- Email: [email protected]
Effect of tolbutamide, glyburide and glipizide administered supraspinally on CA3 hippocampal neuronal cell death and hyperglycemia induced by kainic acid in mice
brain research 1564 (2014) 33–40
Available online at www.sciencedirect.com
www.elsevier.com/locate/brainres
Research Report
Effect of tolbutamide, glyburide and glipizide administered supraspinally on CA3 hippocampal neuronal cell death and hyperglycemia induced by kainic acid in mice Chea-Ha Kim, Soo-Hyun Park, Yun-Beom Sim, Sung-Su Kim, Su-Jin Kim, Su-Min Lim, Jun-Sub Jung, Hong-Won Suhn Department of Pharmacology, Institute of Natural Medicine, College of Medicine Hallym University, 39 Hallymdaehak-gil, Chuncheon 200-702, Gangwon-do, Republic of Korea
art i cle i nfo
ab st rac t
Article history:
Sulfonylureas are widely used oral drugs for the treatment of type II diabetes mellitus. In
Accepted 30 March 2014
the present study, the effects of sulfonylureas administered supraspinally on kainic acid
Available online 5 April 2014
(KA)-induced hippocampal neuronal cell death and hyperglycemia were studied in ICR
Keywords:
mice. Mice were pretreated intracerebroventricularly (i.c.v.) with 30 μg of tolbutamide,
Kainic acid
glyburide or glipizide for 10 min and then, mice were administered i.c.v. with KA (0.1 μg).
Neuronal cell death
The neuronal cell death in the CA3 region in the hippocampus was assessed 24 h after KA
Sulfonylureas
administration and the blood glucose level was measured 30, 60, and 120 min after KA
Blood glucose
administration. We found that i.c.v. pretreatment with tolbutamide, glyburide or glipizide
Tolbutamide
attenuated the KA-induced neuronal cell death in CA3 region of the hippocampus and
Glyburide
hyperglycemia. In addition, KA administered i.c.v. caused an elevation of plasma corti-
Glipizide
costerone level and a reduction of the plasma insulin level. The i.c.v. pretreatment with tolbutamide, glyburide or glipizide attenuated KA-induced increase of plasma corticosterone level. Furthermore, i.c.v. pretreatment with tolbutamide, glyburide or glipizide causes an elevation of plasma insulin level. Glipizide, but not tolbutamide or glyburide, pretreated i.c.v. caused a reversal of KA-induced hypoinsulinemic effect. Our results suggest that supraspinally administered tolbutamide, glyburide and glipizide exert a protective effect against KA-induced neuronal cells death in CA3 region of the hippocampus. The neuroprotective effect of tolbutamide, glyburide and glipizide appears to be mediated by lowering the blood glucose level induced by KA. & 2014 Elsevier B.V. All rights reserved.
n
Corresponding author. Fax: þ82 33 248 2612. E-mail address: [email protected] (H.-W. Suh).
http://dx.doi.org/10.1016/j.brainres.2014.03.046 0006-8993/& 2014 Elsevier B.V. All rights reserved.
34
1.
brain research 1564 (2014) 33–40
Introduction
Kainic acid (KA), an agonist for kainate and alpha-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, causes depolarization of neurons followed by severe status epilepticus (SE), neurodegeneration, plasticity, memory loss and neuronal cell death (Izquierdo et al., 2000; Zagulska-Szymczak et al., 2001; Contractor et al., 2001). KA receptors are encoded by two gene families, GluR5-6 and KA1-2, both of which have significant structural homology to AMPA receptors (Glu1-4). Glu5-7 is all known to form homomeric, non-selective action channels, and heteromers containing KA-1 and KA-2 (Herb et al., 1992; Cui and Mayer, 1999). KA activates AMPA receptors; AMPA receptors containing GluR2 are relatively Ca2þ impermeable (Washburn et al., 1997; Perkinton et al., 1999) and down-regulation of this subunit may lead to formation of Ca2þ-permeable receptors and influx of toxic amounts of Ca2þ in response to endogenous glutamate. This calcium may cause neurotoxic properties of KA (Pollard et al., 1993; Friedman et al., 1994). It is known that seizures result in altered glucose metabolism, the reduction of intracellular energy metabolites such as ATP, ADP and phosphocreatine and the accumulation of metabolic intermediates, such as lactate and adenosine (Schauwecker, 2012). Also the modulation of the glycemic index through glucose rescue greatly aggravates the extent of seizure-induced cell death following KA administration. However, the direct effects of glycemic control on brain metabolism nor the effects of managing systemic glucose concentrations in epilepsy have not been well known. Sulfonylurea is widely used for the treatment of type II diabetes mellitus. Many studies have also reported that the major blood glucose-lowering activity of sulfonylureas appears to be primarily through enhance β-cell responsiveness. Also, increase intracellular cyclic AMP exerts insulinotropic effects by closing ATP-dependent potassium channels (Groop, 1992; Lebovitz and Feinglos, 1978). Its action on blood glucose suppresses glucagon secretion, increasing peripheral insulin sensitivity without affecting insulin binding (Groop et al., 1985). Skillman and Feldman reported that the sulfonylureas potentiate the biologic effect of the insulin, increasing the deficient numbers of insulin receptors and stimulating insulin secretion on skeletal muscle, fat and liver (Skillman and Feldman, 1981). Several lines of evidence have demonstrated that seizure causes elevations of cerebral metabolic rates (Fernandes et al., 1999) and glycolysis (Fray et al., 1997). In addition, Uysal et al. (1996) have reported that insulin reduces KAinduced seizure activity. Furthermore, Koenig and Cho (2005) have shown that hypothalamic KA mRNA levels are increased in insulin-induced hypoglycemic rat, suggesting that KA receptors expression appears to be dynamically regulated depending on the level of the blood glucose. In a recent preliminary study, we found that KA administered supraspinally produces a hyperglycemic effect. In addition, we recently have reported that some of the sulfonylureas administered centrally exert the anti-diabetic effect in oral glucose tolerance test (Sim et al., 2012). However, the central pharmacological actions of sulfonylureas on KA-induced
hippocampal neuronal cell death and hyperglycemia have not been studied yet. Thus, the present study was designed to examine the effects of sulfonylureas administered supraspinally on the neuronal cell death in CA3 region of the hippocampus and hyperglycemia induced by KA administered supraspinally.
2.
Results
2.1. Effects of sulfonylureas administered i.c.v. on hippocampal CA3 neuronal cell death induced by KA We have examined the CA3 neuronal cell death using the cresyl violet stain after i.c.v. administration with KA at the dose of 0.1 μg in the hippocampus. The morphological damage induced by KA in the hippocampus was markedly concentrated in the CA3 pyramidal neurons after 1 day (Figs. 1, 2 and 3). Mice were pretreated i.c.v. with 30 μg of tolbutamide, glyburide and glipizide for 10 min and mice were administered i.c.v. with KA (0.1 μg). Cresyl vilot staining was performed 24 h after i.c.v. KA administration. As revealed in Figs. 1A, 2A, and 3A, KA-induced hippocampal CA3 neuronal death was attenuated by i.c.v. pretreatment with tolbutamide, glyburide or glipizide.
2.2. Effects of sulfonylureas administered i.c.v. on the blood glucose level induced by KA After mice were pretreated i.c.v. with 30 μg of tolbutamide, glyburide or glipizide for 10 min, mice were administered i.c. v. with KA (0.1 μg). The blood glucose level was measured at 30, 60 and 120 min after KA administration. As shown in Figs. 1B, 2B, and 3B, KA produced a hyperglycemia effect. The blood glucose level began to increase at 30 min and a KAinduced hyperglycemic effect was maintained up to 2 h after i.c.v. treatment with KA. Tolbutamide, glyburide or glipizide pretreated i.c.v. attenuated the elevation of the blood glucose level induced by KA (Figs. 1B, 2B, and 3B).
2.3. Effects of sulfonylureas administered i.c.v. on plasma corticosterone and insulin levels induced by KA To examine if the glucocorticoid and insulin systems are involved in a sulfonylureas-induced lowering effect against KA-induced hyperglycemia, effects of sulfonylureas administered i.c.v. on plasma corticosterone and insulin levels are investigated. As shown in Figs. 1C and D, 2C and D, 3C and D, i.c.v. administration with KA caused an elevation of blood corticosterone level, whereas plasma insulin level was decreased by i.c.v. KA administration. The i.c.v. pretreatment with tolbutamide, glyburide or glipizide attenuated KA-induced up-regulation of plasma corticosterone level. Furthermore, i.c.v. administration with tolbutamide, glyburide or glipizide caused an up-regulation of plasma insulin level (Figs. 1D, 2D, and 3D). However, the down-regulation of insulin level induced by KA was significantly reversed by glipizide, but not by tolbutamide or glyburide (Figs. 1D, 2D, and 3D).
