Korean J Physiol Pharmacol.  2016 Mar;20(2):185-192. 10.4196/kjpp.2016.20.2.185.

The neuroprotective mechanism of ampicillin in a mouse model of transient forebrain ischemia

• Lee KE
• Cho KO
• Choi YS
• Kim SY

• KMID: 2388675
• DOI: http://doi.org/10.4196/kjpp.2016.20.2.185

Abstract

Ampicillin, a beta-lactam antibiotic, dose-dependently protects neurons against ischemic brain injury. The present study was performed to investigate the neuroprotective mechanism of ampicillin in a mouse model of transient global forebrain ischemia. Male C57BL/6 mice were anesthetized with halothane and subjected to bilateral common carotid artery occlusion for 40 min. Before transient forebrain ischemia, ampicillin (200 mg/kg, intraperitoneally [i.p.]) or penicillin G (6,000 U/kg or 20,000 U/kg, i.p.) was administered daily for 5 days. The pretreatment with ampicillin but not with penicillin G signifi cantly attenuated neuronal damage in the hippocampal CA1 subfield. Mechanistically, the increased activity of matrix metalloproteinases (MMPs) following forebrain ischemia was also attenuated by ampicillin treatment. In addition, the ampicillin treatment reversed increased immunoreactivities to glial fibrillary acidic protein and isolectin B4, markers of astrocytes and microglia, respectively. Furthermore, the ampicillin treatment significantly increased the level of glutamate transporter-1, and dihydrokainic acid (DHK, 10 mg/kg, i.p.), an inhibitor of glutamate transporter-1 (GLT-1), reversed the neuroprotective effect of ampicillin. Taken together, these data indicate that ampicillin provides neuroprotection against ischemia-reperfusion brain injury, possibly through inducing the GLT-1 protein and inhibiting the activity of MMP in the mouse hippocampus.

Keyword

Ampicillin; Dihydrokainic acid; Glutamate transporter-1; Matrix metalloproteinase; Transient global forebrain ischemia

MeSH Terms

Ampicillin*
Animals
Astrocytes
Brain Injuries
Carotid Artery, Common
Glial Fibrillary Acidic Protein
Glutamic Acid
Halothane
Hippocampus
Humans
Ischemia*
Lectins
Male
Matrix Metalloproteinases
Mice*
Microglia
Neurons
Neuroprotective Agents
Penicillin G
Prosencephalon*
Ampicillin
Glial Fibrillary Acidic Protein
Glutamic Acid
Halothane
Lectins
Matrix Metalloproteinases
Neuroprotective Agents
Penicillin G

Figure

• 

  Fig. 1 Effect of the ampicillin pretreatment (200 mg/kg for 5 days) on delayed neuronal death in the hippocampus of mice after transient global forebrain ischemia.(A) Representative images of cresyl violet-stained brain coronal sections 3 days after transient forebrain ischemia or sham manipulation. Daily treatment with ampicillin protected the medial CA1 pyramidal cells of the hippocampus 3 days after forebrain ischemia. The scale bars in e and f indicate 200 µm and 20 µm, respectively. (B) Quantitative analysis of the neuronal damage in the saline- and ampicillin-treated groups. *p<0.05.

• 

  Fig. 2 Effect of the penicillin G sodium salt pretreatment (6,000, 20,000 U/kg, for 5 days) on delayed hippocampal neuronal death after transient global forebrain ischemia in mice.(A) Representative images of cresyl violet-stained brain coronal sections 3 days after transient forebrain ischemia or sham manipulation. The penicillin G sodium salt did not protect the medial CA1 pyramidal cells of the hippocampus 3 days after ischemia/reperfusion. The scale bars in e and f indicate 200 µm and 20 µm, respectively. (B) Quantitative analysis of the neuronal damage in the saline- and ampicillin-treated groups. There was no significant difference in neuronal damage between the saline- and penicillin G-treated groups.

