JOURNAL OF NEUROCHEMISTRY

| 2015 | 132 | 194–205

,1

doi: 10.1111/jnc.12958

,1

,

*Department of Pathophysiology, Hebei Medical University, Shijiazhuang, China †Aging and Cognition Neuroscience Laboratory of Hebei Province, Shijiazhuang, China

Abstract Ceftriaxone(Cef) selectively increases the expression of glial glutamate transporter-1 (GLT-1), which was thought to be neuroprotective in some circumstances. However, the effect of Cef on glutamate uptake of GLT-1 was mostly assayed using in vitro studies such as primary neuron/astrocyte cultures or brain slices. In addition, the effect of Cef on neurons in different ischemic models was still discrepant. Therefore, this study was undertaken to observe the effect of Cef on neurons in global brain ischemia in rats, and especially to provide direct evidence of the up-regulation of GLT-1 uptake for glutamate contributing to the neuronal protection of Cef against brain ischemia. Neuropathological evaluation indicated that administration of Cef, especially pre-treatment protocols, significantly prevented delayed neuronal death in hippocampal CA1

subregion normally induced by global brain ischemia. Simultaneously, pre-administration of Cef significantly up-regulated the expression of GLT-1. Particularly, GLT-1 uptake assay with 3H-glutamate in living cells from adult rats showed that upregulation in glutamate uptake accompanied up-regulated GLT-1 expression. Inhibition of GLT-1 by antisense oligodeoxynucleotides or dihydrokainate significantly inhibited the Cef-induced up-regulation in GLT-1 uptake and the neuroprotective effect against global ischemia. Thus, we may conclude that Cef protects neurons against global brain ischemia via upregulation of the expression and glutamate uptake of GLT-1. Keywords: antisense oligodeoxynucleotides, ceftriaxone, global brain ischemia, glial glutamate transporter-1, glutamate uptake, rat. J. Neurochem. (2015) 132, 194–205.

Glial glutamate transporter-1 (GLT-1) plays an essential role in terminating glutamate-mediated neurotransmission, maintaining the appropriate level of extracellular glutamate, and then limiting the excitotoxicity of glutamate in the central nervous system (Raghavendra Rao et al. 2000; Namura et al. 2002; Suchak et al. 2003; Ganel et al. 2006). Knockout of GLT-1 gene or antisense knockdown of GLT-1 expression led to higher extracellular glutamate level, delayed neuronal death (DND) and significant increase in infarct areas in brain ischemic models in mice or rats (Rao et al. 2001; Mitani and Tanaka 2003). Recently, we found that cerebral ischemic preconditioning up-regulated the expression of GLT-1 and its

splice variant GLT-1a, inhibited the increase of extracellular glutamate concentration normally induced by lethal global brain ischemia and protected neurons against DND in hippocampal CA1 subregion in rat global brain ischemia model (Zhang et al. 2007; Liu et al. 2011; Liu et al. 2012). These findings suggested that the regulation of GLT-1 might provide protection for neurons against brain ischemia. It was reported that beta-lactam antibiotics, such as ceftriaxone (Cef), could significantly and selectively increase the expression of GLT-1 in vitro and in vivo studies, and Cef was found to be neuronally protective in vitro when used in models of oxygen glucose deprivation (OGD) and motor neuron 1

Received June 20, 2014; revised manuscript received September 23, 2014; accepted September 24, 2014. Address correspondence and reprint requests to Dr Wen-Bin Li, Department of Pathophysiology, Hebei Medical University, 361 Zhongshan East Road, Shijiazhuang 050017, China. E-mail: [email protected] 194

These authors contributed equally to this work. Abbreviations used: AS-ODNs, antisense oligodeoxynucleotides; Cef, ceftriaxone; cpm, counts per minute; DHK, dihydrokainate; DND, delayed neuronal death; DW, distilled water; GLT-1, glial glutamate transporter-1; ND, neuronal density; R-ODNs, random oligodeoxynucleotides.

© 2014 International Society for Neurochemistry, J. Neurochem. (2015) 132, 194--205

Cef up-regulates GLT-1 uptake against ischemia

degeneration (Rothstein et al. 2005). The neuronally protective effect of Cef was also shown in the middle cerebral artery occlusion (MCAO)-induced focal brain ischemia, two-vein occlusion-induced ischemia, and transient forebrain ischemia models (Chu et al. 2007; Ouyang et al. 2007; Th€ one-Reineke et al. 2008; Verma et al. 2010; Inui et al. 2013), and was thought to be related to the up-regulation of GLT-1 (Chu et al. 2007; Verma et al. 2010; Inui et al. 2013). However, there are still some other studies that showed contradictory effect of Cef on neurons in ischemia (Lipski et al. 2007; Beller et al. 2011; Kim and Jones 2013). Especially, considering the reverse release of glutamate as well as the failure of glutamate uptake by GLT-1 or impaired astrocytes were always thought to lead to ischemic neuronal injury (Szatkowski et al. 1990; Swanson et al. 2004), it is important to demonstrate that the upregulated expression of GLT-1 induced by Cef contributes to the up-regulation of GLT-1 uptake activity for glutamate during brain ischemia. Therefore, this study was undertaken, using a global brain ischemia model of rats, to provide direct evidence of the up-regulation of GLT-1 uptake activity contributing to the neuronal protection of Cef against brain ischemic injury.

195

performed according to the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Hebei Medical University. All efforts were made to minimize animal suffering, to reduce the number of animals used, and to utilize alternatives to in vivo techniques, if available. Experiment 1: The effect of Cef on neuronal survival after global brain ischemia Forty-five rats were randomly assigned to the following five groups (Fig. 1): Sham group (n = 5). The rats were subjected to sham operation of global brain ischemia, in which all procedures of global brain ischemia were performed except for the occlusion of the bilateral common carotid arteries. Cef control group (n = 5). The rats were intraperitoneally injected with Cef 200 mg/kg once a day for 5 days, and subjected to the sham operation of global brain ischemia 1 day after the last time of the injection. Ischemia group (n = 5). The rats were subjected to a global brain ischemia for 8 min, and then the reperfusion was recovered. Cef pre-treatment group (n = 15). The rats were intraperitoneally injected with Cef once a day for 5 days, and subjected to the global brain ischemia for 8 min 1 day after the last time of the injection. This group was further divided into 50 mg/kg, 100 mg/kg, and 200 mg/kg subgroups according to the doses of Cef (n = 5 in each subgroup).

Materials and methods Experimental animal and grouping Two hundred and ninety-seven male Wistar rats (280–320 g in weight) provided by The Experimental Animal Center of Hebei Medical University were used. All the animal studies were

Sham

Sham i.p. Cef

Cef control

Sham

Ischemia

BI

Cef pre-treatment

i.p. Cef

BI BI i.p. Cef

Cef post-treatment AS-ODNs+Cef control

i.p. Cef i.c.v. AS-ODNs

AS-ODNs+Cef pre-treatment

i.p. Cef i.c.v. AS-ODNs

BI

AS-ODNs control

i.c.v. AS-ODNs

Sham

DHK+Cef pre-treatment

i.p. Cef

Sham

i.c.v. DHK

BI

i.c.v. DHK

DHK control

i.c.v. AS-ODNs Time point

–5 days –4 days –3 days –2 days –1 days 0 days 1 days 2 days 3 days 4 days 5 days 6 days 7 days

i.p. Cef

i.p. Cef Brain ischemia

Fig. 1 The schematic of experimental protocols in each group. Abbreviations and symbols: Cef, ceftriaxone; DHK, dihydrokainate; AS-ODNs, glial glutamate transporter-1 (GLT-1) antisense oligodeoxynucleotides; i.p., intraperitoneal injection; i.c.v., intracerebroventricular injection; BI, brain ischemia; , western blotting analysis for GLT-1 expression; , western blotting analysis for GLT-1 expression and 3H-

glutamate uptake analysis of GLT-1; , western blotting analysis and immunohistochemistry assay for GLT-1 expression, and 3H-glutamate uptake analysis of GLT-1; , neuropathological evaluation. The time point of intracerebroventricular administration of DHK was 30 min before global brain ischemia.

