Korean J Physiol Pharmacol.  2018 May;22(3):301-309. 10.4196/kjpp.2018.22.3.301.

Atorvastatin pretreatment attenuates kainic acid-induced hippocampal neuronal death via regulation of lipocalin-2-associated neuroinflammation

Affiliations
  • 1Department of Anatomy and Convergence Medical Science, Institute of Health Sciences, College of Medicine, Gyeongsang National University, Jinju 52727, Korea. anaroh@gnu.ac.kr
  • 2Department of Neurology, Changwon Fatima Hospital, Changwon 51394, Korea.
  • 3Department of Thoracic and Cardiovascular Surgery, Institute of Health Sciences, College of Medicine, Gyeongsang National University, Jinju 52727, Korea.
  • 4Department of Neurology, Institute of Health Sciences, College of Medicine, Gyeongsang National University, Jinju 52727, Korea. ncchoi@nate.com

Abstract

Statins mediate vascular protection and reduce the prevalence of cardiovascular diseases. Recent work indicates that statins have anticonvulsive effects in the brain; however, little is known about the precise mechanism for its protective effect in kainic acid (KA)-induced seizures. Here, we investigated the protective effects of atorvastatin pretreatment on KA-induced neuroinflammation and hippocampal cell death. Mice were treated via intragastric administration of atorvastatin for 7 days, injected with KA, and then sacrificed after 24 h. We observed that atorvastatin pretreatment reduced KA-induced seizure activity, hippocampal cell death, and neuroinflammation. Atorvastatin pretreatment also inhibited KA-induced lipocalin-2 expression in the hippocampus and attenuated KA-induced hippocampal cyclooxygenase-2 expression and glial activation. Moreover, AKT phosphorylation in KA-treated hippocampus was inhibited by atorvastatin pretreatment. These findings suggest that atorvastatin pretreatment may protect hippocampal neurons during seizures by controlling lipocalin-2-associated neuroinflammation.

Keyword

Atorvastatin; Hippocampal cell death; Kainic acid; Lipocalin-2; Seizures

MeSH Terms

Animals
Atorvastatin Calcium*
Brain
Cardiovascular Diseases
Cell Death
Cyclooxygenase 2
Hippocampus
Hydroxymethylglutaryl-CoA Reductase Inhibitors
Kainic Acid
Mice
Neurons*
Phosphorylation
Prevalence
Seizures
Atorvastatin Calcium
Cyclooxygenase 2
Kainic Acid

Figure

  • Fig. 1 Effect of KA treatment on hippocampal LCN2 expression in mice.(A) Western blot showing altered LCN2 expression in KA-treated hippocampus. The hippocampal LCN2 expression appeared 6 h after KA treatment. (B) Quantitative expression of LCN2 protein from the western blot analysis. Densitometric values for LCN2 were normalized to β-actin levels. The mean values were obtained from two separate experiments (n=5-6 mice per group). Data are shown as mean±SEM. *p<0.05 vs. KA.

  • Fig. 2 Effect of atorvastatin pretreatment on seizure activity and hippocampal cell death in KA-treated mice.At 24 h after KA systemic injection, seizure activity was scored during the 2 h observation period. Behavioral seizure scores (A) and percentage of survival (B) in KA-treated mice with or without AS are shown. Data (n=15 mice per group) are shown as mean±SEM. *p<0.05 vs. KA. (C) Representative microphotographs of cresyl violet-stained sections and immunofluorescent images of TUNEL-stained sections in CA3 regions. The boxed area of cresyl violet-stained images is magnified in the center panel. Scale bar=50 mm.

  • Fig. 3 Effect of atorvastatin pretreatment on LCN2 expression in KA-treated hippocampus.(A) Western blots show hippocampal LCN2 expression 24 h after KA treatment. Densitometric values of LCN2 expression from three separate experiments (n=6–7 mice per group) were normalized to β-actin levels. *p<0.05 vs. CTL. †p<0.05 vs. KA. Representative images of double-immunofluorescence staining for LCN2 (red) and GFAP (green) (B) and LCN2 (red) and Iba-1 (green) (C) in the CA3 region of CTL, KA, KA+AS, and AS mice. Scale bar=50 µm.

  • Fig. 4 Effect of atorvastatin pretreatment on neuroinflammation in KA-treated hippocampus.(A) Western blots show hippocampal COX-2 expression 24 h after KA treatment. Densitometric values of COX-2 expression from three separate experiments (n=6–7 mice per group) were normalized to β-actin levels. *p<0.05 vs. CTL. †p<0.05 vs. KA. (B) Representative images of immunostained COX-2 in the hippocampus of CTL, KA, KA+AS, and AS mice. The boxed area of each image is magnified in the lower panel. Scale bar=50 mm. (C) Western blots show hippocampal GFAP (C) and Iba-1 (D) expression 24 h after KA treatment. Densitometric values (n=6–7 mice per group) for GFAP and Iba1 were normalized to β-actin levels. *p<0.05 vs. CTL. †p<0.05 vs. KA-treated mice.