• 

  Fig. 3 Glial reaction following transient forebrain ischemia and the effect of ampicillin.Representative images show that the ampicillin treatment apparently reduced the immunoreactivities of isolectin-B4 (A and B) and GFAP (C and D) in the hippocampal CA1 area. The animals were administered saline (A and C) or ampicillin (B and D) and sacrificed 3 days after transient forebrain ischemia. The scale bars indicate 50 µm in B and D.

• 

  Fig. 4 Gelatinase activity in the hippocampus.In the saline-treated group, transient forebrain ischemia induced gelatinase activity in the hippocampus. However, ampicillin treatment reversed this activity. In this study, freshly cut hippocampal sections were used for in situ zymography. Gelatinolytic activity was detected as green fluorescence. The scale bars indicate 200 µm and 50 µm in B and D, respectively.

• 

  Fig. 5 Expression level of the GLT-1 protein in the hippocampus.(A) Representative image of a western blot from the control and ampicillin-treated mice. In this study, normal mice were intraperitoneally administered ampicillin or saline for 5 days. (B) Quantitative data of GLT-1 expression in the hippocampus. The data are presented as the mean±SEM. *p<0.05.

• 

  Fig. 6 Effect of dihydrokainic acid (DHK), a selective antagonist of the glutamate transporter GLT-1, on hippocampal neuronal death following global forebrain ischemia.(A) Representative images from each group indicate that the ampicillin treatment (200 mg/kg for 5 days) reduced neuronal cell death in the medial CA1 of the hippocampus. In addition, the protective effect of ampicillin was reversed by co-administration of DHK (10 mg/kg). The scale bar indicates 20 µm. (B) The quantitative analysis of the neuronal damage indicated that the ampicillin treatment significantly reduced neuronal cell death and that DHK reversed its neuroprotective effect. Animal numbers are 6, 8, 6, and 6 for ampicillin treated-, ampicillin+DHK treated-, DHK treated-, and sham-manipulated groups, respectively. *p<0.05 by χ2 test.

Reference

1. Kirino T. Delayed neuronal death in the gerbil hippocampus following ischemia. Brain Res. 1982; 239:57–69. PMID: 7093691.

2. Lipton P. Ischemic cell death in brain neurons. Physiol Rev. 1999; 79:1431–1568. PMID: 10508238.

3. Chong W, Kim SN, Han SK, Lee SY, Ryu PD. Low non-NMDA receptor current density as possible protection mechanism from neurotoxicity of circulating glutamate on subfornical organ neurons in rats. Korean J Physiol Pharmacol. 2015; 19:177–181. PMID: 25729280.

4. Melani A, Dettori I, Corti F, Cellai L, Pedata F. Time-course of protection by the selective A2A receptor antagonist SCH58261 after transient focal cerebral ischemia. Neurol Sci. 2015; 36:1441–1448. PMID: 25805704.

5. Yu G, Wu F, Wang ES. BQ-869, a novel NMDA receptor antagonist, protects against excitotoxicity and attenuates cerebral ischemic injury in stroke. Int J Clin Exp Pathol. 2015; 8:1213–1225. PMID: 25973006.

6. Kong Q, Takahashi K, Schulte D, Stouffer N, Lin Y, Lin CL. Increased glial glutamate transporter EAAT2 expression reduces epileptogenic processes following pilocarpine-induced status epilepticus. Neurobiol Dis. 2012; 47:145–154. PMID: 22513140.

7. Zumkehr J, Rodriguez-Ortiz CJ, Cheng D, Kieu Z, Wai T, Hawkins C, Kilian J, Lim SL, Medeiros R, Kitazawa M. Ceftriaxone ameliorates tau pathology and cognitive decline via restoration of glial glutamate transporter in a mouse model of Alzheimer's disease. Neurobiol Aging. 2015; 36:2260–2271. PMID: 25964214.

