Neurotoxicity Research

, Volume 35, Issue 3, pp 654–667 | Cite as

Carbenoxolone Reverses the Amyloid Beta 1–42 Oligomer–Induced Oxidative Damage and Anxiety-Related Behavior in Rats

  • Sheetal Sharma
  • Neha Sharma
  • Avneet Saini
  • Bimla NehruEmail author


The characteristic feature of Alzheimer’s disease (AD) is the deposition of amyloid beta inside the brain mainly consisting of Aβ 40 and 42 aggregates. Soluble aggregates of Aβ 42 are reported to be more toxic and exert their neurotoxicity by the induction of oxidative damage and cognitive deficits such as anxiety-like behavior. These alterations emerge due to the induction of gap junction communication through increased activity and expression of connexins such as connexin43 (Cx43) leading to the release of small neurotoxic molecules. In the present study, single intracerebroventricular (icv) injection of Aβ 42 oligomers (10 μl/rat) was used to induce oxidative damage and anxiety-related behavior in rats. Carbenoxolone (Cbx), a gap junction blocker, was tested (20 mg/kg body weight, i.p., for 6 weeks) against these alterations. Cbx supplementation reversed the Aβ 42 oligomer–induced alterations in the antioxidant defense system. The levels ROS, lipid peroxidation, and protein carbonyls were normalized with Cbx co-treatment leading to the decreased DNA fragmentation and pyknosis in different regions of the rat brain. Cbx induced the anxiolytic behavior and ameliorated the cognitive decline in rats post Aβ 42 oligomer injection. The increased expression of Cx43 post Aβ 42 oligomer injection was also reduced with Cbx supplementation, which might have inhibited the release of small neurotoxic molecules. Our results showed that Cbx prevents the Aβ 42 oligomer–induced oxidative damage and anxiety-like behavior partly by blocking the gap junction communication, which suggests that the therapeutic potential of Cbx may be explored in the progression of AD.


Amyloid beta 1–42 Alzheimer’s disease Anxiety-like behavior Oxidative damage Gap junctions Carbenoxolone 



Alzheimer’s disease

Amyloid beta


Amyloid precursor protein










Reactive oxygen species


2, 7-Dichlorofluorescein diacetate


Lipid peroxidation




Thiobarbituric acid


Superoxide dismutase


Cornus ammonis


Funding Information

This work is financially supported by the University Grants Commission (UGC), New Delhi, India.

Compliance with Ethical Standards

The guidelines laid by the Ethics Committee of the Animal Care of Panjab University in accordance with the Indian national law on animal care and use were strictly followed throughout the study.

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

12640_2018_9975_Fig11_ESM.png (23 mb)
Suppl Fig. 1a

Effect of Cbx supplementation on oligomeric Aβ 1–42 induced astrocytic activation in the rat brain as depicted by immunohistochemistry (100X). The estimations were performed after 6 weeks of Cbx supplementation post Aβ 1–42 injection. (PNG 23534 kb)

12640_2018_9975_MOESM1_ESM.tif (75.5 mb)
High resolution image (TIF 77273 kb)
12640_2018_9975_Fig12_ESM.png (11 kb)
Suppl Fig. 1b

Quantitative analysis of the astrocytic activation in different regions of the rat brain. Cbx supplementation normalized the astrocytic activation in hippocampal, cortical and striatal regions. Values are expressed as mean ± SD; n = 3. *, p ≤ 0.05, compared to sham control group; #, p ≤ 0.05, compared to Aβ 1–42 treated group. (PNG 11 kb)

12640_2018_9975_MOESM2_ESM.tif (508 kb)
High resolution image (TIF 508 kb)
12640_2018_9975_Fig13_ESM.png (158.5 mb)
Suppl Fig. 2

Effect of Cbx on oligomeric Aβ 1–42 induced pyknosis in the rat brain as depicted by thionin staining (× 200). The estimations were performed after 6 weeks of Cbx supplementation post Aβ 1–42 injection. Presence of pyknotic cells in hippocampal, cortical and striatal regions (a) confirmed neurodegeneration post oligomeric Aβ 1–42 injection. Cbx supplementation prevented neuronal damage as represented in the histogram (b) showing the number of pyknotic cells in different regions. Black arrows represent the normal neurons and red arrows represent the pyknotic neurons. Values are expressed as mean ± SD; n = 3. *, p ≤ 0.05, compared to sham control group; #, p ≤ 0.05, compared to Aβ 1–42 treated group. (PNG 162264 kb)