35
brain research 1564 (2014) 33–40
***
Tolbutamide ++
Tolbutamide PEC+PBS PEC+KA
100
Tolbutamide+PBS
75
Tolbutamide+KA
50 25 0
Blood glucose level (mg/dl)
%control (# CA3 neuron)
125
300
*
PEC+KA Tolbutamide 30µg+PBS
+++
200 150
Tolbutamide 30µg+KA0.1µg
++
+++
++ 100 0 0
60
plasma corticosterone (ug/dl)
PEC+PBS
***
250
30
60
90
120
+++
***
(hr)
PEC+PBS PEC+KA
50
Tolbutamide 30µg + PBS Tolbutamide 30µg + KA
40 30 20 10 0
30 min +
9
***
Insulin level (ng/ml)
8
PEC+KA
7 6
PEC+PBS
Tolbutamide 30µg + PBS Tolbutamide 30µg + KA
***
5 4 3 2 1 0
30 min
Fig. 1 – Mice were pretreated i.c.v. with 30 μg of tolbutamide for 10 min. Then KA (0.1 μg /5 μl) was administered i.c.v. (A) Hippocampal neuronal death was measured 24 h after KA administration using Cresyl-violet staining. (B) The blood glucose level was measured at 30, 60 and 120 min after KA administration. The blood was collected from tail-vein. Plasma corticosterone (C) and insulin (D) levels were measured 30 min after i.c.v. injection with KA. The vertical bars indicate the standard error of mean (A: nnnPo0.001; compared to PECþPBS, þþPo0.01; compared to PECþKA, B: nnnPo0.001, nPo0.05; compared to PECþPBS, þþþPo0.001, þþPo0.01; compared to PECþKA, C: nnnPo0.001; compared to PECþPBS, þþþPo0.001; compared to PECþKA, D: nnnPo0.001; compared to PECþPBS, þPo0.05; compared to PECþKA). The number of animal used for each group was 8–10.
3.
Discussion
Previous studies have demonstrated that hypoglycemic condition rescues the neuronal cell death in stroke animal model whereas hyperglycemia condition even aggravates neuronal cell death in the animal stroke model (Nadya Kagansky et al., 2001; Tang et al., 2002). We have recently reported that some of orally active anti-diabetic drugs produce a hypoglycemic effect when they are administered centrally (Sim et al., 2012). Taken together those findings, we postulated that centrally administered oral hypoglycemic anti-diabetic drugs may protect KA-induced hippocampal CA3 neuronal death by ameliorating
the KA-induced hyperglycemic effect. To examine this hypothesis, the possible inhibitory actions of anti-diabetic drugs administered supraspinally against the hippocampal neuronal cell death and the elevation of the blood glucose level induced by KA were investigated in the present study. We found in the present study that KA induces neuronal cell death as well as hyperglycemia. In addition, we found that sulfonylureas administered supraspinally not only reduces the KA-induced hyperglycemia but also reduces the KA-induced neuronal cell death. As many studies have previously suggested that hyperglycemia may lead to neuronal cell death and glial damage. Hyperglycemia reported during ischemia may cause anaerobic glycolysis
36
brain research 1564 (2014) 33–40
Glyburide
%control (# CA3 neuron)
+
***
PEC+PBS PEC+KA
100
Glyburide+PBS
75
Glyburide+KA
50 25
Blood glucose level (mg/dl)
Glyburide 125
300
PEC+PBS
***
250
PEC+KA Glyburide 30µg+PBS Glyburide 30µg+KA0.1µg
200
++
150
++ +++
+
100
++
0 0
0
60
plasma corticosterone (ug/dl)
*
30
60
***
120 (hr)
90
*
PEC+PBS PEC+KA
50
Glyburide 30µg + PBS Glyburide 30µg + KA
40 30 20 10 0
Insulin level (ng/ml)
30 min
5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
***
PEC+PBS PEC+KA Glyburide 30µg + PBS Glyburide 30µg + KA
***
30 min
Fig. 2 – Mice were pretreated i.c.v. with 30 μg of glyburide for 10 min. Then KA (0.1 μg/5 μl) was administered i.c.v. (A) Hippocampal neuronal death was measured 24 h after KA administration using Cresyl-violet staining. (B) The blood glucose level was measured at 30, 60 and 120 min after KA administration. The blood was collected from tail-vein. Plasma corticosterone (C) and insulin (D) levels were measured 30 min after i.c.v. injection with KA. The vertical bars indicate the standard error of mean (A: nnnPo0.001; compared to PECþPBS, þPo0.05; compared to PECþKA, B: nnnPo0.001, nPo0.05; compared to PECþPBS, þþþPo0.001, þþPo0.01, þPo0.05; compared to PECþKA, C: nnnPo0.001; compared to PECþPBS, þPo0.05; compared to PECþKA, D: nnnPo0.001; compared to PECþPBS). The number of animal used for each group was 8–10.
which leads to intracellular acidosis and this also cause increase in cerebral lactate concentration resulting in neuronal and glial damage (Hoxworth et al., 1999; Siesjo et al., 1996; Anderson et al., 1999). And second mechanism is the effect of hyperglycemia on excitatory amino acids, mainly glutamate plays central role in neuronal cell death (Siesjo et al., 1995; Benveniste, 1991; Lee et al., 1999). In addition to that American Heart Association guidelines state that during cardio pulmonary resuscitation, drugs should not be given with glucose containing solutions to avoid worsening of the neurological outcome (American Heart Association. JAMA, 1992).
The results of the present study are, in part, in line with some previous studies. For example, one of sulfonylureas, tolbutamide, shows a protection in anoxic preconditioned hippocampal slices (Perez-Pinzon and Born, 1999). In addition, an anti-diabetic drug, metformin, exerts a protective effect against ethanol-induced neurodegeneration in cultured prenatal rat cortical neurons (Ullah et al., 2012). ATP-sensitive potassium channel exists in the neurons glial cells, and the brain vasculature (Sun and Hu, 2010). The opening of ATP-sensitive potassium channel may be responsible for the neuroprotection in several models such as ischemia, stroke, and in vitro model of oxygen glucose
37
brain research 1564 (2014) 33–40
Glipizide
***
+++
PEC+PBS PEC+KA
100
Glipizide+PBS Glipizide+KA
75 50 25 0
Blood glucose level (mg/dl)
%control (# CA3 neuron)
Glipizide 125
300 PEC+PBS
***
250
PEC+KA Glipizide 30µg+PBS Glipizide 30µg+KA 0.1µg
200
++
150
+++ 100
++
+
++
0 0
30
60
120 (hr)
90
+++
***
60
plasma corticosterone (ug/dl)
*
PEC+PBS PEC+KA
50
Glipizide 30µg + PBS Glipizide 30µg + KA
40 30 20 10 0
30 min +++
3.5
***
Insulin level (ng/ml)
3.0
PEC+PBS PEC+KA Glipizide 30µg + PBS
2.5
***
Glipizide 30µg + KA
2.0 1.5 1.0 0.5 0.0
30 min
Fig. 3 – Mice were pretreated i.c.v. with 30 μg of glipizide for 10 min. Then KA (0.1 μg/5 μl) was administered i.c.v. (A) Hippocampal neuronal death was measured 24 h after KA administration using cresyl-violet staining. (B) The blood glucose level was measured at 30, 60 and 120 min after KA administration. The blood was collected from tail-vein. Plasma corticosterone (C) and insulin (D) levels were measured 30 min after i.c.v. injection with KA. The vertical bars indicate the standard error of mean (A: nnnPo0.001; compared to PECþPBS, þþþPo0.001; compared to PECþKA, B: nnnPo0.001, nPo0.05; compared to PECþPBS, þþþPo0.001, þþPo0.01, þPo0.05; compared to PECþKA, C: nnnPo0.001; compared to PECþPBS, þþþ Po0.001; compared to PECþKA, D: nnnPo0.001; compared to PECþPBS, þþþPo0.001; compared to PECþKA). The number of animal used for each group was 8-10.