8. Zhang M, Li WB, Liu YX, Liang CJ, Liu LZ, Cui X, Gong JX, Gong SJ, Hu YY, Xian XH. High expression of GLT-1 in hippocampal CA3 and dentate gyrus subfields contributes to their inherent resistance to ischemia in rats. Neurochem Int. 2011; 59:1019–1028. PMID: 21925558.

9. Fontana AC. Current approaches to enhance glutamate transporter function and expression. J Neurochem. 2015; 134:982–1007. PMID: 26096891.

10. Massie A, Cnops L, Smolders I, McCullumsmith R, Kooijman R, Kwak S, Arckens L, Michotte Y. High-affinity Na⁺/K⁺-dependent glutamate transporter EAAT4 is expressed throughout the rat fore- and midbrain. J Comp Neurol. 2008; 511:155–172. PMID: 18770868.

11. Scofield MD, Kalivas PW. Astrocytic dysfunction and addiction: consequences of impaired glutamate homeostasis. Neuroscientist. 2014; 20:610–622. PMID: 24496610.

12. Mookherjee P, Green PS, Watson GS, Marques MA, Tanaka K, Meeker KD, Meabon JS, Li N, Zhu P, Olson VG, Cook DG. GLT-1 loss accelerates cognitive deficit onset in an Alzheimer's disease animal model. J Alzheimers Dis. 2011; 26:447–455. PMID: 21677376.

13. Soni N, Reddy BV, Kumar P. GLT-1 transporter: an effective pharmacological target for various neurological disorders. Pharmacol Biochem Behav. 2014; 127:70–81. PMID: 25312503.

14. Zhou J, Sutherland ML. Glutamate transporter cluster formation in astrocytic processes regulates glutamate uptake activity. J Neurosci. 2004; 24:6301–6306. PMID: 15254085.

15. Meeker KD, Meabon JS, Cook DG. Partial loss of the glutamate transporter GLT-1 alters brain Akt and insulin signaling in a mouse model of Alzheimer's disease. J Alzheimers Dis. 2015; 45:509–520. PMID: 25589729.

16. Petr GT, Sun Y, Frederick NM, Zhou Y, Dhamne SC, Hameed MQ, Miranda C, Bedoya EA, Fischer KD, Armsen W, Wang J, Danbolt NC, Rotenberg A, Aoki CJ, Rosenberg PA. Conditional deletion of the glutamate transporter GLT-1 reveals that astrocytic GLT-1 protects against fatal epilepsy while neuronal GLT-1 contributes significantly to glutamate uptake into synaptosomes. J Neurosci. 2015; 35:5187–5201. PMID: 25834045.

17. Hu YY, Xu J, Zhang M, Wang D, Li L, Li WB. Ceftriaxone modulates uptake activity of glial glutamate transporter-1 against global brain ischemia in rats. J Neurochem. 2015; 132:194–205. PMID: 25270764.

18. Chu K, Lee ST, Sinn DI, Ko SY, Kim EH, Kim JM, Kim SJ, Park DK, Jung KH, Song EC, Lee SK, Kim M, Roh JK. Pharmacological induction of ischemic tolerance by glutamate transporter-1 (EAAT2) upregulation. Stroke. 2007; 38:177–182. PMID: 17122424.

19. Lee KE, Kim SK, Cho KO, Kim SY. Pre-ischemic treatment with ampicillin reduces neuronal damage in the mouse hippocampus and neostriatum after transient forebrain ischemia. Korean J Physiol Pharmacol. 2008; 12:287–291. PMID: 19967069.

20. Rothstein JD, Patel S, Regan MR, Haenggeli C, Huang YH, Bergles DE, Jin L, Dykes Hoberg M, Vidensky S, Chung DS, Toan SV, Bruijn LI, Su ZZ, Gupta P, Fisher PB. Beta-lactam antibiotics offer neuroprotection by increasing glutamate transporter expression. Nature. 2005; 433:73–77. PMID: 15635412.