  • Fig. 5 Effect of atorvastatin pretreatment on AKT phosphorylation in KA-treated hippocampus.(A) Western blot shows p-AKT and AKT expression in KA-treated hippocampus. (B) Quantitative expression of p-AKT and AKT protein from the western blot analysis. Densitometric values of expression of each protein from three separate experiments (n=6–7 mice per group) were normalized to β-actin levels. Data are shown as mean±SEM. *p<0.05 vs. CTL. †p<0.05 vs. KA-treated mice.


Reference

1. McCord MC, Lorenzana A, Bloom CS, Chancer ZO, Schauwecker PE. Effect of age on kainate-induced seizure severity and cell death. Neuroscience. 2008; 154:1143–1153. PMID: 18479826.
Article
2. Carta M, Fièvre S, Gorlewicz A, Mulle C. Kainate receptors in the hippocampus. Eur J Neurosci. 2014; 39:1835–1844. PMID: 24738709.
Article
3. Ravizza T, Rizzi M, Perego C, Richichi C, Velísková J, Moshé SL, De Simoni MG, Vezzani A. Inflammatory response and glia activation in developing rat hippocampus after status epilepticus. Epilepsia. 2005; 46(Suppl 5):113–117. PMID: 15987264.
Article
4. Kim JE, Ryu HJ, Choi SY, Kang TC. Tumor necrosis factor-α-mediated threonine 435 phosphorylation of p65 nuclear factor-κB subunit in endothelial cells induces vasogenic edema and neutrophil infiltration in the rat piriform cortex following status epilepticus. J Neuroinflammation. 2012; 9:6. PMID: 22240205.
Article
5. Shin HJ, Jeon BT, Kim J, Jeong EA, Kim MJ, Lee DH, Kim HJ, Kang SS, Cho GJ, Choi WS, Roh GS. Effect of the calcineurin inhibitor FK506 on K+-Cl-cotransporter 2 expression in the mouse hippocampus after kainic acid-induced status epilepticus. J Neural Transm (Vienna). 2012; 119:669–677. PMID: 22160488.
6. Xie WJ, Dong M, Liu Q, Meng HM. Early predictors and prevention for post-stroke epilepsy: changes in neurotransmitter levels. Transl Neurosci. 2016; 7:1–5. PMID: 28123814.
Article
7. Ní Chróinín D, Asplund K, Åsberg S, Callaly E, Cuadrado-Godia E, Díez-Tejedor E, Di Napoli M, Engelter ST, Furie KL, Giannopoulos S, Gotto AM Jr, Hannon N, Jonsson F, Kapral MK, Martí-Fàbregas J, Martínez-Sánchez P, Milionis HJ, Montaner J, Muscari A, Pikija S, Probstfield J, Rost NS, Thrift AG, Vemmos K, Kelly PJ. Statin therapy and outcome after ischemic stroke: systematic review and meta-analysis of observational studies and randomized trials. Stroke. 2013; 44:448–456. PMID: 23287777.
Article
8. Li Q, Zhuang QK, Yang JN, Zhang YY. Statins excert neuroprotection on cerebral ischemia independent of their lipid-lowering action: the potential molecular mechanisms. Eur Rev Med Pharmacol Sci. 2014; 18:1113–1126. PMID: 24817284.
9. Pannu R, Christie DK, Barbosa E, Singh I, Singh AK. Post-trauma Lipitor treatment prevents endothelial dysfunction, facilitates neuroprotection, and promotes locomotor recovery following spinal cord injury. J Neurochem. 2007; 101:182–200. PMID: 17217414.
Article
10. Lee JK, Won JS, Singh AK, Singh I. Statin inhibits kainic acid-induced seizure and associated inflammation and hippocampal cell death. Neurosci Lett. 2008; 440:260–264. PMID: 18583044.
Article
11. Wang L, Zhang X, Liu L, Yang R, Cui L, Li M. Atorvastatin protects rat brains against permanent focal ischemia and downregulates HMGB1, HMGB1 receptors (RAGE and TLR4), NF-kappaB expression. Neurosci Lett. 2010; 471:152–156. PMID: 20100543.
12. Goetz DH, Holmes MA, Borregaard N, Bluhm ME, Raymond KN, Strong RK. The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition. Mol Cell. 2002; 10:1033–1043. PMID: 12453412.
Article