deprivation (Moha Ou Maati et al., 2012; Ran and Wang, 2011; Xue et al., 2011; Pérez-Pinzón and Born, 1999). However, the blockade of ATP-sensitive potassium channel appears to be also effective for protection against neuronal death (Ortega et al., 2012), leaving that the roles of centrally located ATP-sensitive potassium channel in the regulation of neuronal death is still controversial. Moreover many studies show that insulin therapy reduces ischemic brain damage and may be protective (Auer, 1998;
Wass et al., 1996). Another study reveals that insulin not only reduces the histological injury but also improves neurobehavioral outcome (Voll et al., 1989; Wass et al., 1996). To delineate the action of tolbutamide, glyburide, or glipizide against KA-induced neurotoxicity, plasma corticosterone or insulin levels were measured after KA administration. In our present study, we found that KA attenuates insulin levels and increased corticosterone level which may cause KA-induced hyperglycemia. In addition, we found in the present study
38
brain research 1564 (2014) 33–40
that supraspinal treatment with tolbutamide, glyburide, or glipizide attenuates KA-induced blood corticosterone level, suggesting that supraspinally administered tolbutamide, glyburide, or glipizide ameliorate the KA-induced hyperglycemia by reducing KA-induced up-regulation of plasma corticosterone level. Furthermore, we found in the present study that supraspinally administered tolbutamide, glyburide, or glipizide cause an elevation of blood insulin level. However, we observed that supraspinal pretreatment with tolbutamide, glyburide, or glipizide modulates KA-induced blood insulin level differentially. For example, glipizide, but not tolbutamide and glyburide, causes a reversal of KA-induced lowering insulin level, suggesting that glipizide exerts an anti-diabetic action against KA-induced hyperglycemia by producing a hyperinsulimic effect as well as reversing KA-induced hypoinsulinemic action. In contrast, both tolbutamide and glyburide exert an anti-diabetic action against KA-induced hyperglycemia by producing the hyperinsulimic effect. Moreover, a neuroprotective effect was observed in all sulfonylureas treated groups, like glipizide, tolbutamide and glyburide. In conclusion, we found in the present study that tolbutamide, glyburide and glipizide administered centrally exert a neuroprotective effect against KA-induced hippocampal CA3 neuronal death. In addition, KA-induced hyperglycemia is also reduced by centrally administered glyburide, glipizide, or tolbutamide. These findings suggest that the neuroprotective effect induced by glyburide, glipizide, or tolbutamide administered centrally against hippocampal CA3 neuronal death appears to be mediated by lowering the blood glucose level induced by KA.
4.
Experimental procedures
These experiments were approved by the Hallym University Animal Care and Use Committee (Registration number: Hallym 2013-72). All procedures were conducted in accordance with the ‘Guide for Care and Use of Laboratory Animals’ published by the National Institutes of Health.
4.1.
Experimental animals
Male ICR mice (MJ Co., Seoul, Korea) weighing 20–25 g were used for all the experiments. Animals were housed 5 per cage in a room maintained at 2270.5 1C with an alternating 12 h light–dark cycle. Food and water were available ad libitum. The animals were allowed to adapt to the laboratory for at least 2 h before testing and were only used once. Experiments were performed during the light phase of the cycle (10:00– 17:00).
4.2.
Intracerebroventricular (i.c.v.) injection
The i.c.v. injection volumes were 5 μl, and the injection sites were verified by injecting a similar volume of 1% methylene blue solution and determining the distribution of the injected dye in the ventricular space. The success rate for prior injections with this technique was over 95%. I.c.v. administration followed the method described by Haley (1957). Each mouse was grasped firmly without anesthesia by the loose
skin behind the head. The skin was pulled taut. A 30-guage needle attached to a 25 μl syringe was inserted perpendicularly through the skull into the brain and solution was injected.
4.3. Cresyl violet staining method and histological analysis After injection of KA, all mice were transcardially perfused at 30 min, 1 day post-fixed for 4 h in 4% paraformaldehyde. Brains were cryoprotected in 30% sucrose, sectioned coronally (45 μm) on a freezing micro-tome and collected inn cryoprotectant for storage at 20 1C until processed. Sections were rinsed 3 10 min in phosphate-buffered saline (PBS) to remove in cryoprotectant. Sections were mounted on microscope slides (Fair Lawn, NJ, Fisher) and dried in air. The slidemounted brain sections were soaked in cresyl violet working solution (0.02% in buffer solution; 0.2% sodium acetate, and 0.3% glacial acetic acid) for 2 min. Then, the slides were dehydrated through graded ethanol, cleared in histoclear, and cover slipped using Permount (Fair Lawn, NJ, Fisher). A histological analysis method in a pyramidal layer of hippocampal CA3 region was performed following under procedures. The number of cresyl violet-positive neurons was counted by two blinded observers at the same time using an image analyzing system equipped with a computerbased CCD camera (Olympus AX70; Center Valley, PA, USA).
4.4.
Measurement of blood glucose level
The blood glucose level was measured at 30, 60 and 120 min after i.c.v. KA administration. The blood was collected shortly as much as possible with a minimum volume (1 μl) from the tail-vein. The glucose level was measured using an AccuChek Performa blood glucose monitoring system (glucometer; Mannheim, Baden-Württemberg, Germany).
4.5.
Corticosterone assay and blood sampling
Plasma and adrenal gland corticosterone levels were determined fluorometrically according to a method published previously (Levine et al., 1967). Briefly, the blood sample from retro-orbital venous plexus was collected into a heparin treated microcentrifuge tube and centrifuged. The adrenal medulla sample was blended and centrifuged. Supernatant (50 ml) was added to 5 ml of methylene chloride and incubated at room temperature for 10 min. After filtration with cheese cloth, the mixture was combined with 2.5 ml of fluorescence reagent (7:3, sulfuric acid/absolute ethanol), vortexed vigorously, and incubated for 30 min at room temperature. After centrifugation, the lower layer was fluorometrically measured (excited wavelength¼ 475 nm; emission wavelength¼ 530 nm).
4.6.
Insulin ELISA assay
In Mouse Insulin ELISA, biotin conjugated anti insulin, and standard or samples are incubated in monoclonal antiinsulin-coated wells to capture insulin bound with biotin conjugated anti insulin. After 2 h incubation and washing,
brain research 1564 (2014) 33–40
HRP (horse radish peroxidase) conjugated streptavidin is added, and incubated for 30 min. After washing, HRP conjugated streptavidin remaining in wells is reacted with a substrate chromogen reagent (TMB) for 20 min, and reaction is stopped by addition of acidic solution, and absorbance of yellow product is measured spectrophotometrically at 450 nm. The absorbance is proportional to insulin concentration. The standard curve is prepared by plotting absorbance against standard insulin concentrations. Insulin concentrations in unknown samples are determined using this standard curve.
4.7.
Drugs
Tolbutamide, glyburide and glipizide were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Tolbutamide, glyburide and glipizide were prepared by following steps: (A) 1 g of tolbutamide, glyburide and glipizide was dissolved in 0.5 ml of ethanol plus 0.5 ml of polyethylene glycol 400. (B) Separately, 100 mg of sodium carboxymethylcellulose was dissolved in 9 ml of distilled water. (C) Finally, Solution (A) and Solution (B) were vigorously mixed. This solution (PEC) excluding tolbutamide, glyburide and glipizide were used as vehicle control. All drugs were prepared just before use. Blood glucose meter, lancing device and strips were purchased from Roche Diagnostics (Accu-Chek Performa, Germany).
4.8.
Statistical analysis
Statistical analysis was carried out by student t test GraphPad Prism Version 4.0 for Windows (GraphPad Software, San Diego, CA, USA). P-values less than 0.05 were considered to indicate statistical significance. All values were expressed as the mean7S.E.M. In our study, we established the mean blood glucose value of the control group through many experiments under matching conditions. Selected mice of established blood glucose level were then used in replication experiments.
Acknowledgments This research was supported by Priority Research Centers (NRF-2009-0094071) Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology.
r e f e r e n c e s
American Heart Association. Guidelines for cardiopulmonary resuscitation and emergency cardiac care, iii: adult advanced life support. JAMA. 1992; 26, pp. 2199–2241. Anderson, R.E., Tan, W.K., Martin, H.S., Meyer, F.B., 1999. Effects of glucose and PaO2 modulation on cortical intracellular acidosis, NADH redox state, and infarction in the ischemic penumbra. Stroke 30, 160–170. Auer, R.N., 1998. Insulin, blood glucose levels, and ischemic brain damage. Neurology 51 (S3), S39–S43. Benvensite, H., 1991. The ecxitotoxin hypothesis in relation to cerebral ischemia. Cerebrovasc. Metab. Rev. 3, 213–245.
