Abstract
Recent preclinical and clinical reports suggest that cerebrolysin shows neuroprotective properties similar to endogenous neurotrophic factors in neurodegenerative disorders including ischemic stroke. However, little is known about its underlying antiexcitotoxic action. Adult male Wistar rats were intraperitoneally treated with cerebrolysin (0.15 or 0.30 mg/kg) or vehicle at 3, 6 and 12 h after ischemic reperfusion and were assessed 24 h after reperfusion in ischemic rats. We added cerebrolysin (2.5 or 5 mg/ml) or vehicle in primary cortical culture cells at 3, 6 and 12 h of post-glutamate exposure and performed cell viability assays at 24 h. Our in-vivo and in-vitro findings showed that cerebrolysin substantially reduced neuronal cell death in delayed hours of post ischemic- and glutamate-insult conditions respectively. Further, we have assessed the influence of NR-2 A/-2B receptor antagonism on neuroprotective action of cerebrolysin at 6 h in in-vivo as well as in-vitro conditions. Neuroprotective effect of cerebrolysin at 6 h of reperfusion was enhanced by pretreatment of NR2B antagonist RO25-6981.We found that cerebrolysin restrained upregulation of extrasynaptic NR2B responsible for triggering apoptotic pathways. Cerebrolysin reduced expression of important cell death proteins such as, JNK, PTEN, Calpain and Caspase-3 components. Importantly, we also found that cerebrolysin reduced SREBP1 expression, which gets activated only after 6 h of ischemia. These results demonstrate that cerebrolysin reduces excitotoxicity and protect neuronal cells in delayed hours of ischemic reperfusion injuries by decreasing cell death proteins.
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All data generated or analyzed during this study are included in this article.
Abbreviations
- ANOVA:
-
Analysis of variance
- JNK:
-
c-Jun N-terminal kinase
- LDH:
-
Lactate dehydrogenase
- MCAO:
-
Middle cerebral artery occlusion
- MTT:
-
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
- NMDAR:
-
N-methyl-D-aspartate receptor
- PTEN:
-
Phosphatase and tensin homolog
- SREBP1:
-
Sterol regulatory element-binding protein 1
- TTC:
-
2,3,5-triphenyltetrazolium chloride
References
Amiri-Nikpour MR, Nazarbaghi S, Ahmadi-Salmasi B et al (2014) Cerebrolysin effects on neurological outcomes and cerebral blood flow in acute ischemic stroke. Neuropsychiatr Dis Treat 10:2299–2306. https://doi.org/10.2147/NDT.S75304
Barakat W, Fahmy A, Askar M, El-Kannishy S (2018) Effectiveness of arginase inhibitors against experimentally induced stroke. Naunyn Schmiedebergs Arch Pharmacol 391(6):603–612. https://doi.org/10.1007/s00210-018-1489-1
Benveniste H, Drejer J, Schousboe A, Diemer NH (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. https://doi.org/10.1111/j.1471-4159.1984.tb05396.x
Borsello T, Clarkel PGH, Hirt L et al (2003) A peptide inhibitor of c-Jun N-terminal kinase protects against excitotoxicity and cerebral ischemia. Nat Med 9:1180–1186. https://doi.org/10.1038/nm911
Chang WH, Park C, Kim DY et al (2016) Cerebrolysin combined with rehabilitation promotes motor recovery in patients with severe motor impairment after stroke. BMC Neurol 16:31. https://doi.org/10.1186/s12883-016-0553-z
DeRidder MN, Simon MJ, Siman R et al (2006) Traumatic mechanical injury to the hippocampus in vitro causes regional caspase-3 and calpain activation that is influenced by NMDA receptor subunit composition. Neurobiol Dis 22:165–176. https://doi.org/10.1016/j.nbd.2005.10.011
Dirnagl U, Iadecola C, Moskowitz MA (1999) Pathobiology of ischaemic stroke: an integrated view. Trends Neurosci 22(9):391–397. https://doi.org/10.1016/S0166-2236(99)01401-0