© 2014 International Society for Neurochemistry, J. Neurochem. (2015) 132, 194--205

196

Y.-Y. Hu et al.

Cef post-treatment group (n = 15). The rats were subjected to the global brain ischemia for 8 min first, and Cef was intraperitoneally injected once a day for 5 days beginning immediately after the global brain ischemia. This group was also further divided into 50, 100, and 200 mg/kg subgroups according to the doses of Cef (n = 5 in each subgroup). The animals in all groups were killed 7 day after the sham operation (in sham and Cef control groups) or global brain ischemia (in other groups), and DND in hippocampal CA1 subregion was observed. Experiment 2: The effect of Cef on the expression and glutamate uptake of GLT-1 in the hippocampal CA1 subregion of sham and global brain ischemic rats Ninety-two rats were randomly assigned to sham, Cef control, ischemia, and Cef pre-treatment groups (n = 23 in each group). The treatments in each group were the same as those mentioned in the corresponding groups in experiment 1, except that only the dose of 200 mg/kg of Cef was selected in the Cef pre-treatment group. The expression and glutamate uptake of GLT-1 in each group were observed 12 h (n = 10, five of them were used for western blotting analysis and five for glutamate uptake) and 24 h (n = 13, five of them were used for western blotting analysis, three for immunohistochemisty and five for glutamate uptake) after the sham operation or ischemia (Fig. 1). Experiment 3: The effect of GLT-1 AS-ODNs on the Cef-induced upregulation of glutamate uptake and neuronal protection against global brain ischemia This experiment was designed to examine the effect of knockdown of the expression of GLT-1 using GLT-1 antisense oligodeoxynucleotides (AS-ODNs) on Cef-induced up-regulation of glutamate uptake and neuronal protection against global brain ischemia in rats. The results obtained from each group in experiment 2 and the corresponding groups in experiment 1 were used as control of this experiment. Based on the groups in experiment 2, the following groups were designed (Fig. 1). AS-ODNs+Cef control group (n = 40). To confirm the validity of the inhibition effect of AS-ODNs on Cef-induced GLT-1 expression, this group was designed. On the basis of the treatments in Cef control group, GLT-1 AS-ODNs solution of 10 lL was injected into the right lateral cerebral ventricle of rats 3.5, 2 day and 12 h before the sham operation. According to the doses of GLT-1 AS-ODNs, this group was further divided into 9 nmol (n = 10) and 18 nmol (n = 10) subgroups. In each subgroup, the animals were killed 12 h (n = 5) and 24 h (n = 5) after the sham operation to observe the expression of GLT-1 by western blotting analysis. Meanwhile, distilled water (solvent of AS-ODNs) group (n = 10) and GLT-1 random oligodeoxynucleotides (R-ODNs, 18 nmol) group (n = 10) were designed as control. AS-ODNs+Cef pre-treatment group (n = 95). On the basis of the treatments in Cef pre-treatment group, GLT-1 AS-ODNs solution of 10 lL was injected before the global brain ischemia in the same protocol as those in AS-ODNs+Cef control group. Two doses of 9 nmol (n = 25) and 18 nmol (n = 25) were used. In each dose group, the animals were killed 12 h (n = 10, 5 of them were used

for western blotting analysis and five for glutamate uptake) and 24 h (n = 10, five of them were used for western blotting analysis and five for glutamate uptake) after the global brain ischemia to observe the expression and glutamate uptake of GLT-1, and 7 day (n = 5) after the global brain ischemia to examine the DND in hippocampal CA1 subregion. Meanwhile, distilled water (solvent of AS-ODNs) group (n = 20) and R-ODNs (18 nmol) group (n = 25) were designed as control. AS-ODNs control group (n = 5). To observe whether the administration of GLT-1 AS-ODNs alone would induce DND in hippocampal CA1 subregion or not, GLT-1 AS-ODNs solution of 10 lL (18 nmol) was injected before the sham operation in the same protocol mentioned above. The animals were killed 7 day after the sham operation to observe the DND in hippocampal CA1 subregion. Experiment 4: The effect of DHK, a selective inhibitor of GLT-1, on the Cef-induced neuronal protection against global brain ischemia This experiment was designed to examine the effect of inhibiting GLT-1 function by dihydrokainate (DHK), a selective inhibitor of GLT-1, on the Cef-induced neuronal protection against global brain ischemia. The results obtained from the corresponding groups in experiment 1 were used as control of this experiment. Based on the groups in experiment 1, the following groups were designed (Fig. 1). DHK+Cef pre-treatment group (n = 15). On the basis of the treatments in Cef pre-treatment group, DHK solution of 20 lL was injected into the right lateral cerebral ventricle of rats 30 min before the global brain ischemia. This group was further divided into 50 nmol, 100 nmol, and 200 nmol subgroups according to the doses of DHK (n = 5 in each subgroup). The animals were killed 7 day after the global brain ischemia to observe the DND in hippocampal CA1 subregion. DHK control group (n = 5). To observe whether the administration of DHK alone would induce the DND in hippocampal CA1 subregion or not, DHK solution of 20 lL (200 nmol) was injected into sham rats in the same protocol mentioned above. The animals were killed 7 day after the sham operation to observe the DND in hippocampal CA1 subregion. Establishment of Global brain Ischemic Model The global brain ischemic model was established according to the methods of four-vessel occlusion described by Pulsinelli and Brierley (Pulsinelli and Brierley 1979). Briefly, after the exposure of the bilateral alar foramina of the first cervical vertebra under chloral hydrate anesthesia (i.p. 350 mg/kg), a heated electrocautery needle was inserted into the alar foramen and the bilateral vertebral arteries were permanently electrocauterized. Two days after the electrocauterization, the bilateral common carotid arteries of rats were exposed under ether anesthesia and local anesthesia with 1% procaine solution. After the rats recovered from ether anesthesia, the bilateral common carotid arteries were clamped using clips for 8 min to produce global brain ischemia, which has been proved to be lethal for CA1 pyramidal neurons and usually results in DND (Zhang et al. 2007). Procaine was added to the wound for pain relief in the process. Changes in consciousness, righting reflex, and

© 2014 International Society for Neurochemistry, J. Neurochem. (2015) 132, 194--205