21. Cho KO, Kim SK, Cho YJ, Sung KW, Kim SY. A simple method for predicting hippocampal neurodegeneration in a mouse model of transient global forebrain ischemia. Korean J Physiol Pharmacol. 2006; 10:167–172.

22. Zhang M, Li WB, Geng JX, Li QJ, Sun XC, Xian XH, Qi J, Li SQ. The upregulation of glial glutamate transporter-1 participates in the induction of brain ischemic tolerance in rats. J Cereb Blood Flow Metab. 2007; 27:1352–1368. PMID: 17228332.

23. Murakami K, Kondo T, Kawase M, Chan PH. The development of a new mouse model of global ischemia: focus on the relationships between ischemia duration, anesthesia, cerebral vasculature, and neuronal injury following global ischemia in mice. Brain Res. 1998; 780:304–310. PMID: 9507171.

24. Lee SR, Tsuji K, Lee SR, Lo EH. Role of matrix metalloproteinases in delayed neuronal damage after transient global cerebral ischemia. J Neurosci. 2004; 24:671–678. PMID: 14736853.

25. Pandey AK, Bhattacharya P, Shukla SC, Paul S, Patnaik R. Resveratrol inhibits matrix metalloproteinases to attenuate neuronal damage in cerebral ischemia: a molecular docking study exploring possible neuroprotection. Neural Regen Res. 2015; 10:568–575. PMID: 26170816.

26. Asahi M, Asahi K, Jung JC, del Zoppo GJ, Fini ME, Lo EH. Role for matrix metalloproteinase 9 after focal cerebral ischemia: effects of gene knockout and enzyme inhibition with BB-94. J Cereb Blood Flow Metab. 2000; 20:1681–1689. PMID: 11129784.

27. Spector R. Nature and consequences of mammalian brain and CSF efflux transporters: four decades of progress. J Neurochem. 2010; 112:13–23. PMID: 19860860.

28. Hu YY, Xu J, Zhang M, Wang D, Li L, Li WB. Ceftriaxone modulates uptake activity of glial glutamate transporter-1 against global brain ischemia in rats. J Neurochem. 2015; 132:194–205. PMID: 25270764.

29. Kelsey JE, Neville C. The effects of the β-lactam antibiotic, ceftriaxone, on forepaw stepping and L-DOPA-induced dyskinesia in a rodent model of Parkinson's disease. . Psychopharmacology (Berl). 2014; 231:2405–2415. PMID: 24402134.

30. Gao D, Huang T, Jiang X, Hu S, Zhang L, Fei Z. Resveratrol protects primary cortical neuron cultures from transient oxygen-glucose deprivation by inhibiting MMP-9. Mol Med Rep. 2014; 9:2197–2204. PMID: 24682241.

31. Kim SJ, Lee SR. Protective effect of melatonin against transient global cerebral ischemia-induced neuronal cell damage via inhibition of matrix metalloproteinase-9. Life Sci. 2014; 94:8–16. PMID: 24269215.

32. Harkness KA, Adamson P, Sussman JD, Davies-Jones GA, Greenwood J, Woodroofe MN. Dexamethasone regulation of matrix metalloproteinase expression in CNS vascular endothelium. Brain. 2000; 123:698–709. PMID: 10734001.

33. Wang X, Jung J, Asahi M, Chwang W, Russo L, Moskowitz MA, Dixon CE, Fini ME, Lo EH. Effects of matrix metalloproteinase-9 gene knock-out on morphological and motor outcomes after traumatic brain injury. J Neurosci. 2000; 20:7037–7042. PMID: 10995849.

34. Asahi M, Sumii T, Fini ME, Itohara S, Lo EH. Matrix metalloproteinase 2 gene knockout has no effect on acute brain injury after focal ischemia. Neuroreport. 2001; 12:3003–3007. PMID: 11588620.