12640_2018_9975_MOESM3_ESM.tif (70.5 mb)
High resolution image (TIF 72239 kb)


  1. Best TM, Fiebig R, Corr DT, Brickson S, Ji L (1999) Free radical activity, antioxidant enzyme, and glutathione changes with muscle stretch injury in rabbits. J Appl Physiol 87:74–82CrossRefPubMedGoogle Scholar
  2. Brouillette J, Caillierez R, Zommer N, Alves-Pires C, Benilova I (2012) Neurotoxicity and memory deficits induced by soluble low-molecular-weight amyloid-β1-42 oligomers are revealed in vivo by using a novel animal model. J Neurosci 32:7852–7861. CrossRefPubMedGoogle Scholar
  3. Brureau A, Zussy C, Delair B, Ogier C, Ixart G (2013) Deregulation of hypothalamic-pituitary-adrenal axis functions in an Alzheimer’s disease rat model. Neurobiol Aging 34:1426–1439. CrossRefPubMedGoogle Scholar
  4. Butterfield DA, Lauderback CM (2002) Lipid peroxidation and protein oxidation in Alzheimer’s disease brain: potential causes and consequences involving amyloid beta-peptide-associated free radical oxidative stress. Free Radic Biol Med 32:1050–1060CrossRefPubMedGoogle Scholar
  5. Butterfield DA, Sultana R (2011) Methionine-35 of aβ(1-42): importance for oxidative stress in Alzheimer disease. J Amino Acids 2011:198430. CrossRefPubMedPubMedCentralGoogle Scholar
  6. Butterfield DA, Drake J, Pocernich C, Castegna A (2001) Evidence of oxidative damage in Alzheimer’s disease brain: central role for amyloid beta-peptide. Trends Mol Med 7:548–554CrossRefPubMedGoogle Scholar
  7. Butterfield DA, Swomley AM, Sultana R (2013) Amyloid β-peptide (1–42)-induced oxidative stress in Alzheimer disease: importance in disease pathogenesis and progression. Antioxid Redox Signal 19:823–835. CrossRefPubMedPubMedCentralGoogle Scholar
  8. Caraci F, Copani A, Nicoletti F, Drago F (2010) Depression and Alzheimer’s disease: neurobiological links and common pharmacological targets. Eur J Pharmacol 626:64–71. CrossRefPubMedGoogle Scholar
  9. Cioanca O, Hancianu M, Mihasan M, Hritcu L (2015) Anti-acetylcholinesterase and antioxidant activities of inhaled juniper oil on amyloid beta (1-42)-induced oxidative stress in the rat hippocampus. Neurochem Res 40:952–960. CrossRefPubMedGoogle Scholar
  10. Epelbaum S, Youssef I, Lacor PN, Chaurand P, Duplus E (2015) Acute amnestic encephalopathy in amyloid-β oligomer-injected mice is due to their widespread diffusion in vivo. Neurobiol Aging 36:2043–2052. CrossRefPubMedGoogle Scholar
  11. Faucher P, Mons N, Micheau J, Louis C, Beracochea DJ (2015) Hippocampal injections of oligomeric amyloid β-peptide (1-42) induce selective working memory deficits and long-lasting alterations of ERK signaling pathway. Front Aging Neurosci 7:245. PubMedGoogle Scholar
  12. Ferreira ST, Vieira MNN, De Felice FG (2007) Soluble protein oligomers as emerging toxins in Alzheimer’s and other amyloid diseases. IUBMB Life 59:332–345. CrossRefPubMedGoogle Scholar
  13. Figueiredo CP, Clarke JR, Ledo JH, Ribeiro FC, Costa CV (2013) Memantine rescues transient cognitive impairment caused by high-molecular-weight aβ oligomers but not the persistent impairment induced by low-molecular-weight oligomers. J Neurosci 33:9626–9634. CrossRefPubMedGoogle Scholar
  14. Forny-Germano L, Lyra e Silva NM, Batista AF, Brito-Moreira J, Gralle M (2014) Alzheimer’s disease-like pathology induced by amyloid-β oligomers in nonhuman primates. J Neurosci 34:13629–13643. CrossRefPubMedGoogle Scholar
  15. Gradinariu V, Cioanca O, Hritcu L, Trifan A (2015) Comparative efficacy of Ocimum sanctum L. and Ocimum basilicum L. essential oils against amyloid beta (1–42)-induced anxiety and depression in laboratory rats. Phytochem Rev 14:567.