Cef up-regulates GLT-1 uptake against ischemia

diameter of pupils were observed to determine if global brain ischemia was induced. Only animals, in which the consciousness lost, the righting reflex disappeared, and the pupils dilated obviously after four-vessel occlusion, were selected for further experiments. The elimination rate was approximately 20% of total animals and the labeled numbers in each group (i.e. ‘n’) did not include the eliminated animals. The wounds of animals were sutured after each operation. The body temperature of animals was kept at about 37°C with a heating pad and the whole body of animals was irradiated with an electric light during the above operations and treatments until normal activity of the rats recovered. The rats in sham group were subjected to all the procedures of global brain ischemia except for the occlusion of bilateral common carotid arteries. Intracerebroventricular injection The heads of rats were secured in a stereoscopic frame under general anesthesia by chloral hydrate (350 mg/kg, i.p.). A needle of 0.4 mm out-diameter connected with a microsyringe was inserted into the right lateral cerebral ventricle. The orientation was 1.5 mm lateral and 0.8 mm posterior to the bregma. Ten microliter of ODNs or 20 lL of DHK solution was injected into the ventricle over 2 min. And the needle was kept in the ventricle for an additional 2 min after the injection and then drawn out slowly. Antisense oligodeoxynucleotides (AS-ODNs) Sequences of GLT-1 AS-ODNs used in this study were as follows: 50 -ATATTGTTGGCACCCTCGGTTGAT-30 , which had been proved to be successful in inhibiting GLT-1 expression in the previous study (Rothstein et al. 1996; Rao et al. 2001). These ASODNs were synthesized with a phosphorothioate backbone. RODNs, in which the proportion of each nucleotide was identical to that of the AS-ODNs, were used as negative controls. Sequences of GLT-1 R-ODNs were as follows: 50 -AATTGTGT0 TAGCCCCCTCTGTTGA-3 (Rothstein et al. 1996; Rao et al. 2001). According to the total amount of the microinfusion used in the Rao’s study (Rao et al. 2001) and the times of our drug administration, the lyophilized oligodeoxynucleotides were diluted to a final concentration of 0.9 nmol/lL or 1.8 nmol/lL by distilled water and 10 lL of each time for total three times was administered as described above. The AS-ODNs and R-ODNs were synthesized by Sangon, Inc. Shanghai, China (Lot: M10269, M10270). Neuropathological evaluation At the determined time point of each group, the animals were deeply anesthetized and perfused through the ascending aorta with normal saline followed by 4% paraformaldehyde. The brain was then removed and a brain slice in 3 mm thick including the bilateral hippocampus was excised coronally 1 mm behind the optical chiasm. The slices were embedded in paraffin after postfixation overnight with 4% paraformaldehyde. The brain sections of 6 lm thickness were prepared and stained with thionin. DND of pyramidal neurons in CA1 hippocampal subregion was evaluated under microscope and presented quantitatively by neuronal density (ND) (Kitagawa et al. 1990; Kato et al. 1991). The value of ND was determined by counting the number of the pyramidal neurons, which possessed intact outline, full and clear

197

nucleus, within 1 mm length area in each side of the CA1 subregion. Total five sections of each rat were counted. The average number of pyramidal neurons was calculated from five rats as the final value of ND. Immunohistochemistry Sections were prepared with the same methods described in the part of Neuropathological Evaluation. After deparaffinized with xylene and hydrated in descending alcohol solutions, the sections were treated with 3% H2O2 diluted by methanol for 15 min, and then heated in 0.01 mol/L citrate buffer (pH 6.0) using microwave for 18 min to repair the antigen. After incubation of 10% normal goat serum (Lot: 060915, Zhongshan, Bejing, China) for 1 h at 37°C, the sections were incubated at 4°C over night with the primary antibody against GLT-1 (guinea pig polyclonal antibody, 1 : 2000 dilution with 0.01 M phosphate-buffered saline (PBS), Lot: LV1458674, Chemicon, Billerica, MA, USA). After rinsing, the sections were incubated in biotin-labeled anti-guinea pig IgG (1 : 500 dilution with PBS, Lot: 25010395, Chemicon) for 1 h at 37°C. Then, after rinsing, the sections were incubated in horseradish peroxidaseconjugated streptavidin working solution (Lot: s0801, Zhongshan) for 50 min at 37°C. Peroxidase of the sections was demonstrated with DAB substrate kit (Lot: 290575, Zhongshan). A computerassisted image analysis system (JEDA801D, Jieda Science and Technology Company Limited, Nanjing, China) was used for quantitative analysis of the immunohistochemical staining of GLT1. Five sections were randomly selected for the quantification of GLT-1 expression in each rat. Since GLT-1 displayed a rather diffuse staining pattern, we chose the integral optical density (IOD) for the determination of GLT-1 expression. Western blot analysis Rats were decapitated at determined time points. The hippocampal CA1 subregion was quickly separated and homogenized in fivevolume lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 12 mM C24H40O4Na, 0.1% sodium dodecyl sulfate (SDS), 1 mM EDTA, 1 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 2 lg/mL leupeptin. The homogenates were centrifuged at 14811 g for 20 min at 4°C and the supernatants were analyzed. The protein concentration was determined using Coomassie assay. Fifty-microgram protein of each sample was loaded with loading buffer containing 60 mM Tris-HCl (pH 6.8), 25% glycerol, 2% SDS, 14.4 mM b-mercaptoethanol, and 0.1% bromophenol blue. The samples were electrophoresed on 12% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride filters (Millipore Corporation, Billerica, MA, USA). The filters were blocked in 5% defatted milk for 1 h and then incubated overnight at 4°C with primary antibody against GLT-1 (guinea pig polyclonal antibody, 1 : 2000 dilution with 0.01 M PBS, Lot: LV1458674, Chemicon) and b-actin (mouse monoclonal antibody, 1 : 10 000 dilution with 0.01 M PBS, Lot: 247643, Abcam, UK). After washing with TPBS (Tween-20 and PBS mixed solution), the filters were incubated with biotin-labeled anti-guinea pig IgG for GLT-1 (1 : 4000 dilution, Lot: 25010395, Chemicon) and biotin-labeled anti-mouse IgG for b-actin (1 : 4000 dilution, Lot: 060752, KPL, Gaithersburg, MD, USA) for 1 h at 37°C. After washing with TPBS again, the filters were incubated with horseradish peroxidase-conjugated streptavidin (1 : 4000 dilution, Zymed Laboratories Inc., South San Francisco, CA, USA) for 1 h at

© 2014 International Society for Neurochemistry, J. Neurochem. (2015) 132, 194--205

Cef up-regulates GLT-1 uptake against ischemia

diameter of pupils were observed to determine if global brain ischemia was induced. Only animals, in which the consciousness lost, the righting reflex disappeared, and the pupils dilated obviously after four-vessel occlusion, were selected for further experiments. The elimination rate was approximately 20% of total animals and the labeled numbers in each group (i.e. ‘n’) did not include the eliminated animals. The wounds of animals were sutured after each operation. The body temperature of animals was kept at about 37°C with a heating pad and the whole body of animals was irradiated with an electric light during the above operations and treatments until normal activity of the rats recovered. The rats in sham group were subjected to all the procedures of global brain ischemia except for the occlusion of bilateral common carotid arteries. Intracerebroventricular injection The heads of rats were secured in a stereoscopic frame under general anesthesia by chloral hydrate (350 mg/kg, i.p.). A needle of 0.4 mm out-diameter connected with a microsyringe was inserted into the right lateral cerebral ventricle. The orientation was 1.5 mm lateral and 0.8 mm posterior to the bregma. Ten microliter of ODNs or 20 lL of DHK solution was injected into the ventricle over 2 min. And the needle was kept in the ventricle for an additional 2 min after the injection and then drawn out slowly. Antisense oligodeoxynucleotides (AS-ODNs) Sequences of GLT-1 AS-ODNs used in this study were as follows: 50 -ATATTGTTGGCACCCTCGGTTGAT-30 , which had been proved to be successful in inhibiting GLT-1 expression in the previous study (Rothstein et al. 1996; Rao et al. 2001). These ASODNs were synthesized with a phosphorothioate backbone. RODNs, in which the proportion of each nucleotide was identical to that of the AS-ODNs, were used as negative controls. Sequences of GLT-1 R-ODNs were as follows: 50 -AATTGTGT0 TAGCCCCCTCTGTTGA-3 (Rothstein et al. 1996; Rao et al. 2001). According to the total amount of the microinfusion used in the Rao’s study (Rao et al. 2001) and the times of our drug administration, the lyophilized oligodeoxynucleotides were diluted to a final concentration of 0.9 nmol/lL or 1.8 nmol/lL by distilled water and 10 lL of each time for total three times was administered as described above. The AS-ODNs and R-ODNs were synthesized by Sangon, Inc. Shanghai, China (Lot: M10269, M10270). Neuropathological evaluation At the determined time point of each group, the animals were deeply anesthetized and perfused through the ascending aorta with normal saline followed by 4% paraformaldehyde. The brain was then removed and a brain slice in 3 mm thick including the bilateral hippocampus was excised coronally 1 mm behind the optical chiasm. The slices were embedded in paraffin after postfixation overnight with 4% paraformaldehyde. The brain sections of 6 lm thickness were prepared and stained with thionin. DND of pyramidal neurons in CA1 hippocampal subregion was evaluated under microscope and presented quantitatively by neuronal density (ND) (Kitagawa et al. 1990; Kato et al. 1991). The value of ND was determined by counting the number of the pyramidal neurons, which possessed intact outline, full and clear