39
Contractor, A., Swanson, G., Heinemann, S.F., 2001. Kainate receptors are involved in short- and long-term plasticity at mossy fiber synapses in the hippocampus. Neuron 29, 209–216. Cui, C., Mayer, M.L., 1999. Heteromeric kainate receptors formed by the coassembly of GluR5, GluR6, and GluR7. J. Neurosci. 19, 8281–8291. Fernandes, M.J., Dube´, C., Boyet, S., Marescaux, C., Nehlig, A., 1999. Correlation between hypermetabolism and neuronal damage during status epilepticus induced by lithium and pilocarpine in immature and adult rats. J. Cereb. Blood Flow Metab. 19, 195–209. Fray, A.E., Boutelle, M., Fillenz, M., 1997. Extracellular glucose turnover in the striatum of unanaesthetized rats measured by quantitative microdialysis. J. Physiol. 504, 721–726. Friedman, L.K., Pellegrini-Giampietro, D.E., Sperber, E.F., Bennett, M.V., Moshe´, S.L., Zukin, R.S., 1994. Kainate-induced status epilepticus alters glutamate and GABAA receptor gene expression in adult rat hippocampus: an in situ hybridization study. J. Neurosci. 14, 2697–2707. Groop, L., Wa˚hlin-Boll, E., Groop, P.H., To¨tterman, K.J., Melander, A., Tolppanen, E.M., Fyhrqvist, F., 1985. Pharmacokinetics and metabolic effects of glibenclamide and glipizide in type 2 diabetics. Eur. J. Clin. Pharmacol. 28, 697–704. Groop, L.C., 1992. Sulfonylureas in NIDDM. Diabetes Care 15, 737–754. Haley, T.J., 1957. Pharmacological effects produced by intracerebral administration of drugs of unrelated structure to conscious mice. Arch. Int. Pharmacodyn. Ther. 110, 239–244. Herb, A., Burnashev, N., Werner, P., Sakmann, B., Wisden, W., Seeburg, P.H., 1992. The KA-2 subunit of excitatory amino acid receptors shows widespread expression in brain and forms ion channels with distantly related subunits. Neuron 8, 775–785. Hoxworth, J.M., Xu, K., Zhou, Y., Lust, W.D., LaManna, J., 1999. Cerebral metabolic profile, selective neuron loss,and survival of acute and chronic hyperglycemic rats following cardiac arrest and resuscitation. Brain Res. 821, 467–479. Izquierdo, L.A., Barros, D.M., Ardenghi, P.G., Pereira, P., Rodrigues, C., Choi, H., Medina, J.H., Izquierdo, I., 2000. Different hippocampal molecular requirements for short- and longterm retrieval of one-trial avoidance learning. Behav. Brain Res. 111, 93–98. Koenig, J.I., Cho, J.Y., 2005. Provocation of kainic acid receptor mRNA changes in the rat paraventricular nucleus by insulininduced hypoglycaemia. J. Neuroendocrinol. 17, 111–118. Lebovitz, H.E., Feinglos, M.N., 1978. Sulfonylurea drugs: mechanism of antidiabetic action and therapeutic usefulness. Diabetes Care 1, 189–198. Lee, J.M., Zipfel, G.J., Choi, D.W., 1999. The changing landscape of ischaemic brain injury mechanisms. Nature 399, A7–A14. Levine, S., Glick, D., Nakane, P.K., 1967. Adrenal and plasma corticosterone and vitamin A in rat adrenal glands during postnatal development. Endocrinology 80, 910–914. Moha Ou Maati, H., Borsotto, M., Chatelain, F., Widmann, C., Lazdunski, M., Heurteaux, C., 2012. Activation of ATP-sensitive potassium channels as an element of the neuroprotective effects of the Traditional Chinese Medicine MLC901 against oxygen glucose deprivation. Neuropharmacology 63, 692–700. Nadya Kagansky, M.D., Shmuel Levy, M.D., Hilla Knobler, M.D., 2001. The role of hyperglycemia in acute stroke. Arch. Neurol. 58, 1209–1212. Ortega, F.J., Gimeno-Bayon, J., Espinosa-Parrilla, J.F., Carrasco, J.L., Batlle, M., Pugliese, M., Mahy, N., Rodrı´guez, M.J., 2012. ATPdependent potassium channel blockade strengthens microglial neuroprotection after hypoxia-ischemia in rats. Exp. Neurol. 235, 282–296.
40
brain research 1564 (2014) 33–40
Pe´rez-Pinzo´n, M.A., Born, J.G., 1999. Rapid preconditioning neuroprotection following anoxia in hippocampal slices role of the Kþ ATP channel and protein kinase C. Neuroscience 89, 453–459. Perkinton, M.S., Sihra, T.S., Williams, R.J., 1999. Ca(2þ)-permeable AMPA receptors induce phosphorylation of cAMP response element-binding protein through a phosphatidylinositol 3kinase-dependent stimulation of the mitogen-activated protein kinase signaling cascade in neurons. J. Neurosci. 19, 5861–5874. Pollard, H., He´ron, A., Moreau, J., Ben-Ari, Y., Khrestchatisky, M., 1993. Alterations of the GluR-B AMPA receptor subunit flip/ flop expression in kainate-induced epilepsy and ischemia. Neuroscience 57, 545–554. Ran, Y.H., Wang, H., 2011. Iptakalim, an ATP-sensitive potassium channel opener, confers neuroprotection against cerebral ischemia/reperfusion injury in rats by protecting neurovascular unit cells. J. Zhejiang Univ. Sci. B 12, 835–845. Schauwecker, P.E., 2012. The effects of glycemic control on seizures and seizure-induced excitotoxic cell death. BMC Neurosci. 13, 94. Siesjo¨, B.K., Zhao, Q., Pahlmark, K., Siesjo¨, P., Katsura, K., Folbergrova´, J., 1995. Glutamate, calcium, and free radicals as mediators of ischemic brain damage. Ann. Thorac. Surg. 59, 1316–1320. Siesjo¨, B.K., Katsura, K.I., Kristia´n, T., Li, P.A., Siesjo¨, P., 1996. Molecular mechanisms of acidosis-mediated damage. Acta Neurochir. Suppl. 66, 8–14. Sim, Y.B., Park, S.H., Kang, Y.J., Kim, S.S., Kim, C.H., Kim, S.J., Jung, J.S., Ryu, O.H., Choi, M.G., Suh, H.W., 2012. Central antidiabetic action of biguanide and thizolidinediones in D-glucose fed and streptozotocin-treated mouse models. Neurosci. Lett. 528, 73–77. Skillman, T.G., Feldman, J.M., 1981. The pharmacology of sulfonylureas. Am. J. Med. 70, 361–372.
Sun, X.L., Hu, G., 2010. ATP-sensitive potassium channels: a promising target for protecting neurovascular unit function in stroke. Clin. Exp. Pharmacol. Physiol. 37, 243–252. Tang, Y., Lu, A., Aronow, B.J., Wagner, K.R., Sharp, F.R., 2002. Genomic responses of the brain to ischemic stroke, intracerebral haemorrhage, kainate seizures, hypoglycemia, and hypoxia 15, 1937–1952Eur. J. Neurosci. 15, 1937–1952. Ullah, I., Ullah, N., Naseer, M.I., Lee, H.Y., Kim, M.O., 2012. Neuroprotection with metformin and thymoquinone against ethanol-induced apoptotic neurodegeneration in prenatal rat cortical neurons. BMC Neurosci. 13, 11. Uysal, H., Kuli, P., Cag˘lar, S., Inan, L.E., Akarsu, E.S., Palaog˘lu, O., Ayhan, I.H., 1996. Antiseizure activity of insulin: insulin inhibits pentylenetetrazole, penicillin and kainic acid-induced seizures in rats. Epilepsy Res. 25, 185–190. Voll, C.L., Whishaw, I.Q., Auer, R.N., 1989. Postischemic insulin reduces spatial learning deficit following transient forebrain ischemia in rats. Stroke 20, 646–651. Washburn, M.S., Numberger, M., Zhang, S., Dingledine, R., 1997. Differential dependence on GluR2 expression of three characteristic features of AMPA receptors. J. Neurosci. 17, 9393–9406. Wass, C.T., Scheithauer, B.W., Bronk, J.T., Wilson, R.M., Lanier, W.L., 1996. Insulin treatment of corticosteroid-associated hyperglycemia and its effect on outcome after forebrain ischemia in rats. Anesthesiology 84, 644–651. Xue, Y., Xie, N., Cao, L., Zhao, X., Jiang, H., Chi, Z., 2011. Diazoxide preconditioning against seizure-induced oxidative injury is via the PI3K/Akt pathway in epileptic rat. Neurosci. Lett. 495, 130–134. Zagulska-Szymczak, S., Filipkowski, R.K., Kaczmarek, L., 2001. Kainate-induced genes in the hippocampus: lessons from expression patterns. Neurochem. Int. 38, 485–501.