Elango C, Jayachandaran KS, Niranjali Devaraj S (2009) Hawthorn extract reduces infarct volume and improves neurological score by reducing oxidative stress in rat brain following middle cerebral artery occlusion. Int J Dev Neurosci 27:799–803. https://doi.org/10.1016/j.ijdevneu.2009.08.008
Ferrer I (2006) Apoptosis: future targets for neuroprotective strategies. Cerebrovasc Dis 21:9–20. https://doi.org/10.1159/000091699
Gary DS, Mattson MP (2002) PTEN regulates akt kinase activity in hippocampal neurons and increases their sensitivity to glutamate and apoptosis. Neuro Mol Med 2:261–269. https://doi.org/10.1385/NMM:2:3:261
Gascón S, Sobrado M, Roda JM et al (2008) Excitotoxicity and focal cerebral ischemia induce truncation of the NR2A and NR2B subunits of the NMDA receptor and cleavage of the scaffolding protein PSD-95. Mol Psychiatry 13:99–114. https://doi.org/10.1038/sj.mp.4002017
Ge Y, Chen W, Axerio-Cilies P et al (2020) NMDARs in cell survival and death: implications in stroke pathogenesis and treatment. Trends Mol Med 26(6):533–551. https://doi.org/10.1016/j.molmed.2020.03.001
Glassford A, Lee J-E, Xu L, Giffard RG (2002) Caspase inhibitors reduce the apoptotic but not necrotic component of kainate injury in primary murine cortical neuronal cultures. Neurol Res 24:796–800. https://doi.org/10.1179/016164102101200915
Guo J-Y, Ding J, Yuan F et al (2013) Dose-dependent protective effect of bisperoxovanadium against acute cerebral ischemia in a rat model of ischemia/reperfusion injury. Int J Mol Sci 14:12013–12022. https://doi.org/10.3390/ijms140612013
Gutmann B, Hutter-Paier B, Skofitsch G et al (2002) In vitro models of brain ischemia: the peptidergic drug cerebrolysin protects cultured chick cortical neurons from cell death. Neurotox Res 4:59–65. https://doi.org/10.1080/10298420290007637
Hardingham GE, Fukunaga Y, Bading H (2002) Extrasynaptic NMDARs oppose synaptic NMDARs by triggering CREB shut-off and cell death pathways. Nat Neurosci 5:405–414. https://doi.org/10.1038/nn835
Hartbauer M, Hutter-Paier B, Skofitsch G, Windisch M (2001) Antiapoptotic effects of the peptidergic drug cerebrolysin on primary cultures of embryonic chick cortical neurons. J Neural Transm 108:459–473. https://doi.org/10.1007/s007020170067
Hartwig K, Fackler V, Jaksch-Bogensperger H et al (2014) Cerebrolysin protects PC12 cells from CoCl2-induced hypoxia employing GSK3β signaling. Int J Dev Neurosci 38:52–58. https://doi.org/10.1016/j.ijdevneu.2014.07.005
Heiss WD, Brainin M, Bornstein NM et al (2012) Cerebrolysin in patients with acute ischemic stroke in Asia: results of a double-blind, placebo-controlled randomized trial. Stroke 43:630–636. https://doi.org/10.1161/STROKEAHA.111.628537
Hutter-Paier B, Grygar E, Windisch M (1996) Death of cultured telencephalon neurons induced by glutamate is reduced by the peptide derivative cerebrolysin. J Neural Transm Suppl 47:267–273. https://doi.org/10.1007/978-3-7091-6892-9_19
Jang JY, Choi YW, Kim HN et al (2014) Neuroprotective effects of a novel single compound 1-methoxyoctadecan-1-ol isolated from Uncaria sinensis in primary cortical neurons and a photothrombotic ischemia model. PLoS ONE 9:e85322. https://doi.org/10.1371/journal.pone.0085322
Jolkkonen J, Puurunen K, Rantakömi S et al (2000) Behavioral effects of the α2-adrenoceptor antagonist, atipamezole, after focal cerebral ischemia in rats. Eur J Pharmacol 400:211–219. https://doi.org/10.1016/S0014-2999(00)00409-X
Kokare DM, Shelkar GP, Borkar CD et al (2011) A simple and inexpensive method to fabricate a cannula system for intracranial injections in rats and mice. J Pharmacol Toxicol Methods 64:246–250. https://doi.org/10.1016/j.vascn.2011.08.002