197

nucleus, within 1 mm length area in each side of the CA1 subregion. Total five sections of each rat were counted. The average number of pyramidal neurons was calculated from five rats as the final value of ND. Immunohistochemistry Sections were prepared with the same methods described in the part of Neuropathological Evaluation. After deparaffinized with xylene and hydrated in descending alcohol solutions, the sections were treated with 3% H2O2 diluted by methanol for 15 min, and then heated in 0.01 mol/L citrate buffer (pH 6.0) using microwave for 18 min to repair the antigen. After incubation of 10% normal goat serum (Lot: 060915, Zhongshan, Bejing, China) for 1 h at 37°C, the sections were incubated at 4°C over night with the primary antibody against GLT-1 (guinea pig polyclonal antibody, 1 : 2000 dilution with 0.01 M phosphate-buffered saline (PBS), Lot: LV1458674, Chemicon, Billerica, MA, USA). After rinsing, the sections were incubated in biotin-labeled anti-guinea pig IgG (1 : 500 dilution with PBS, Lot: 25010395, Chemicon) for 1 h at 37°C. Then, after rinsing, the sections were incubated in horseradish peroxidaseconjugated streptavidin working solution (Lot: s0801, Zhongshan) for 50 min at 37°C. Peroxidase of the sections was demonstrated with DAB substrate kit (Lot: 290575, Zhongshan). A computerassisted image analysis system (JEDA801D, Jieda Science and Technology Company Limited, Nanjing, China) was used for quantitative analysis of the immunohistochemical staining of GLT1. Five sections were randomly selected for the quantification of GLT-1 expression in each rat. Since GLT-1 displayed a rather diffuse staining pattern, we chose the integral optical density (IOD) for the determination of GLT-1 expression. Western blot analysis Rats were decapitated at determined time points. The hippocampal CA1 subregion was quickly separated and homogenized in fivevolume lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 12 mM C24H40O4Na, 0.1% sodium dodecyl sulfate (SDS), 1 mM EDTA, 1 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 2 lg/mL leupeptin. The homogenates were centrifuged at 14811 g for 20 min at 4°C and the supernatants were analyzed. The protein concentration was determined using Coomassie assay. Fifty-microgram protein of each sample was loaded with loading buffer containing 60 mM Tris-HCl (pH 6.8), 25% glycerol, 2% SDS, 14.4 mM b-mercaptoethanol, and 0.1% bromophenol blue. The samples were electrophoresed on 12% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride filters (Millipore Corporation, Billerica, MA, USA). The filters were blocked in 5% defatted milk for 1 h and then incubated overnight at 4°C with primary antibody against GLT-1 (guinea pig polyclonal antibody, 1 : 2000 dilution with 0.01 M PBS, Lot: LV1458674, Chemicon) and b-actin (mouse monoclonal antibody, 1 : 10 000 dilution with 0.01 M PBS, Lot: 247643, Abcam, UK). After washing with TPBS (Tween-20 and PBS mixed solution), the filters were incubated with biotin-labeled anti-guinea pig IgG for GLT-1 (1 : 4000 dilution, Lot: 25010395, Chemicon) and biotin-labeled anti-mouse IgG for b-actin (1 : 4000 dilution, Lot: 060752, KPL, Gaithersburg, MD, USA) for 1 h at 37°C. After washing with TPBS again, the filters were incubated with horseradish peroxidase-conjugated streptavidin (1 : 4000 dilution, Zymed Laboratories Inc., South San Francisco, CA, USA) for 1 h at

© 2014 International Society for Neurochemistry, J. Neurochem. (2015) 132, 194--205

Cef up-regulates GLT-1 uptake against ischemia

(b)

(c)

Sham

Cef control (200 mg/kg)

Ischemia

(d)

(e)

(f)

Cef pre-treatment (50 mg/kg)

Cef pre-treatment (100 mg/kg)

Cef pre-treatment (200 mg/kg)

(g)

(h)

(i)

Cef post-treatment (50 mg/kg)

Cef post-treatment (100 mg/kg)

Cef post-treatment (200 mg/kg)

*

(j) 250

Neuronal density

Fig. 2 The neuronal protection of Cef against delayed neuronal death (DND) of pyramidal neurons in the hippocampal CA1 subregion normally induced by global brain ischemia. The upper (a–i) are representative microphotographs (thionin staining, scale bar: 20 lm) of each group, and the lower bar graph (j) shows the neuronal density (ND) of each group. *p < 0.05. It could be found that pre-treatment with Cef significantly prevented the DND in CA1 subregion normally induced by the global brain ischemia in a dose-dependent manner; while post-treatment with Cef only showed a mild but significant protective effect against the DND induced by the global brain ischemia in the maximal dose.

(a)

200

*

150 100 50 0

GLT-1 AS-ODNs inhibits the Cef-induced up-regulation of glutamate uptake and neuronal protection against global brain ischemia First, we confirmed the effectiveness of GLT-1 AS-ODNs on the inhibition of the up-regulation of GLT-1 expression

* *

Sham Cef pre-treatment (mg/kg) Ischemia Cef post-treatment (mg/kg)

hippocampal CA1 subregion in Cef pre-treatment group (200 mg/kg) [Fig. 3e(iv)] in a similar feature to those in the Cef control group, and the IOD was significantly increased [Fig. 3e(v)]. Accompanied by the up-regulation of GLT-1 expression, Cef also up-regulated the glutamate uptake of GLT-1 in hippocampal CA1 subregion in sham and global brain ischemic rats. Compared with sham group, pre-treatment with Cef significantly increased the glutamate uptake of GLT-1 in Cef control group. The uptake activity of GLT-1 had no significant changes in ischemia group (Fig. 4). Compared with ischemia group, Cef pre-treatment significantly increased the glutamate uptake of GLT-1 at both observed time points of 12 h and 24 h after the global brain ischemia (Fig. 4).

199

+

+

200

+

50

100

200

+

+

+

+

+

+

50

100

200

induced by Cef on sham rats. GLT-1 expression was significantly down-regulated after the administration of ASODNs in the dose of 18 nmol in AS-ODNs+Cef control group compared with Cef control group, while there was no significant influence on the up-regulation after administration of either distilled water, GLT-1 AS-ODNs (9 nmol) or RODNs (18 nmol) (Fig. 3a and b). The results indicated that the GLT-1 AS-ODNs in the dose of 18 nmol effectively inhibited the up-regulation of GLT-1 expression induced by Cef. Based on the above results, we further observed the effect of the GLT-1 AS-ODNs on the up-regulation of GLT-1 induced by Cef during global brain ischemia. Compared with Cef pre-treatment group, the up-regulation of the GLT-1 expression induced by Cef was significantly decreased after administration of AS-ODNs in the dose of 18 nmol in ASODNs+Cef pre-treatment group, while there was no significant influence on the up-regulation after administration of either distilled water, GLT-1 AS-ODNs 9 nmol or R-ODNs (18 nmol) (Fig. 3c and d). The results indicated that the GLT-1 AS-ODNs in the dose of 18 nmol inhibited the

© 2014 International Society for Neurochemistry, J. Neurochem. (2015) 132, 194--205

200

Y.-Y. Hu et al.

