia. Cell type specific expression was confirmed by double label immunohistochemistry. When the expression constructs were injected two days before transient forebrain ischemia, the loss of CA1 hippocampal neurons observed seven days later was significantly reduced on the injected side compared with controls. This neuroprotection was associated with significantly better preservation of astrocyte glutamate transporter‐1 immunoreactivity at 5‐h reperfusion and reduced oxidative stress. Improving the resistance of astrocytes to ischemic stress by targeting either the cytosolic or mitochondrial compartment was thus associated with preservation of CA1 neurons following forebrain ischemia. Targeting astrocytes is a promising strategy for neuronal preservation following cardiac arrest and resuscitation. © 2010 Wiley‐Liss, Inc.     

Ischemia-induced changes in glucagon-like peptide-1 receptor and neuroprotective effect of its agonist, exendin-4, in experimental transient cerebral ischemia

Glucagon-like peptide-1 receptor (GLP-1R) protects against neuronal damages in the brain. In the present study, ischemia-induced changes in GLP-1R immunoreactivity in the gerbil hippocampal CA1 region were evaluated after transient cerebral ischemia; in addition, the neuroprotective effect of the GLP-1R agonist exendin-4 (EX-4) against ischemic damage was studied. GLP-1R immunoreactivity and its protein levels in the ischemic CA1 region were highest at 1 day after ischemia/reperfusion (I/R). At 4 days after I/R, GLP-1R immunoreactivity was hardly detected in CA1 pyramidal neurons, and its protein level was lowest. GLP-1R protein level was increased again at 10 days after I/R, and GLP-1R immunoreactivity was found in astrocytes and GABAergic interneurons. In addition, EX-4 treatment attenuated ischemia-induced hyperactivity, neuronal damage, and microglial activation in the ischemic CA1 region in a dose-dependent manner. EX-4 treatment also induced the elevation of GLP-1R immunoreactivity and protein levels in the ischemic CA1 region. These results indicate that GLP-1R is altered in the ischemic region after an ischemic insult and that EX-4 protects against ischemia-induced neuronal death possibly by increasing GLP-1R expression and attenuating microglial activation against transient cerebral ischemic damage.