Available online at www.sciencedirect.com
www.elsevier.com/locate/brainres
Research Report
Effect of tolbutamide, glyburide and glipizide administered supraspinally on CA3 hippocampal neuronal cell death and hyperglycemia induced by kainic acid in mice Chea-Ha Kim, Soo-Hyun Park, Yun-Beom Sim, Sung-Su Kim, Su-Jin Kim, Su-Min Lim, Jun-Sub Jung, Hong-Won Suhn Department of Pharmacology, Institute of Natural Medicine, College of Medicine Hallym University, 39 Hallymdaehak-gil, Chuncheon 200-702, Gangwon-do, Republic of Korea
art i cle i nfo
ab st rac t
Article history:
Sulfonylureas are widely used oral drugs for the treatment of type II diabetes mellitus. In
Accepted 30 March 2014
the present study, the effects of sulfonylureas administered supraspinally on kainic acid
Available online 5 April 2014
(KA)-induced hippocampal neuronal cell death and hyperglycemia were studied in ICR
Keywords:
mice. Mice were pretreated intracerebroventricularly (i.c.v.) with 30 μg of tolbutamide,
Kainic acid
glyburide or glipizide for 10 min and then, mice were administered i.c.v. with KA (0.1 μg).
Neuronal cell death
The neuronal cell death in the CA3 region in the hippocampus was assessed 24 h after KA
Sulfonylureas
administration and the blood glucose level was measured 30, 60, and 120 min after KA
Blood glucose
administration. We found that i.c.v. pretreatment with tolbutamide, glyburide or glipizide
Tolbutamide
attenuated the KA-induced neuronal cell death in CA3 region of the hippocampus and
Glyburide
hyperglycemia. In addition, KA administered i.c.v. caused an elevation of plasma corti-
Glipizide
costerone level and a reduction of the plasma insulin level. The i.c.v. pretreatment with tolbutamide, glyburide or glipizide attenuated KA-induced increase of plasma corticosterone level. Furthermore, i.c.v. pretreatment with tolbutamide, glyburide or glipizide causes an elevation of plasma insulin level. Glipizide, but not tolbutamide or glyburide, pretreated i.c.v. caused a reversal of KA-induced hypoinsulinemic effect. Our results suggest that supraspinally administered tolbutamide, glyburide and glipizide exert a protective effect against KA-induced neuronal cells death in CA3 region of the hippocampus. The neuroprotective effect of tolbutamide, glyburide and glipizide appears to be mediated by lowering the blood glucose level induced by KA. & 2014 Elsevier B.V. All rights reserved.
n
Corresponding author. Fax: þ82 33 248 2612. E-mail address: [email protected] (H.-W. Suh).
http://dx.doi.org/10.1016/j.brainres.2014.03.046 0006-8993/& 2014 Elsevier B.V. All rights reserved.
34
1.
brain research 1564 (2014) 33–40
Introduction
Kainic acid (KA), an agonist for kainate and alpha-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, causes depolarization of neurons followed by severe status epilepticus (SE), neurodegeneration, plasticity, memory loss and neuronal cell death (Izquierdo et al., 2000; Zagulska-Szymczak et al., 2001; Contractor et al., 2001). KA receptors are encoded by two gene families, GluR5-6 and KA1-2, both of which have significant structural homology to AMPA receptors (Glu1-4). Glu5-7 is all known to form homomeric, non-selective action channels, and heteromers containing KA-1 and KA-2 (Herb et al., 1992; Cui and Mayer, 1999). KA activates AMPA receptors; AMPA receptors containing GluR2 are relatively Ca2þ impermeable (Washburn et al., 1997; Perkinton et al., 1999) and down-regulation of this subunit may lead to formation of Ca2þ-permeable receptors and influx of toxic amounts of Ca2þ in response to endogenous glutamate. This calcium may cause neurotoxic properties of KA (Pollard et al., 1993; Friedman et al., 1994). It is known that seizures result in altered glucose metabolism, the reduction of intracellular energy metabolites such as ATP, ADP and phosphocreatine and the accumulation of metabolic intermediates, such as lactate and adenosine (Schauwecker, 2012). Also the modulation of the glycemic index through glucose rescue greatly aggravates the extent of seizure-induced cell death following KA administration. However, the direct effects of glycemic control on brain metabolism nor the effects of managing systemic glucose concentrations in epilepsy have not been well known. Sulfonylurea is widely used for the treatment of type II diabetes mellitus. Many studies have also reported that the major blood glucose-lowering activity of sulfonylureas appears to be primarily through enhance β-cell responsiveness. Also, increase intracellular cyclic AMP exerts insulinotropic effects by closing ATP-dependent potassium channels (Groop, 1992; Lebovitz and Feinglos, 1978). Its action on blood glucose suppresses glucagon secretion, increasing peripheral insulin sensitivity without affecting insulin binding (Groop et al., 1985). Skillman and Feldman reported that the sulfonylureas potentiate the biologic effect of the insulin, increasing the deficient numbers of insulin receptors and stimulating insulin secretion on skeletal muscle, fat and liver (Skillman and Feldman, 1981). Several lines of evidence have demonstrated that seizure causes elevations of cerebral metabolic rates (Fernandes et al., 1999) and glycolysis (Fray et al., 1997). In addition, Uysal et al. (1996) have reported that insulin reduces KAinduced seizure activity. Furthermore, Koenig and Cho (2005) have shown that hypothalamic KA mRNA levels are increased in insulin-induced hypoglycemic rat, suggesting that KA receptors expression appears to be dynamically regulated depending on the level of the blood glucose. In a recent preliminary study, we found that KA administered supraspinally produces a hyperglycemic effect. In addition, we recently have reported that some of the sulfonylureas administered centrally exert the anti-diabetic effect in oral glucose tolerance test (Sim et al., 2012). However, the central pharmacological actions of sulfonylureas on KA-induced
hippocampal neuronal cell death and hyperglycemia have not been studied yet. Thus, the present study was designed to examine the effects of sulfonylureas administered supraspinally on the neuronal cell death in CA3 region of the hippocampus and hyperglycemia induced by KA administered supraspinally.
2.
Results
2.1. Effects of sulfonylureas administered i.c.v. on hippocampal CA3 neuronal cell death induced by KA We have examined the CA3 neuronal cell death using the cresyl violet stain after i.c.v. administration with KA at the dose of 0.1 μg in the hippocampus. The morphological damage induced by KA in the hippocampus was markedly concentrated in the CA3 pyramidal neurons after 1 day (Figs. 1, 2 and 3). Mice were pretreated i.c.v. with 30 μg of tolbutamide, glyburide and glipizide for 10 min and mice were administered i.c.v. with KA (0.1 μg). Cresyl vilot staining was performed 24 h after i.c.v. KA administration. As revealed in Figs. 1A, 2A, and 3A, KA-induced hippocampal CA3 neuronal death was attenuated by i.c.v. pretreatment with tolbutamide, glyburide or glipizide.
2.2. Effects of sulfonylureas administered i.c.v. on the blood glucose level induced by KA After mice were pretreated i.c.v. with 30 μg of tolbutamide, glyburide or glipizide for 10 min, mice were administered i.c. v. with KA (0.1 μg). The blood glucose level was measured at 30, 60 and 120 min after KA administration. As shown in Figs. 1B, 2B, and 3B, KA produced a hyperglycemia effect. The blood glucose level began to increase at 30 min and a KAinduced hyperglycemic effect was maintained up to 2 h after i.c.v. treatment with KA. Tolbutamide, glyburide or glipizide pretreated i.c.v. attenuated the elevation of the blood glucose level induced by KA (Figs. 1B, 2B, and 3B).
2.3. Effects of sulfonylureas administered i.c.v. on plasma corticosterone and insulin levels induced by KA To examine if the glucocorticoid and insulin systems are involved in a sulfonylureas-induced lowering effect against KA-induced hyperglycemia, effects of sulfonylureas administered i.c.v. on plasma corticosterone and insulin levels are investigated. As shown in Figs. 1C and D, 2C and D, 3C and D, i.c.v. administration with KA caused an elevation of blood corticosterone level, whereas plasma insulin level was decreased by i.c.v. KA administration. The i.c.v. pretreatment with tolbutamide, glyburide or glipizide attenuated KA-induced up-regulation of plasma corticosterone level. Furthermore, i.c.v. administration with tolbutamide, glyburide or glipizide caused an up-regulation of plasma insulin level (Figs. 1D, 2D, and 3D). However, the down-regulation of insulin level induced by KA was significantly reversed by glipizide, but not by tolbutamide or glyburide (Figs. 1D, 2D, and 3D).