(a)

(b) (24 h)

(12 h)

GLT-1

72 kDa

GLT-1/β-actin

β-actin

43 kDa

*

0.8

*

0.8

0.6

0.6

0.4

0.4

0.2

0.2

0 Sham Cef pre-treatment DW control AS-ODNs (nmol) R-ODNs (nmol)

(c)

*

*

0

+

+ + +

+ +

+ +

+ +

9

18

+

+ +

+ + +

+ +

+ +

+ +

9

18

+ +

18

18

(d) (24 h)

(12 h)

72 kDa

β-actin

43 kDa

*

*

0.8 0.6

0.6

0.4

0.4

0.2

0.2

0 Sham Ischemia Cef pre-treatment DW control AS-ODNs (nmol) R-ODNs (nmol)

*

*

0.8

0

+

+

(e) i

+ +

+ + +

+ +

+ +

9

18

iii

+

+ +

+ + +

+ +

+ +

9

18

v

Cef control 24 h

+ + 18

18

ii

Sham 24 h

+

+ +

*

*

120 IOD

GLT-1/β-actin

GLT-1

80 40

iv 0

Ischemia 24 h

Cef pre-treatment 24 h (200 mg/kg)

Sham Cef pre-treatment Ischemia

+

up-regulation of GLT-1 expression induced by Cef in rats subjected to the global brain ischemia as well. Having confirmed the effectiveness of the GLT-1 ASODNs above, we observed the effect of GLT-1 AS-ODNs on the Cef-induced up-regulation in glutamate uptake of GLT-1 and neuronal protection against global brain ischemia in rats. It was shown that, compared with Cef pre-treatment group, administration of GLT-1 AS-ODNs significantly inhibited the Cef-induced up-regulation in the glutamate uptake of GLT-1 at the two observed time points of 12 and 24 h after the global brain ischemia in AS-ODNs+Cef pre-treatment group, especially in the larger dose of 18 nmol group; while there was no significant change in the uptake activity in distilled water and R-ODNs control groups compared with the Cef pre-treatment group (Fig. 4). The results indicated that the Cef-induced up-regulation in the glutamate uptake of GLT-1 was at least partly resulted from the Cef-induced upregulation of GLT-1 expression. Accompanied by the inhibition of the Cef-induced upregulation in the glutamate uptake of GLT-1, obvious

+ +

+

+ +

Fig. 3 The effect of Cef on the expression of glial glutamate transporter-1 (GLT-1) in hippocampal CA1 subregion and the effect of GLT-1 AS-ODNs on the Cef-induced upregulation of GLT-1 expression assayed with western blotting analysis (a–d) and immunohistochemistry (e). a and b show the results in sham rats, and the time points indicate the time after sham operation. c and d show the results in brain ischemic rats, and the time points indicate the time after brain ischemia. In a to d, the upper is representative immunoblot bands of the western blotting analysis in each group; the lower is a quantitative presentation of the immunoblots in each group with integral optical density (IOD). The ratio of the IOD of immunoblot of the aim protein to that of bactin is used for statistical analysis. The representative microphotographs [e(i-iv)] show the changes of GLT-1 expression in each group (Scale bar: 20 lm). The bar graph [e(v)] is the quantitative presentation of the immunohistochemical staining in e(i– iv) with IOD. *p < 0.05. It could be found that Cef significantly up-regulated the GLT-1 expression in hippocampal CA1 subregion of sham and global brain ischemic rats. The GLT-1 AS-ODNs in the dose of 18 nmol effectively inhibited the up-regulation of GLT-1 expression induced by Cef.

destruction and significant decrease in the value of ND in the CA1 subregion were observed in AS-ODNs (18 nmol)+Cef pre-treatment group (Fig. 5e and i), compared with Cef pre-treatment group. The GLT-1 AS-ODNs in lower dose of 9 nmol and R-ODNs in the dose of 18 nmol had no significant effect on the neuronal protection of Cef (Fig. 5f, g and i). This result indicated that the AS-ODNs significantly inhibited the neuronal protection of Cef against DND normally induced by global brain ischemia. In addition, administration of GLT-1 AS-ODNs in the dose of 18 nmol to sham rats (AS-ODNs control group) had no significant influence on the histological morphology of the CA1 subregion compared with sham group (Fig. 5h and i). This indicated that the GLT-1 AS-ODNs alone did not induce DND of pyramidal neurons in the CA1 subregion. DHK, a selective inhibitor of GLT-1, inhibits the Cef-induced neuronal protection against global brain ischemia To further examine the mechanism underlying the Cefinduced neuronal protection against global brain ischemia,

© 2014 International Society for Neurochemistry, J. Neurochem. (2015) 132, 194--205

Cef up-regulates GLT-1 uptake against ischemia

(b)

(c)

Sham

Cef control (200 mg/kg)

Ischemia

(d)

(e)

(f)

Cef pre-treatment (50 mg/kg)

Cef pre-treatment (100 mg/kg)

Cef pre-treatment (200 mg/kg)

(g)

(h)

(i)

Cef post-treatment (50 mg/kg)

Cef post-treatment (100 mg/kg)

Cef post-treatment (200 mg/kg)

*

(j) 250

Neuronal density

Fig. 2 The neuronal protection of Cef against delayed neuronal death (DND) of pyramidal neurons in the hippocampal CA1 subregion normally induced by global brain ischemia. The upper (a–i) are representative microphotographs (thionin staining, scale bar: 20 lm) of each group, and the lower bar graph (j) shows the neuronal density (ND) of each group. *p < 0.05. It could be found that pre-treatment with Cef significantly prevented the DND in CA1 subregion normally induced by the global brain ischemia in a dose-dependent manner; while post-treatment with Cef only showed a mild but significant protective effect against the DND induced by the global brain ischemia in the maximal dose.

(a)

200

*

150 100 50 0

GLT-1 AS-ODNs inhibits the Cef-induced up-regulation of glutamate uptake and neuronal protection against global brain ischemia First, we confirmed the effectiveness of GLT-1 AS-ODNs on the inhibition of the up-regulation of GLT-1 expression

* *

Sham Cef pre-treatment (mg/kg) Ischemia Cef post-treatment (mg/kg)

hippocampal CA1 subregion in Cef pre-treatment group (200 mg/kg) [Fig. 3e(iv)] in a similar feature to those in the Cef control group, and the IOD was significantly increased [Fig. 3e(v)]. Accompanied by the up-regulation of GLT-1 expression, Cef also up-regulated the glutamate uptake of GLT-1 in hippocampal CA1 subregion in sham and global brain ischemic rats. Compared with sham group, pre-treatment with Cef significantly increased the glutamate uptake of GLT-1 in Cef control group. The uptake activity of GLT-1 had no significant changes in ischemia group (Fig. 4). Compared with ischemia group, Cef pre-treatment significantly increased the glutamate uptake of GLT-1 at both observed time points of 12 h and 24 h after the global brain ischemia (Fig. 4).

199

+

+

200

+

50

100

200

+

+

+

+

+

+

50

100

200

induced by Cef on sham rats. GLT-1 expression was significantly down-regulated after the administration of ASODNs in the dose of 18 nmol in AS-ODNs+Cef control group compared with Cef control group, while there was no significant influence on the up-regulation after administration of either distilled water, GLT-1 AS-ODNs (9 nmol) or RODNs (18 nmol) (Fig. 3a and b). The results indicated that the GLT-1 AS-ODNs in the dose of 18 nmol effectively inhibited the up-regulation of GLT-1 expression induced by Cef. Based on the above results, we further observed the effect of the GLT-1 AS-ODNs on the up-regulation of GLT-1 induced by Cef during global brain ischemia. Compared with Cef pre-treatment group, the up-regulation of the GLT-1 expression induced by Cef was significantly decreased after administration of AS-ODNs in the dose of 18 nmol in ASODNs+Cef pre-treatment group, while there was no significant influence on the up-regulation after administration of either distilled water, GLT-1 AS-ODNs 9 nmol or R-ODNs (18 nmol) (Fig. 3c and d). The results indicated that the GLT-1 AS-ODNs in the dose of 18 nmol inhibited the

© 2014 International Society for Neurochemistry, J. Neurochem. (2015) 132, 194--205

202

Y.-Y. Hu et al.