Ladurner G, Kalvach P, Moessler H (2005) Neuroprotective treatment with cerebrolysin in patients with acute stroke: a randomised controlled trial. J Neural Transm 112:415–428. https://doi.org/10.1007/s00702-004-0248-2
Lai TW, Zhang S, Wang YT (2014) Excitotoxicity and stroke: identifying novel targets for neuroprotection. Prog Neurobiol 115:157–188. https://doi.org/10.1016/j.pneurobio.2013.11.006
Lang W, Stadler CH, Poljakovic Z et al (2013) A prospective, randomized, placebo-controlled, double-blind trial about safety and efficacy of combined treatment with alteplase (rt-PA) and cerebrolysin in acute ischaemic hemispheric stroke. Int J Stroke 8:95–104. https://doi.org/10.1111/j.1747-4949.2012.00901.x
Li K, Ding D, Zhang M (2016) Neuroprotection of osthole against cerebral ischemia/reperfusion injury through an anti-apoptotic pathway in rats. Biol Pharm Bull 39:336–342. https://doi.org/10.1248/bpb.b15-00699
Longa EZ, Weinstein PR, Carlson S, Cummins R (1989) Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke 20:84–91. https://doi.org/10.1161/01.STR.20.1.84
Meade AJ, Meloni BP, Cross J et al (2010) AP-1 inhibitory peptides are neuroprotective following acute glutamate excitotoxicity in primary cortical neuronal cultures. J Neurochem 112:258–270. https://doi.org/10.1111/j.1471-4159.2009.06459.x
Nijboer CH, Heijnen CJ, Van Der Kooij MA et al (2011) Targeting the p53 pathway to protect the neonatal ischemic brain. Ann Neurol 70:255–264. https://doi.org/10.1002/ana.22413
Ning K, Pei L, Liao M et al (2004) Dual neuroprotective signaling mediated by downregulating two distinct phosphatase activities of PTEN. J Neurosci 24:4052–4060. https://doi.org/10.1523/JNEUROSCI.5449-03.2004
Paxinos G, Franklin KBJ (2001) The mouse brain in stereotaxic coordinates. 2nd edition, Academic Press, San Diego
Ren J, Sietsma D, Qiu S et al (2007) Cerebrolysin enhances functional recovery following focal cerebral infarction in rats. Restor Neurol Neurosci 25:25–31
Riley C, Hutter-Paier B, Windisch M et al (2006) A peptide preparation protects cells in organotypic brain slices against cell death after glutamate intoxication. J Neural Transm 113:103–110. https://doi.org/10.1007/s00702-005-0302-8
Rockenstein E, Mante M, Adame A et al (2007) Effects of Cerebrolysin™ on neurogenesis in an APP transgenic model of Alzheimer’s disease. Acta Neuropathol 113:265–275. https://doi.org/10.1007/s00401-006-0166-5
Salvador E, Burek M, Förster CY (2015) Stretch and/or oxygen glucose deprivation (OGD) in an in vitro traumatic brain injury (TBI) model induces calcium alteration and inflammatory cascade. Front Cell Neurosci 9:323. https://doi.org/10.3389/fncel.2015.00323
Schauer E, Wronski R, Patockova J et al (2006) Neuroprotection of cerebrolysin in tissue culture models of brain ischemia: post lesion application indicates a wide therapeutic window. J Neural Transm 113:855–868. https://doi.org/10.1007/s00702-005-0384-3
Shah SA, Lee HY, Bressan RA et al (2014) Novel osmotin attenuates glutamate-induced synaptic dysfunction and neurodegeneration via the JNK/PI3K/Akt pathway in postnatal rat brain. Cell Death Dis 5:e1026–e1010. https://doi.org/10.1038/cddis.2013.538
Sharma HS, Zimmermann-Meinzingen S, Johanson CE (2010) Cerebrolysin reduces blood-cerebrospinal fluid barrier permeability change, brain pathology, and functional deficits following traumatic brain injury in the rat. Ann N Y Acad Sci 1199:125–137. https://doi.org/10.1111/j.1749-6632.2009.05329.x
Siman R, Carl Noszek J (1988) Excitatory amino acids activate calpain I and induce structural protein breakdown in vivo. Neuron 1:279–287. https://doi.org/10.1016/0896-6273(88)90076-1
Squier MKT, Miller ACK, Malkinson AM, Cohen JJ (1994) Calpain activation in apoptosis. J Cell Physiol 159:229–237. https://doi.org/10.1002/jcp.1041590206