35
brain research 1564 (2014) 33–40
***
Tolbutamide ++
Tolbutamide PEC+PBS PEC+KA
100
Tolbutamide+PBS
75
Tolbutamide+KA
50 25 0
Blood glucose level (mg/dl)
%control (# CA3 neuron)
125
300
*
PEC+KA Tolbutamide 30µg+PBS
+++
200 150
Tolbutamide 30µg+KA0.1µg
++
+++
++ 100 0 0
60
plasma corticosterone (ug/dl)
PEC+PBS
***
250
30
60
90
120
+++
***
(hr)
PEC+PBS PEC+KA
50
Tolbutamide 30µg + PBS Tolbutamide 30µg + KA
40 30 20 10 0
30 min +
9
***
Insulin level (ng/ml)
8
PEC+KA
7 6
PEC+PBS
Tolbutamide 30µg + PBS Tolbutamide 30µg + KA
***
5 4 3 2 1 0
30 min
Fig. 1 – Mice were pretreated i.c.v. with 30 μg of tolbutamide for 10 min. Then KA (0.1 μg /5 μl) was administered i.c.v. (A) Hippocampal neuronal death was measured 24 h after KA administration using Cresyl-violet staining. (B) The blood glucose level was measured at 30, 60 and 120 min after KA administration. The blood was collected from tail-vein. Plasma corticosterone (C) and insulin (D) levels were measured 30 min after i.c.v. injection with KA. The vertical bars indicate the standard error of mean (A: nnnPo0.001; compared to PECþPBS, þþPo0.01; compared to PECþKA, B: nnnPo0.001, nPo0.05; compared to PECþPBS, þþþPo0.001, þþPo0.01; compared to PECþKA, C: nnnPo0.001; compared to PECþPBS, þþþPo0.001; compared to PECþKA, D: nnnPo0.001; compared to PECþPBS, þPo0.05; compared to PECþKA). The number of animal used for each group was 8–10.
3.
Discussion
Previous studies have demonstrated that hypoglycemic condition rescues the neuronal cell death in stroke animal model whereas hyperglycemia condition even aggravates neuronal cell death in the animal stroke model (Nadya Kagansky et al., 2001; Tang et al., 2002). We have recently reported that some of orally active anti-diabetic drugs produce a hypoglycemic effect when they are administered centrally (Sim et al., 2012). Taken together those findings, we postulated that centrally administered oral hypoglycemic anti-diabetic drugs may protect KA-induced hippocampal CA3 neuronal death by ameliorating
the KA-induced hyperglycemic effect. To examine this hypothesis, the possible inhibitory actions of anti-diabetic drugs administered supraspinally against the hippocampal neuronal cell death and the elevation of the blood glucose level induced by KA were investigated in the present study. We found in the present study that KA induces neuronal cell death as well as hyperglycemia. In addition, we found that sulfonylureas administered supraspinally not only reduces the KA-induced hyperglycemia but also reduces the KA-induced neuronal cell death. As many studies have previously suggested that hyperglycemia may lead to neuronal cell death and glial damage. Hyperglycemia reported during ischemia may cause anaerobic glycolysis
36
brain research 1564 (2014) 33–40
Glyburide
%control (# CA3 neuron)
+
***
PEC+PBS PEC+KA
100
Glyburide+PBS
75
Glyburide+KA
50 25
Blood glucose level (mg/dl)
Glyburide 125
300
PEC+PBS
***
250
PEC+KA Glyburide 30µg+PBS Glyburide 30µg+KA0.1µg
200
++
150
++ +++
+
100
++
0 0
0
60
plasma corticosterone (ug/dl)
*
30
60
***
120 (hr)
90
*
PEC+PBS PEC+KA
50
Glyburide 30µg + PBS Glyburide 30µg + KA
40 30 20 10 0
Insulin level (ng/ml)
30 min
5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
***
PEC+PBS PEC+KA Glyburide 30µg + PBS Glyburide 30µg + KA
***
30 min
Fig. 2 – Mice were pretreated i.c.v. with 30 μg of glyburide for 10 min. Then KA (0.1 μg/5 μl) was administered i.c.v. (A) Hippocampal neuronal death was measured 24 h after KA administration using Cresyl-violet staining. (B) The blood glucose level was measured at 30, 60 and 120 min after KA administration. The blood was collected from tail-vein. Plasma corticosterone (C) and insulin (D) levels were measured 30 min after i.c.v. injection with KA. The vertical bars indicate the standard error of mean (A: nnnPo0.001; compared to PECþPBS, þPo0.05; compared to PECþKA, B: nnnPo0.001, nPo0.05; compared to PECþPBS, þþþPo0.001, þþPo0.01, þPo0.05; compared to PECþKA, C: nnnPo0.001; compared to PECþPBS, þPo0.05; compared to PECþKA, D: nnnPo0.001; compared to PECþPBS). The number of animal used for each group was 8–10.
which leads to intracellular acidosis and this also cause increase in cerebral lactate concentration resulting in neuronal and glial damage (Hoxworth et al., 1999; Siesjo et al., 1996; Anderson et al., 1999). And second mechanism is the effect of hyperglycemia on excitatory amino acids, mainly glutamate plays central role in neuronal cell death (Siesjo et al., 1995; Benveniste, 1991; Lee et al., 1999). In addition to that American Heart Association guidelines state that during cardio pulmonary resuscitation, drugs should not be given with glucose containing solutions to avoid worsening of the neurological outcome (American Heart Association. JAMA, 1992).
The results of the present study are, in part, in line with some previous studies. For example, one of sulfonylureas, tolbutamide, shows a protection in anoxic preconditioned hippocampal slices (Perez-Pinzon and Born, 1999). In addition, an anti-diabetic drug, metformin, exerts a protective effect against ethanol-induced neurodegeneration in cultured prenatal rat cortical neurons (Ullah et al., 2012). ATP-sensitive potassium channel exists in the neurons glial cells, and the brain vasculature (Sun and Hu, 2010). The opening of ATP-sensitive potassium channel may be responsible for the neuroprotection in several models such as ischemia, stroke, and in vitro model of oxygen glucose
37
brain research 1564 (2014) 33–40
Glipizide
***
+++
PEC+PBS PEC+KA
100
Glipizide+PBS Glipizide+KA
75 50 25 0
Blood glucose level (mg/dl)
%control (# CA3 neuron)
Glipizide 125
300 PEC+PBS
***
250
PEC+KA Glipizide 30µg+PBS Glipizide 30µg+KA 0.1µg
200
++
150
+++ 100
++
+
++
0 0
30
60
120 (hr)
90
+++
***
60
plasma corticosterone (ug/dl)
*
PEC+PBS PEC+KA
50
Glipizide 30µg + PBS Glipizide 30µg + KA
40 30 20 10 0
30 min +++
3.5
***
Insulin level (ng/ml)
3.0
PEC+PBS PEC+KA Glipizide 30µg + PBS
2.5
***
Glipizide 30µg + KA
2.0 1.5 1.0 0.5 0.0
30 min
Fig. 3 – Mice were pretreated i.c.v. with 30 μg of glipizide for 10 min. Then KA (0.1 μg/5 μl) was administered i.c.v. (A) Hippocampal neuronal death was measured 24 h after KA administration using cresyl-violet staining. (B) The blood glucose level was measured at 30, 60 and 120 min after KA administration. The blood was collected from tail-vein. Plasma corticosterone (C) and insulin (D) levels were measured 30 min after i.c.v. injection with KA. The vertical bars indicate the standard error of mean (A: nnnPo0.001; compared to PECþPBS, þþþPo0.001; compared to PECþKA, B: nnnPo0.001, nPo0.05; compared to PECþPBS, þþþPo0.001, þþPo0.01, þPo0.05; compared to PECþKA, C: nnnPo0.001; compared to PECþPBS, þþþ Po0.001; compared to PECþKA, D: nnnPo0.001; compared to PECþPBS, þþþPo0.001; compared to PECþKA). The number of animal used for each group was 8-10.