(a)

(b)

(c)

(d)

Sham

Cef control

Ischemia

Cef pre-treatment (200 mg/kg)

(e)

(f)

(g)

(h)

DHK(50 nmol) +Cef pre-treatment

DHK(100 nmol) +Cef pre-treatment

DHK(200 nmol) +Cef pre-treatment

DHK control (200 nmol)

Neuronal density

*

*

(i) 250

*

200 150 100 50 0

Sham Cef pre-treatment Ischemia DHK (nmol)

+

+ +

+

+ +

+ 200

+ +

+ +

+ +

50

100

200

Ouyang et al. 2007; Th€ one-Reineke et al. 2008; Verma et al. 2010; Inui et al. 2013); while Cef treatment initially worsened motor performance besides the absence of reducing the ischemic injury size in an endothelin-1-induced focal ischemic model (Kim and Jones 2013). This negative effect of Cef was also shown in in vitro studies. For example, high dose of Cef was toxic to both astrocytes and neurons even in the absence of a glutamate challenge in a neuron/astrocyte coculture model (Beller et al. 2011). More interestingly, in acute hippocampal slices, Cef treatment was neuronally protective against OGD and increased the activity of glutamate transporters; while in chronic organotypic hippocampal slice cultures, neither the neuronal protection against OGD or glutamate-evoked cell death nor the increase of GLT-1 protein was found after Cef treatment (Lipski et al. 2007). Our study, using a global brain ischemia model combined with GLT-1 AS-ODNs and GLT-1 selective inhibitor DHK, further demonstrated the neuronally protective effect of Cef against brain ischemic injury. These results supports the notion mentioned above that Cef has a neuronally protective effect against brain ischemic injury. Thus, the discrepancy effect of Cef on neurons in ischemia might be related to models of ischemia, specific developmental stages/age, or the timing/dose of Cef treatment (Lipski et al. 2007). Furthermore, our study provided evidence to demonstrate the important role of GLT-1 up-regulation in the neuronally protective effect of Cef. First, we found that administration of Cef significantly up-regulated GLT-1 expression in the

Fig. 6 The effect of DHK, a selective inhibitor of glial glutamate transporter-1 (GLT-1), on the Cef-induced neuronal protection against global brain ischemia in rats. The upper (a–h) are representative microphotographs (thionin staining, scale bar: 20 lm) of each group, and the lower bar graph shows the neuronal density (ND) of each group. *p < 0.05. It could be found that DHK significantly inhibited the neuronal protection of Cef against delayed neuronal death (DND) in the hippocampal CA1 subregion induced by global brain ischemia in a dose-dependent manner; while DHK alone did not induce DND of pyramidal neurons in the hippocampal CA1 subregion.

hippocampal CA1 subregion of rats subjected to global brain ischemia. It is well known that the overflow of glutamate plays a critical role in ischemic injury (Benveniste et al. 1984; Rothman and Olney 1986; Torp et al. 1993; Liu et al. 2012). A large concentration gradient of glutamate is maintained across the plasma membrane by sodium-dependent glutamate transporters, primarily by GLT-1, in normal condition (Danbolt 2001; Doyle et al. 2008; Kim et al. 2011). After brain ischemia, GLT-1 mRNA and protein levels significantly decreased and the maximal amplitude of glutamate transporter currents significantly reduced, which all preceded the initiation of DND (Yeh et al. 2005; Ketheeswaranathan et al. 2011). The inherent high expression of GLT-1 in hippocampal CA3 and dentate gyrus subfield contributed to their native resistance to ischemic insult (Zhang et al. 2011). These reports suggested that the abnormality in expression and function of GLT-1 played an essential role in the development of ischemic insult. Therefore, the Cef-induced up-regulation of GLT-1 expression in this study might, in turn, change the function of GLT-1 (i.e., increase the glutamate uptake), reduce the accumulation of glutamate and then protect neurons from excitotoxicity. Thus, the determination of functional changes of GLT-1 is necessary for interpreting the neuronally protective effect of Cef against brain ischemia. Although the effect of Cef on glutamate uptake of GLT-1 has been reported in some previous studies, most of the uptake was observed in primary neuron/astrocyte cultures, brain slices, gliosomes, or via microdialysis. Primary neuron/astrocyte cultures or

© 2014 International Society for Neurochemistry, J. Neurochem. (2015) 132, 194--205

204

Y.-Y. Hu et al.

up-regulation might resist the down-regulation of GLT-1 expression in the early stage after the brain ischemic insult, and made the GLT-1 expression to be not obviously decreased until 5 days after the global brain ischemia (Zhang et al. 2007). Although the GLT-1 expression and function are not significantly decreased in the early stage after the global brain ischemia in this study, the basal expression of GLT-1 is not sufficient to remove the rapid elevated extracellular glutamate induced by the ischemia. Thus, the neurons would still be injured because of the excitotoxicity. Our previous findings that the inherent higher expression of GLT-1 in hippocampal CA3 and dentate gyrus subfield contributed to their native resistance to ischemic insult (Zhang et al. 2011) support the insufficient elimination of glutamate by the inherent GLT-1 in CA1 subfield after the global brain ischemic insult for 8 min. As to the administration of GLT-1 AS-ODNs, we referred to the previous study of Rao et al. (2001) with some modifications, in which the continuous perfusion of the GLT1 AS-ODNs to the lateral cerebroventricle was replaced by three times injection to the lateral cerebroventricle with the approximately equal dose. In our previous studies, we have observed the inhibitory effect of this antisense delivery approach (three times injection), and found that this delivery approach effectively knocked down the expression of GLT-1 by approximately 70–80% in sham rats (Geng et al. 2008). In this study, this delivery approach also effectively knocked down the expression of GLT-1 by approximately 60% in Cef-treated sham or ischemia rats whose GLT-1 had been upregulated by Cef. Furthermore, the antisense delivery did not induce neuronal deficit in hippocampal CA1 subfield in our previous (Geng et al. 2008) and the present studies. These results indicated that this antisense delivery approach is effective in knocking down GLT-1 and safe to hippocampal neurons.

Acknowledgments and conflict of interest disclosure This work was supported by Key Basic Research Project in Application Plan of Hebei Province, China (No: 11966121D); National Natural Science Foundation of China (No: 81271454, No: 31271149, No: 81000477, and No: 31100781); Special Foundation for Doctoral Education in University from Ministry of Education, China (No: 20111323110005). No author has a conflict of interest in this study. All experiments were conducted in compliance with the ARRIVE guidelines.

References Apric o K., Beart P. M., Lawrence A. J., Crawford D. and O’Shea R. D. (2001) [(3)H](2S,4R)-4-Methylglutamate: a novel ligand for the characterization of glutamate transporters. J. Neurochem. 77, 1218–1225.