Taghibiglou C, Martin HGS, Lai TW et al (2009) Role of NMDA receptor-dependent activation of SREBP1 in excitotoxic and ischemic neuronal injuries. Nat Med 15:1399–1406. https://doi.org/10.1038/nm.2064
Veinbergs I, Mante M, Mallory M, Masliah E (2000) Neurotrophic effects of Cerebrolysin® in animal models of excitotoxicity. Neural Transm Suppl 59:273–80. https://doi.org/10.1007/978-3-7091-6781-6_29
Wang Z, Shi L, Xu S, Zhang J (2017) Cerebrolysin for functional recovery in patients with acute ischemic stroke: a meta-analysis of randomized controlled trials. Drug Des Devel Ther 11:1273–1282. https://doi.org/10.2147/DDDT.S124273
Wang LW, Tu YF, Huang CC, Ho CJ (2012) JNK signaling is the shared pathway linking neuroinflammation, blood-brain barrier disruption, and oligodendroglial apoptosis in the white matter injury of the immature brain. J Neuroinflammation 9:175. https://doi.org/10.1186/1742-2094-9-175
Woodruff TM, Thundyil J, Tang SC et al (2011) Pathophysiology, treatment, and animal and cellular models of human ischemic stroke. Mol Neurodegener 6:11. https://doi.org/10.1186/1750-1326-6-11
Xing S, Zhang J, Dang C et al (2014) Cerebrolysin reduces amyloid-β deposits, apoptosis and autophagy in the thalamus and improves functional recovery after cortical infarction. J Neurol Sci 337:104–111. https://doi.org/10.1016/j.jns.2013.11.028
Xu SY, Wu YM, Ji Z et al (2012) A modified technique for culturing primary fetal rat cortical neurons. J Biomed Biotechnol 2012:803930. https://doi.org/10.1155/2012/803930
Zhang YM, Bhavnani BR (2005) Glutamate-induced apoptosis in primary cortical neurons is inhibited by equine estrogens via down-regulation of caspase-3 and prevention of mitochondrial cytochrome c release. BMC Neurosci 6:13. https://doi.org/10.1186/1471-2202-6-13
Zhang C, Chopp M, Cui Y et al (2010) Cerebrolysin enhances neurogenesis in the ischemic brain and improves functional outcome after stroke. J Neurosci Res 88:3275–3281. https://doi.org/10.1002/jnr.22495
Zhang L, Chopp M, Lu M et al (2016) Cerebrolysin dose-dependently improves neurological outcome in rats after acute stroke: a prospective, randomized, blinded, and placebo-controlled study. Int J Stroke 11(3):347–355. https://doi.org/10.1177/1747493015625645
Zhang J, Zhang P, Wei Y et al (2013a) Deubiquitylation and stabilization of PTEN by USP13. Nat Cell Biol 15:1486–1494. https://doi.org/10.1038/ncb2874
Zhang L, Chopp M, Meier DH et al (2013b) Sonic hedgehog signaling pathway mediates Cerebrolysin-improved neurological function after stroke. Stroke 44:1965–1972. https://doi.org/10.1161/STROKEAHA.111.000831
Zhu H, Zhang Y, Shi Z et al (2016) The neuroprotection of liraglutide against ischaemia-induced apoptosis through the activation of the PI3K/AKT and MAPK pathways. Sci Rep 6:26859. https://doi.org/10.1038/srep26859
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This study has been financially supported by Science and Engineering Research Board, Department of Science and Technology, Government of India. (EMR/2014/001015)
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AP and RRU designed the study. LPS, TG and VM performed experiments, analyzed and interpreted the data presented in this study. LPS and RRU prepared manuscript. BV performed the experiments and revised the manuscript.
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Sarode, L.P., Ghatage, T., Mardhekar, V. et al. Cerebrolysin reduces excitotoxicity by modulation of cell-death proteins in delayed hours of ischemic reperfusion injury. Metab Brain Dis 38, 2401–2416 (2023). https://doi.org/10.1007/s11011-023-01240-4
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DOI: https://doi.org/10.1007/s11011-023-01240-4