13. Devireddy LR, Gazin C, Zhu X, Green MR. A cell-surface receptor for lipocalin 24p3 selectively mediates apoptosis and iron uptake. Cell. 2005; 123:1293–1305. PMID: 16377569.
Article
14. Flo TH, Smith KD, Sato S, Rodriguez DJ, Holmes MA, Strong RK, Akira S, Aderem A. Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron. Nature. 2004; 432:917–921. PMID: 15531878.
Article
15. Lee S, Kim JH, Kim JH, Seo JW, Han HS, Lee WH, Mori K, Nakao K, Barasch J, Suk K. Lipocalin-2 is a chemokine inducer in the central nervous system: role of chemokine ligand 10 (CXCL10) in lipocalin-2-induced cell migration. J Biol Chem. 2011; 286:43855–43870. PMID: 22030398.
16. Jin M, Kim JH, Jang E, Lee YM, Soo Han H, Woo DK, Park DH, Kook H, Suk K. Lipocalin-2 deficiency attenuates neuroinflammation and brain injury after transient middle cerebral artery occlusion in mice. J Cereb Blood Flow Metab. 2014; 34:1306–1314. PMID: 24780901.
Article
17. Lee S, Park JY, Lee WH, Kim H, Park HC, Mori K, Suk K. Lipocalin-2 is an autocrine mediator of reactive astrocytosis. J Neurosci. 2009; 29:234–249. PMID: 19129400.
Article
18. Sehar N, Agarwal NB, Vohora D, Raisuddin S. Atorvastatin prevents development of kindling by modulating hippocampal levels of dopamine, glutamate, and GABA in mice. Epilepsy Behav. 2015; 42:48–53. PMID: 25499163.
Article
19. Funck VR, de Oliveira CV, Pereira LM, Rambo LM, Ribeiro LR, Royes LF, Ferreira J, Guerra GP, Furian AF, Oliveira MS, Mallmann CA, de Mello CF, Oliveira MS. Differential effects of atorvastatin treatment and withdrawal on pentylenetetrazol-induced seizures. Epilepsia. 2011; 52:2094–2104. PMID: 21906051.
Article
20. Hu RQ, Koh S, Torgerson T, Cole AJ. Neuronal stress and injury in C57/BL mice after systemic kainic acid administration. Brain Res. 1998; 810:229–240. PMID: 9813346.
Article
21. Kang DH, Heo RW, Yi CO, Kim H, Choi CH, Roh GS. High-fat diet-induced obesity exacerbates kainic acid-induced hippocampal cell death. BMC Neurosci. 2015; 16:72. PMID: 26518260.
Article
22. Mucha M, Skrzypiec AE, Schiavon E, Attwood BK, Kucerova E, Pawlak R. Lipocalin-2 controls neuronal excitability and anxiety by regulating dendritic spine formation and maturation. Proc Natl Acad Sci U S A. 2011; 108:18436–18441. PMID: 21969573.
Article
23. Skrzypiec AE, Shah RS, Schiavon E, Baker E, Skene N, Pawlak R, Mucha M. Stress-induced lipocalin-2 controls dendritic spine formation and neuronal activity in the amygdala. PLoS One. 2013; 8:e61046. PMID: 23593384.
Article
24. Jeong EA, Jeon BT, Shin HJ, Kim N, Lee DH, Kim HJ, Kang SS, Cho GJ, Choi WS, Roh GS. Ketogenic diet-induced peroxisome proliferator-activated receptor-γ activation decreases neuroinflammation in the mouse hippocampus after kainic acid-induced seizures. Exp Neurol. 2011; 232:195–202. PMID: 21939657.
Article
25. Chen J, Zhang ZG, Li Y, Wang Y, Wang L, Jiang H, Zhang C, Lu M, Katakowski M, Feldkamp CS, Chopp M. Statins induce angiogenesis, neurogenesis, and synaptogenesis after stroke. Ann Neurol. 2003; 53:743–751. PMID: 12783420.
Article
26. Piermartiri TC, Vandresen-Filho S, de Araújo Herculano B, Martins WC, Dal'agnolo D, Stroeh E, Carqueja CL, Boeck CR, Tasca CI. Atorvastatin prevents hippocampal cell death due to quinolinic acid-induced seizures in mice by increasing Akt phosphorylation and glutamate uptake. Neurotox Res. 2009; 16:106–115. PMID: 19526287.
Article
27. Pursnani A, Massaro JM, D'Agostino RB Sr, O'Donnell CJ, Hoffmann U. Guideline-based statin eligibility, coronary artery calcification, and cardiovascular events. JAMA. 2015; 314:134–141. PMID: 26172893.