Aprico K., Beart P. M., Crawford D. and O’Shea R. D. (2004) Binding and transport of [3H](2S,4R)-4-methylglutamate, a new ligand for glutamate transporters, demonstrate labeling of EAAT1 in cultured murine astrocytes. J. Neurosci. Res. 75, 751–759. Beller J. A., Gurkoff G. G., Berman R. F. and Lyeth B. G. (2011) Pharmacological enhancement of glutamate transport reduces excitotoxicity in vitro. Restor. Neurol. Neurosci. 29, 331–346. Benveniste H., Drejer J., Schousboe A. and Diemer N. H. (1984) Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis. J. Neurochem. 43, 1369– 1374. Chu K., Lee S. T., Sinn D. I. et al. (2007) Pharmacological induction of ischemic tolerance by glutamate transporter-1 (EAAT2) upregulation. Stroke 38, 177–182. Danbolt N. C. (2001) Glutamate uptake. Prog. Neurobiol. 65, 1–105. Doyle K. P., Simon R. P. and Stenzel-Poore M. P. (2008) Mechanisms of ischemic brain damage. Neuropharmacology 55, 310–318. Fekety F. R. (1990) Safety of parenteral third-generation cephalosporins. Am. J. Med. 88, 38S–44S. Ganel R., Ho T., Maragakis N. J., Jackson M., Steiner J. P. and Rothstein J. D. (2006) Selective upregulation of the glial Na+-dependent glutamate transporter GLT1 by a neuroimmunophilin ligand results in neuroprotection. Neurobiol. Dis. 21, 556–567. Geng J. X., Cai J. S., Zhang M., Li S. Q., Sun X. C., Xian X. H., Hu Y. Y., Li W. B. and Li Q. J. (2008) Antisense oligodeoxynucleotides of glial glutamate transporter-1 inhibits the neuro-protection of cerebral ischemic preconditioning in rats. Sheng Li Xue Bao 60, 497–503. Harvey B. K., Airavaara M., Hinzman J., Wires E. M., Chiocco M. J., Howard D. B., Shen H., Gerhardt G., Hoffer B. J. and Wang Y. (2011) Targeted over-expression of glutamate transporter 1 (GLT1) reduces ischemic brain injury in a rat model of stroke. PLoS ONE 6, e22135. Inui T., Alessandri B., Heimann A., Nishimura F., Frauenknecht K., Sommer C. and Kempski O. (2013) Neuroprotective effect of ceftriaxone on the penumbra in a rat venous ischemia model. Neuroscience 242, 1–10. Kato H., Liu Y., Araki T. and Kogure K. (1991) Temporal profile of the effects of pretreatment with brief cerebral ischemia on the neuronal damage following secondary ischemic insult in the gerbil: cumulative damage and protective effects. Brain Res. 553, 238– 242. Ketheeswaranathan P., Turner N. A., Spary E. J., Batten T. F., McColl B. W. and Saha S. (2011) Changes in glutamate transporter expression in mouse forebrain areas following focal ischemia. Brain Res. 1418, 93–103. Kim S. Y. and Jones T. A. (2013) The effects of ceftriaxone on skill learning and motor functional outcome after ischemic cortical damage in rats. Restor. Neurol. Neurosci. 31, 87–97. Kim K., Lee S. G., Kegelman T. P. et al. (2011) Role of excitatory amino acid transporter-2 (EAAT2) and glutamate in neurodegeneration: opportunities for developing novel therapeutics. J. Cell. Physiol. 226, 2484–2493. Kitagawa K., Matsumoto M., Tagaya M. et al. (1990) ‘Ischemic tolerance’ phenomenon found in the brain. Brain Res. 528, 21–24. Lipski J., Wan C. K., Bai J. Z., Pi R., Li D. and Donnelly D. (2007) Neuroprotective potential of ceftriaxone in in vitro models of stroke. Neuroscience 146, 617–629. Liu A. J., Hu Y. Y., Li W. B., Xu J. and Zhang M. (2011) Cerebral ischemic pre-conditioning enhances the binding characteristics and glutamate uptake of glial glutamate transporter-1 in hippocampal CA1 subfield of rats. J. Neurochem. 119, 202–209.

© 2014 International Society for Neurochemistry, J. Neurochem. (2015) 132, 194--205

204

Y.-Y. Hu et al.

up-regulation might resist the down-regulation of GLT-1 expression in the early stage after the brain ischemic insult, and made the GLT-1 expression to be not obviously decreased until 5 days after the global brain ischemia (Zhang et al. 2007). Although the GLT-1 expression and function are not significantly decreased in the early stage after the global brain ischemia in this study, the basal expression of GLT-1 is not sufficient to remove the rapid elevated extracellular glutamate induced by the ischemia. Thus, the neurons would still be injured because of the excitotoxicity. Our previous findings that the inherent higher expression of GLT-1 in hippocampal CA3 and dentate gyrus subfield contributed to their native resistance to ischemic insult (Zhang et al. 2011) support the insufficient elimination of glutamate by the inherent GLT-1 in CA1 subfield after the global brain ischemic insult for 8 min. As to the administration of GLT-1 AS-ODNs, we referred to the previous study of Rao et al. (2001) with some modifications, in which the continuous perfusion of the GLT1 AS-ODNs to the lateral cerebroventricle was replaced by three times injection to the lateral cerebroventricle with the approximately equal dose. In our previous studies, we have observed the inhibitory effect of this antisense delivery approach (three times injection), and found that this delivery approach effectively knocked down the expression of GLT-1 by approximately 70–80% in sham rats (Geng et al. 2008). In this study, this delivery approach also effectively knocked down the expression of GLT-1 by approximately 60% in Cef-treated sham or ischemia rats whose GLT-1 had been upregulated by Cef. Furthermore, the antisense delivery did not induce neuronal deficit in hippocampal CA1 subfield in our previous (Geng et al. 2008) and the present studies. These results indicated that this antisense delivery approach is effective in knocking down GLT-1 and safe to hippocampal neurons.

Acknowledgments and conflict of interest disclosure This work was supported by Key Basic Research Project in Application Plan of Hebei Province, China (No: 11966121D); National Natural Science Foundation of China (No: 81271454, No: 31271149, No: 81000477, and No: 31100781); Special Foundation for Doctoral Education in University from Ministry of Education, China (No: 20111323110005). No author has a conflict of interest in this study. All experiments were conducted in compliance with the ARRIVE guidelines.

References Apric o K., Beart P. M., Lawrence A. J., Crawford D. and O’Shea R. D. (2001) [(3)H](2S,4R)-4-Methylglutamate: a novel ligand for the characterization of glutamate transporters. J. Neurochem. 77, 1218–1225.