deprivation (Moha Ou Maati et al., 2012; Ran and Wang, 2011; Xue et al., 2011; Pérez-Pinzón and Born, 1999). However, the blockade of ATP-sensitive potassium channel appears to be also effective for protection against neuronal death (Ortega et al., 2012), leaving that the roles of centrally located ATP-sensitive potassium channel in the regulation of neuronal death is still controversial. Moreover many studies show that insulin therapy reduces ischemic brain damage and may be protective (Auer, 1998;
Wass et al., 1996). Another study reveals that insulin not only reduces the histological injury but also improves neurobehavioral outcome (Voll et al., 1989; Wass et al., 1996). To delineate the action of tolbutamide, glyburide, or glipizide against KA-induced neurotoxicity, plasma corticosterone or insulin levels were measured after KA administration. In our present study, we found that KA attenuates insulin levels and increased corticosterone level which may cause KA-induced hyperglycemia. In addition, we found in the present study
38
brain research 1564 (2014) 33–40
that supraspinal treatment with tolbutamide, glyburide, or glipizide attenuates KA-induced blood corticosterone level, suggesting that supraspinally administered tolbutamide, glyburide, or glipizide ameliorate the KA-induced hyperglycemia by reducing KA-induced up-regulation of plasma corticosterone level. Furthermore, we found in the present study that supraspinally administered tolbutamide, glyburide, or glipizide cause an elevation of blood insulin level. However, we observed that supraspinal pretreatment with tolbutamide, glyburide, or glipizide modulates KA-induced blood insulin level differentially. For example, glipizide, but not tolbutamide and glyburide, causes a reversal of KA-induced lowering insulin level, suggesting that glipizide exerts an anti-diabetic action against KA-induced hyperglycemia by producing a hyperinsulimic effect as well as reversing KA-induced hypoinsulinemic action. In contrast, both tolbutamide and glyburide exert an anti-diabetic action against KA-induced hyperglycemia by producing the hyperinsulimic effect. Moreover, a neuroprotective effect was observed in all sulfonylureas treated groups, like glipizide, tolbutamide and glyburide. In conclusion, we found in the present study that tolbutamide, glyburide and glipizide administered centrally exert a neuroprotective effect against KA-induced hippocampal CA3 neuronal death. In addition, KA-induced hyperglycemia is also reduced by centrally administered glyburide, glipizide, or tolbutamide. These findings suggest that the neuroprotective effect induced by glyburide, glipizide, or tolbutamide administered centrally against hippocampal CA3 neuronal death appears to be mediated by lowering the blood glucose level induced by KA.
4.
Experimental procedures
These experiments were approved by the Hallym University Animal Care and Use Committee (Registration number: Hallym 2013-72). All procedures were conducted in accordance with the ‘Guide for Care and Use of Laboratory Animals’ published by the National Institutes of Health.
4.1.
Experimental animals
Male ICR mice (MJ Co., Seoul, Korea) weighing 20–25 g were used for all the experiments. Animals were housed 5 per cage in a room maintained at 2270.5 1C with an alternating 12 h light–dark cycle. Food and water were available ad libitum. The animals were allowed to adapt to the laboratory for at least 2 h before testing and were only used once. Experiments were performed during the light phase of the cycle (10:00– 17:00).
4.2.
Intracerebroventricular (i.c.v.) injection
The i.c.v. injection volumes were 5 μl, and the injection sites were verified by injecting a similar volume of 1% methylene blue solution and determining the distribution of the injected dye in the ventricular space. The success rate for prior injections with this technique was over 95%. I.c.v. administration followed the method described by Haley (1957). Each mouse was grasped firmly without anesthesia by the loose
skin behind the head. The skin was pulled taut. A 30-guage needle attached to a 25 μl syringe was inserted perpendicularly through the skull into the brain and solution was injected.
4.3. Cresyl violet staining method and histological analysis After injection of KA, all mice were transcardially perfused at 30 min, 1 day post-fixed for 4 h in 4% paraformaldehyde. Brains were cryoprotected in 30% sucrose, sectioned coronally (45 μm) on a freezing micro-tome and collected inn cryoprotectant for storage at 20 1C until processed. Sections were rinsed 3 10 min in phosphate-buffered saline (PBS) to remove in cryoprotectant. Sections were mounted on microscope slides (Fair Lawn, NJ, Fisher) and dried in air. The slidemounted brain sections were soaked in cresyl violet working solution (0.02% in buffer solution; 0.2% sodium acetate, and 0.3% glacial acetic acid) for 2 min. Then, the slides were dehydrated through graded ethanol, cleared in histoclear, and cover slipped using Permount (Fair Lawn, NJ, Fisher). A histological analysis method in a pyramidal layer of hippocampal CA3 region was performed following under procedures. The number of cresyl violet-positive neurons was counted by two blinded observers at the same time using an image analyzing system equipped with a computerbased CCD camera (Olympus AX70; Center Valley, PA, USA).
4.4.
Measurement of blood glucose level
The blood glucose level was measured at 30, 60 and 120 min after i.c.v. KA administration. The blood was collected shortly as much as possible with a minimum volume (1 μl) from the tail-vein. The glucose level was measured using an AccuChek Performa blood glucose monitoring system (glucometer; Mannheim, Baden-Württemberg, Germany).
4.5.
Corticosterone assay and blood sampling
Plasma and adrenal gland corticosterone levels were determined fluorometrically according to a method published previously (Levine et al., 1967). Briefly, the blood sample from retro-orbital venous plexus was collected into a heparin treated microcentrifuge tube and centrifuged. The adrenal medulla sample was blended and centrifuged. Supernatant (50 ml) was added to 5 ml of methylene chloride and incubated at room temperature for 10 min. After filtration with cheese cloth, the mixture was combined with 2.5 ml of fluorescence reagent (7:3, sulfuric acid/absolute ethanol), vortexed vigorously, and incubated for 30 min at room temperature. After centrifugation, the lower layer was fluorometrically measured (excited wavelength¼ 475 nm; emission wavelength¼ 530 nm).
4.6.
Insulin ELISA assay
In Mouse Insulin ELISA, biotin conjugated anti insulin, and standard or samples are incubated in monoclonal antiinsulin-coated wells to capture insulin bound with biotin conjugated anti insulin. After 2 h incubation and washing,
brain research 1564 (2014) 33–40
HRP (horse radish peroxidase) conjugated streptavidin is added, and incubated for 30 min. After washing, HRP conjugated streptavidin remaining in wells is reacted with a substrate chromogen reagent (TMB) for 20 min, and reaction is stopped by addition of acidic solution, and absorbance of yellow product is measured spectrophotometrically at 450 nm. The absorbance is proportional to insulin concentration. The standard curve is prepared by plotting absorbance against standard insulin concentrations. Insulin concentrations in unknown samples are determined using this standard curve.
4.7.
Drugs
Tolbutamide, glyburide and glipizide were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Tolbutamide, glyburide and glipizide were prepared by following steps: (A) 1 g of tolbutamide, glyburide and glipizide was dissolved in 0.5 ml of ethanol plus 0.5 ml of polyethylene glycol 400. (B) Separately, 100 mg of sodium carboxymethylcellulose was dissolved in 9 ml of distilled water. (C) Finally, Solution (A) and Solution (B) were vigorously mixed. This solution (PEC) excluding tolbutamide, glyburide and glipizide were used as vehicle control. All drugs were prepared just before use. Blood glucose meter, lancing device and strips were purchased from Roche Diagnostics (Accu-Chek Performa, Germany).
4.8.
Statistical analysis
Statistical analysis was carried out by student t test GraphPad Prism Version 4.0 for Windows (GraphPad Software, San Diego, CA, USA). P-values less than 0.05 were considered to indicate statistical significance. All values were expressed as the mean7S.E.M. In our study, we established the mean blood glucose value of the control group through many experiments under matching conditions. Selected mice of established blood glucose level were then used in replication experiments.
Acknowledgments This research was supported by Priority Research Centers (NRF-2009-0094071) Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology.
r e f e r e n c e s
American Heart Association. Guidelines for cardiopulmonary resuscitation and emergency cardiac care, iii: adult advanced life support. JAMA. 1992; 26, pp. 2199–2241. Anderson, R.E., Tan, W.K., Martin, H.S., Meyer, F.B., 1999. Effects of glucose and PaO2 modulation on cortical intracellular acidosis, NADH redox state, and infarction in the ischemic penumbra. Stroke 30, 160–170. Auer, R.N., 1998. Insulin, blood glucose levels, and ischemic brain damage. Neurology 51 (S3), S39–S43. Benvensite, H., 1991. The ecxitotoxin hypothesis in relation to cerebral ischemia. Cerebrovasc. Metab. Rev. 3, 213–245.