Article
28. Castilla-Guerra L, Fernández-Moreno Mdel C, López-Chozas JM. Statins in the secondary prevention of stroke: New evidence from the SPARCL Study. Clin Investig Arterioscler. 2016; 28:202–208.
29. Takemoto M, Yokote K. Prevention, treatment and management of inflammation in atherosclerosis. Nihon Rinsho. 2011; 69:18–24. PMID: 21226254.
30. Wood WG, Mΰller WE, Eckert GP. Statins and neuroprotection: basic pharmacology needed. Mol Neurobiol. 2014; 50:214–220. PMID: 24474514.
Article
31. Ramirez C, Tercero I, Pineda A, Burgos JS. Simvastatin is the statin that most efficiently protects against kainate-induced excitotoxicity and memory impairment. J Alzheimers Dis. 2011; 24:161–174.
Article
32. Gouveia TL, Scorza FA, Silva MJ, Bandeira Tde A, Perosa SR, Argañaraz GA, Silva Mde P, Araujo TR, Frangiotti MI, Amado D, Cavalheiro EA, Silva JA Jr, Naffah-Mazzacoratti Mda G. Lovastatin decreases the synthesis of inflammatory mediators in the hippocampus and blocks the hyperthermia of rats submitted to longlasting status epilepticus. Epilepsy Behav. 2011; 20:1–5. PMID: 21130693.
Article
33. Saito T, Nito C, Ueda M, Inaba T, Kamiya F, Muraga K, Katsura K, Katayama Y. Continuous oral administration of atorvastatin ameliorates brain damage after transient focal ischemia in rats. Life Sci. 2014; 94:106–114. PMID: 24333133.
Article
34. Chia WJ, Dawe GS, Ong WY. Expression and localization of the iron-siderophore binding protein lipocalin 2 in the normal rat brain and after kainate-induced excitotoxicity. Neurochem Int. 2011; 59:591–599. PMID: 21683107.
Article
35. Jang E, Lee S, Kim JH, Kim JH, Seo JW, Lee WH, Mori K, Nakao K, Suk K. Secreted protein lipocalin-2 promotes microglial M1 polarization. FASEB J. 2013; 27:1176–1190. PMID: 23207546.
Article
36. Lee S, Lee J, Kim S, Park JY, Lee WH, Mori K, Kim SH, Kim IK, Suk K. A dual role of lipocalin 2 in the apoptosis and deramification of activated microglia. J Immunol. 2007; 179:3231–3241. PMID: 17709539.
Article
37. Xing C, Wang X, Cheng C, Montaner J, Mandeville E, Leung W, van Leyen K, Lok J, Wang X, Lo EH. Neuronal production of lipocalin-2 as a help-me signal for glial activation. Stroke. 2014; 45:2085–2092. PMID: 24916903.
Article
38. Fukunaga K, Kawano T. Akt is a molecular target for signal transduction therapy in brain ischemic insult. J Pharmacol Sci. 2003; 92:317–327. PMID: 12939516.
Article
39. Dunleavy M, Provenzano G, Henshall DC, Bozzi Y. Kainic acid-induced seizures modulate Akt (SER473) phosphorylation in the hippocampus of dopamine D2 receptor knockout mice. J Mol Neurosci. 2013; 49:202–210. PMID: 23188702.
Article
40. Noshita N, Lewén A, Sugawara T, Chan PH. Akt phosphorylation and neuronal survival after traumatic brain injury in mice. Neurobiol Dis. 2002; 9:294–304. PMID: 11950275.
Article
41. Kawano T, Fukunaga K, Takeuchi Y, Morioka M, Yano S, Hamada J, Ushio Y, Miyamoto E. Neuroprotective effect of sodium orthovanadate on delayed neuronal death after transient forebrain ischemia in gerbil hippocampus. J Cereb Blood Flow Metab. 2001; 21:1268–1280. PMID: 11702042.
Article
42. Kim YS, Choi MY, Lee DH, Jeon BT, Roh GS, Kim HJ, Kang SS, Cho GJ, Choi WS. Decreased interaction between FoxO3a and Akt correlates with seizure-induced neuronal death. Epilepsy Res. 2014; 108:367–378. PMID: 24518891.
Article
43. Guo J, Guo J, Li J, Zhou M, Qin F, Zhang S, Wu B, He L, Zhou D. Statin treatment reduces the risk of poststroke seizures. Neurology. 2015; 85:701–707. PMID: 26203092.
Article
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