Aprico K., Beart P. M., Crawford D. and O’Shea R. D. (2004) Binding and transport of [3H](2S,4R)-4-methylglutamate, a new ligand for glutamate transporters, demonstrate labeling of EAAT1 in cultured murine astrocytes. J. Neurosci. Res. 75, 751–759. Beller J. A., Gurkoff G. G., Berman R. F. and Lyeth B. G. (2011) Pharmacological enhancement of glutamate transport reduces excitotoxicity in vitro. Restor. Neurol. Neurosci. 29, 331–346. Benveniste H., Drejer J., Schousboe A. and Diemer N. H. (1984) Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis. J. Neurochem. 43, 1369– 1374. Chu K., Lee S. T., Sinn D. I. et al. (2007) Pharmacological induction of ischemic tolerance by glutamate transporter-1 (EAAT2) upregulation. Stroke 38, 177–182. Danbolt N. C. (2001) Glutamate uptake. Prog. Neurobiol. 65, 1–105. Doyle K. P., Simon R. P. and Stenzel-Poore M. P. (2008) Mechanisms of ischemic brain damage. Neuropharmacology 55, 310–318. Fekety F. R. (1990) Safety of parenteral third-generation cephalosporins. Am. J. Med. 88, 38S–44S. Ganel R., Ho T., Maragakis N. J., Jackson M., Steiner J. P. and Rothstein J. D. (2006) Selective upregulation of the glial Na+-dependent glutamate transporter GLT1 by a neuroimmunophilin ligand results in neuroprotection. Neurobiol. Dis. 21, 556–567. Geng J. X., Cai J. S., Zhang M., Li S. Q., Sun X. C., Xian X. H., Hu Y. Y., Li W. B. and Li Q. J. (2008) Antisense oligodeoxynucleotides of glial glutamate transporter-1 inhibits the neuro-protection of cerebral ischemic preconditioning in rats. Sheng Li Xue Bao 60, 497–503. Harvey B. K., Airavaara M., Hinzman J., Wires E. M., Chiocco M. J., Howard D. B., Shen H., Gerhardt G., Hoffer B. J. and Wang Y. (2011) Targeted over-expression of glutamate transporter 1 (GLT1) reduces ischemic brain injury in a rat model of stroke. PLoS ONE 6, e22135. Inui T., Alessandri B., Heimann A., Nishimura F., Frauenknecht K., Sommer C. and Kempski O. (2013) Neuroprotective effect of ceftriaxone on the penumbra in a rat venous ischemia model. Neuroscience 242, 1–10. Kato H., Liu Y., Araki T. and Kogure K. (1991) Temporal profile of the effects of pretreatment with brief cerebral ischemia on the neuronal damage following secondary ischemic insult in the gerbil: cumulative damage and protective effects. Brain Res. 553, 238– 242. Ketheeswaranathan P., Turner N. A., Spary E. J., Batten T. F., McColl B. W. and Saha S. (2011) Changes in glutamate transporter expression in mouse forebrain areas following focal ischemia. Brain Res. 1418, 93–103. Kim S. Y. and Jones T. A. (2013) The effects of ceftriaxone on skill learning and motor functional outcome after ischemic cortical damage in rats. Restor. Neurol. Neurosci. 31, 87–97. Kim K., Lee S. G., Kegelman T. P. et al. (2011) Role of excitatory amino acid transporter-2 (EAAT2) and glutamate in neurodegeneration: opportunities for developing novel therapeutics. J. Cell. Physiol. 226, 2484–2493. Kitagawa K., Matsumoto M., Tagaya M. et al. (1990) ‘Ischemic tolerance’ phenomenon found in the brain. Brain Res. 528, 21–24. Lipski J., Wan C. K., Bai J. Z., Pi R., Li D. and Donnelly D. (2007) Neuroprotective potential of ceftriaxone in in vitro models of stroke. Neuroscience 146, 617–629. Liu A. J., Hu Y. Y., Li W. B., Xu J. and Zhang M. (2011) Cerebral ischemic pre-conditioning enhances the binding characteristics and glutamate uptake of glial glutamate transporter-1 in hippocampal CA1 subfield of rats. J. Neurochem. 119, 202–209.

© 2014 International Society for Neurochemistry, J. Neurochem. (2015) 132, 194--205

Cef up-regulates GLT-1 uptake against ischemia

Liu Y. X., Zhang M., Liu L. Z., Cui X., Hu Y. Y. and Li W. B. (2012) The role of glutamate transporter-1a in the induction of brain ischemic tolerance in rats. Glia 60, 112–124. Mitani A. and Tanaka K. (2003) Functional changes of glial glutamate transporter GLT-1 during ischemia: an in vivo study in the hippocampal CA1 of normal mice and mutant mice lacking GLT-1. J. Neurosci. 23, 7176–7182. Namura S., Maeno H., Takami S., Jiang X. F., Kamichi S., Wada K. and Nagata I. (2002) Inhibition of glial glutamate transporter GLT-1 augments brain edema after transient focal cerebral ischemia in mice. Neurosci. Lett. 324, 117–120. Ouyang Y. B., Voloboueva L. A., Xu L. J. and Giffard R. G. (2007) Selective dysfunction of hippocampal CA1 astrocytes contributes to delayed neuronal damage after transient forebrain ischemia. J. Neurosci. 27, 4253–4260. Pulsinelli W. A. and Brierley J. B. (1979) A new model of bilateral hemispheric ischemia in the unanesthetized rat. Stroke 10, 267– 272. Raghavendra Rao V. L., Rao A. M., Dogan A., Bowen K. K., Hatcher J., Rothstein J. D. and Dempsey R. J. (2000) Glial glutamate transporter GLT-1 down-regulation precedes delayed neuronal death in gerbil hippocampus following transient global cerebral ischemia. Neurochem. Int. 36, 531–537. Rao V. L., Dogan A., Todd K. G., Bowen K. K., Kim B. T., Rothstein J. D. and Dempsey R. J. (2001) Antisense knockdown of the glial glutamate transporter GLT-1, but not the neuronal glutamate transporter EAAC1, exacerbates transient focal cerebral ischemiainduced neuronal damage in rat brain. J. Neurosci. 21, 1876–1883. Rothman S. M. and Olney J. W. (1986) Glutamate and the pathophysiology of hypoxic–ischemic brain damage. Ann. Neurol. 19, 105–111. Rothstein J. D., Dykes-Hoberg M., Pardo C. A. et al. (1996) Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate. Neuron 16, 675–686. Rothstein J. D., Patel S., Regan M. R. et al. (2005) Beta-lactam antibiotics offer neuroprotection by increasing glutamate transporter expression. Nature. 433, 73–77. Suchak S. K., Baloyianni N. V., Perkinton M. S., Williams R. J., Meldrum B. S. and Rattray M. (2003) The ‘glial’ glutamate transporter, EAAT2 (Glt-1) accounts for high affinity glutamate

205

uptake into adult rodent nerve endings. J. Neurochem. 84, 522– 532. Swanson R. A., Ying W. and Kauppinen T. M. (2004) Astrocyte influences on ischemic neuronal death. Curr. Mol. Med. 4, 193– 205. Szatkowski M., Barbour B. and Attwell D. (1990) Non-vesicular release of glutamate from glial cells by reversed electrogenic glutamate uptake. Nature 348, 443–446. Tanaka K., Watase K., Manabe T. et al. (1997) Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1. Science 276, 1699–1702. Th€one-Reineke C., Neumann C., Namsolleck P. et al. (2008) The betalactam antibiotic, ceftriaxone, dramatically improves survival, increases glutamate uptake and induces neurotrophins in stroke. J. Hypertens. 26, 2426–2435. Torp R., Arvin B., Le Peillet E., Chapman A. G., Ottersen O. P. and Meldrum B. S. (1993) Effect of ischaemia and reperfusion on the extra- and intracellular distribution of glutamate, glutamine, aspartate and GABA in the rat hippocampus, with a note on the effect of the sodium channel blocker BW1003C87. Exp. Brain Res. 96, 365–376. Verma R., Mishra V., Sasmal D. and Raghubir R. (2010) Pharmacological evaluation of glutamate transporter 1 (GLT-1) mediated neuroprotection following cerebral ischemia/reperfusion injury. Eur. J. Pharmacol. 638, 65–71. Yang X., He Z., Zhang Q. et al. (2012) Pre-Ischemic treadmill training for prevention of ischemic brain injury via regulation of glutamate and its transporter GLT-1. Int. J. Mol. Sci. 13, 9447–9459. Yeh T. H., Hwang H. M., Chen J. J., Wu T., Li A. H. and Wang H. L. (2005) Glutamate transporter function of rat hippocampal astrocytes is impaired following the global ischemia. Neurobiol. Dis. 18, 476–483. Zhang M., Li W. B., Geng J. X., Li Q. J., Sun X. C., Xian X. H., Qi J. and Li S. Q. (2007) The upregulation of glial glutamate transporter1 participates in the induction of brain ischemic tolerance in rats. J. Cereb. Blood Flow Metab. 27, 1352–1368. Zhang M., Li W. B., Liu Y. X., Liang C. J., Liu L. Z., Cui X., Gong J. X., Gong S. J., Hu Y. Y. and Xian X. H. (2011) High expression of GLT-1 in hippocampal CA3 and dentate gyrus subfields contributes to their inherent resistance to ischemia in rats. Neurochem. Int. 59, 1019–1028.

© 2014 International Society for Neurochemistry, J. Neurochem. (2015) 132, 194--205