39
Contractor, A., Swanson, G., Heinemann, S.F., 2001. Kainate receptors are involved in short- and long-term plasticity at mossy fiber synapses in the hippocampus. Neuron 29, 209–216. Cui, C., Mayer, M.L., 1999. Heteromeric kainate receptors formed by the coassembly of GluR5, GluR6, and GluR7. J. Neurosci. 19, 8281–8291. Fernandes, M.J., Dube´, C., Boyet, S., Marescaux, C., Nehlig, A., 1999. Correlation between hypermetabolism and neuronal damage during status epilepticus induced by lithium and pilocarpine in immature and adult rats. J. Cereb. Blood Flow Metab. 19, 195–209. Fray, A.E., Boutelle, M., Fillenz, M., 1997. Extracellular glucose turnover in the striatum of unanaesthetized rats measured by quantitative microdialysis. J. Physiol. 504, 721–726. Friedman, L.K., Pellegrini-Giampietro, D.E., Sperber, E.F., Bennett, M.V., Moshe´, S.L., Zukin, R.S., 1994. Kainate-induced status epilepticus alters glutamate and GABAA receptor gene expression in adult rat hippocampus: an in situ hybridization study. J. Neurosci. 14, 2697–2707. Groop, L., Wa˚hlin-Boll, E., Groop, P.H., To¨tterman, K.J., Melander, A., Tolppanen, E.M., Fyhrqvist, F., 1985. Pharmacokinetics and metabolic effects of glibenclamide and glipizide in type 2 diabetics. Eur. J. Clin. Pharmacol. 28, 697–704. Groop, L.C., 1992. Sulfonylureas in NIDDM. Diabetes Care 15, 737–754. Haley, T.J., 1957. Pharmacological effects produced by intracerebral administration of drugs of unrelated structure to conscious mice. Arch. Int. Pharmacodyn. Ther. 110, 239–244. Herb, A., Burnashev, N., Werner, P., Sakmann, B., Wisden, W., Seeburg, P.H., 1992. The KA-2 subunit of excitatory amino acid receptors shows widespread expression in brain and forms ion channels with distantly related subunits. Neuron 8, 775–785. Hoxworth, J.M., Xu, K., Zhou, Y., Lust, W.D., LaManna, J., 1999. Cerebral metabolic profile, selective neuron loss,and survival of acute and chronic hyperglycemic rats following cardiac arrest and resuscitation. Brain Res. 821, 467–479. Izquierdo, L.A., Barros, D.M., Ardenghi, P.G., Pereira, P., Rodrigues, C., Choi, H., Medina, J.H., Izquierdo, I., 2000. Different hippocampal molecular requirements for short- and longterm retrieval of one-trial avoidance learning. Behav. Brain Res. 111, 93–98. Koenig, J.I., Cho, J.Y., 2005. Provocation of kainic acid receptor mRNA changes in the rat paraventricular nucleus by insulininduced hypoglycaemia. J. Neuroendocrinol. 17, 111–118. Lebovitz, H.E., Feinglos, M.N., 1978. Sulfonylurea drugs: mechanism of antidiabetic action and therapeutic usefulness. Diabetes Care 1, 189–198. Lee, J.M., Zipfel, G.J., Choi, D.W., 1999. The changing landscape of ischaemic brain injury mechanisms. Nature 399, A7–A14. Levine, S., Glick, D., Nakane, P.K., 1967. Adrenal and plasma corticosterone and vitamin A in rat adrenal glands during postnatal development. Endocrinology 80, 910–914. Moha Ou Maati, H., Borsotto, M., Chatelain, F., Widmann, C., Lazdunski, M., Heurteaux, C., 2012. Activation of ATP-sensitive potassium channels as an element of the neuroprotective effects of the Traditional Chinese Medicine MLC901 against oxygen glucose deprivation. Neuropharmacology 63, 692–700. Nadya Kagansky, M.D., Shmuel Levy, M.D., Hilla Knobler, M.D., 2001. The role of hyperglycemia in acute stroke. Arch. Neurol. 58, 1209–1212. Ortega, F.J., Gimeno-Bayon, J., Espinosa-Parrilla, J.F., Carrasco, J.L., Batlle, M., Pugliese, M., Mahy, N., Rodrı´guez, M.J., 2012. ATPdependent potassium channel blockade strengthens microglial neuroprotection after hypoxia-ischemia in rats. Exp. Neurol. 235, 282–296.
40
brain research 1564 (2014) 33–40
Pe´rez-Pinzo´n, M.A., Born, J.G., 1999. Rapid preconditioning neuroprotection following anoxia in hippocampal slices role of the Kþ ATP channel and protein kinase C. Neuroscience 89, 453–459. Perkinton, M.S., Sihra, T.S., Williams, R.J., 1999. Ca(2þ)-permeable AMPA receptors induce phosphorylation of cAMP response element-binding protein through a phosphatidylinositol 3kinase-dependent stimulation of the mitogen-activated protein kinase signaling cascade in neurons. J. Neurosci. 19, 5861–5874. Pollard, H., He´ron, A., Moreau, J., Ben-Ari, Y., Khrestchatisky, M., 1993. Alterations of the GluR-B AMPA receptor subunit flip/ flop expression in kainate-induced epilepsy and ischemia. Neuroscience 57, 545–554. Ran, Y.H., Wang, H., 2011. Iptakalim, an ATP-sensitive potassium channel opener, confers neuroprotection against cerebral ischemia/reperfusion injury in rats by protecting neurovascular unit cells. J. Zhejiang Univ. Sci. B 12, 835–845. Schauwecker, P.E., 2012. The effects of glycemic control on seizures and seizure-induced excitotoxic cell death. BMC Neurosci. 13, 94. Siesjo¨, B.K., Zhao, Q., Pahlmark, K., Siesjo¨, P., Katsura, K., Folbergrova´, J., 1995. Glutamate, calcium, and free radicals as mediators of ischemic brain damage. Ann. Thorac. Surg. 59, 1316–1320. Siesjo¨, B.K., Katsura, K.I., Kristia´n, T., Li, P.A., Siesjo¨, P., 1996. Molecular mechanisms of acidosis-mediated damage. Acta Neurochir. Suppl. 66, 8–14. Sim, Y.B., Park, S.H., Kang, Y.J., Kim, S.S., Kim, C.H., Kim, S.J., Jung, J.S., Ryu, O.H., Choi, M.G., Suh, H.W., 2012. Central antidiabetic action of biguanide and thizolidinediones in D-glucose fed and streptozotocin-treated mouse models. Neurosci. Lett. 528, 73–77. Skillman, T.G., Feldman, J.M., 1981. The pharmacology of sulfonylureas. Am. J. Med. 70, 361–372.
Sun, X.L., Hu, G., 2010. ATP-sensitive potassium channels: a promising target for protecting neurovascular unit function in stroke. Clin. Exp. Pharmacol. Physiol. 37, 243–252. Tang, Y., Lu, A., Aronow, B.J., Wagner, K.R., Sharp, F.R., 2002. Genomic responses of the brain to ischemic stroke, intracerebral haemorrhage, kainate seizures, hypoglycemia, and hypoxia 15, 1937–1952Eur. J. Neurosci. 15, 1937–1952. Ullah, I., Ullah, N., Naseer, M.I., Lee, H.Y., Kim, M.O., 2012. Neuroprotection with metformin and thymoquinone against ethanol-induced apoptotic neurodegeneration in prenatal rat cortical neurons. BMC Neurosci. 13, 11. Uysal, H., Kuli, P., Cag˘lar, S., Inan, L.E., Akarsu, E.S., Palaog˘lu, O., Ayhan, I.H., 1996. Antiseizure activity of insulin: insulin inhibits pentylenetetrazole, penicillin and kainic acid-induced seizures in rats. Epilepsy Res. 25, 185–190. Voll, C.L., Whishaw, I.Q., Auer, R.N., 1989. Postischemic insulin reduces spatial learning deficit following transient forebrain ischemia in rats. Stroke 20, 646–651. Washburn, M.S., Numberger, M., Zhang, S., Dingledine, R., 1997. Differential dependence on GluR2 expression of three characteristic features of AMPA receptors. J. Neurosci. 17, 9393–9406. Wass, C.T., Scheithauer, B.W., Bronk, J.T., Wilson, R.M., Lanier, W.L., 1996. Insulin treatment of corticosteroid-associated hyperglycemia and its effect on outcome after forebrain ischemia in rats. Anesthesiology 84, 644–651. Xue, Y., Xie, N., Cao, L., Zhao, X., Jiang, H., Chi, Z., 2011. Diazoxide preconditioning against seizure-induced oxidative injury is via the PI3K/Akt pathway in epileptic rat. Neurosci. Lett. 495, 130–134. Zagulska-Szymczak, S., Filipkowski, R.K., Kaczmarek, L., 2001. Kainate-induced genes in the hippocampus: lessons from expression patterns. Neurochem. Int. 38, 485–501.