  16. Hellmich HL, Rojo DR, Micci MA, Sell SL, Boone DR (2013) Pathway analysis reveals common pro-survival mechanisms of metyrapone and carbenoxolone after traumatic brain injury. PLoS One 8:e53230. CrossRefPubMedPubMedCentralGoogle Scholar
  17. Hritcu L, Noumedem JA, Cioanca O, Hancianu M, Kuete V (2014) Methanolic extract of Piper nigrum fruits improves memory impairment by decreasing brain oxidative stress in amyloid beta(1-42) rat model of Alzheimer’s disease. Cell Mol Neurobiol 34:437–449. CrossRefPubMedGoogle Scholar
  18. Jin M, Selkoe DJ (2015) Systematic analysis of time-dependent neural effects of soluble amyloid β oligomers in culture and in vivo: prevention by scyllo-inositol. Neurobiol Dis 82:152–163. CrossRefPubMedPubMedCentralGoogle Scholar
  19. Kono Y (1978) Generation of superoxide radical during autoxidation of hydroxylamine and an assay for superoxide dismutase. Arch Biochem Biophys 186:189–195CrossRefPubMedGoogle Scholar
  20. Koulakoff A, Mei X, Orellana JA, Sáez JC, Giaume C (2012) Glial connexin expression and function in the context of Alzheimer’s disease. Biochim Biophys Acta Biomembr 1818:2048–2057. CrossRefGoogle Scholar
  21. LaFerla FM, Green KN, Oddo S (2007) Intracellular amyloid-β in Alzheimer’s disease. Nat Rev Neurosci 8:499–509. CrossRefPubMedGoogle Scholar
  22. Leggio GM, Catania MV, Puzzo D, Spatuzza M, Pellitteri R (2016) The antineoplastic drug flavopiridol reverses memory impairment induced by Amyloid-ß1-42 oligomers in mice. Pharmacol Res 106:10–20. CrossRefPubMedGoogle Scholar
  23. Lioudyno MI, Broccio M, Sokolov Y, Rasool S, Wu J (2012) Effect of synthetic Aβ peptide oligomers and fluorinated solvents on Kv1.3 channel properties and membrane conductance. PLoS One 7:e35090. CrossRefPubMedPubMedCentralGoogle Scholar
  24. Lok K, Zhao H, Zhang C, He N, Shen H (2013) Effects of accelerated senescence on learning and memory, locomotion and anxiety-like behavior in APP/PS1 mouse model of Alzheimer’s disease. J Neurol Sci 335:145–154. CrossRefPubMedGoogle Scholar
  25. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  26. Lück H (1965) Catalase. In: Bergmeyer HU (ed) Methods of enzymatic analysis. Academic, New York, pp 885–894CrossRefGoogle Scholar
  27. Masilamoni JG, Jesudason EP, Dhandayuthapani S, Ashok BS, Vignesh S (2008) The neuroprotective role of melatonin against amyloid beta peptide injected mice. Free Radic Res 42:661–673. CrossRefPubMedGoogle Scholar
  28. Mucke L, Selkoe DJ (2012) Neurotoxicity of amyloid β-protein: synaptic and network dysfunction. Cold Spring Harb Perspect Med 2:a006338. CrossRefPubMedPubMedCentralGoogle Scholar
  29. Nehru B, Bhalla P, Garg A (2006) Evidence for centrophenoxine as a protective drug in aluminium induced behavioral and biochemical alteration in rat brain. Mol Cell Biochem 290:33–42. CrossRefPubMedGoogle Scholar
  30. Orellana JA, Froger N, Ezan P, Jiang JX, Bennett MVL (2011a) ATP and glutamate released via astroglial connexin 43 hemichannels mediate neuronal death through activation of pannexin 1 hemichannels. J Neurochem 118:826–840. CrossRefPubMedPubMedCentralGoogle Scholar
  31. Orellana JA, Shoji KF, Abudara V, Ezan P, Amigou E (2011b) Amyloid β-induced death in neurons involves glial and neuronal hemichannels. J Neurosci 31:4962–4977. CrossRefPubMedGoogle Scholar
  32. Orellana JA, Moraga-Amaro R, Díaz-Galarce R, Rojas S, Maturana CJ (2015) Restraint stress increases hemichannel activity in hippocampal glial cells and neurons. Front Cell Neurosci 9:102. PubMedPubMedCentralGoogle Scholar
  33. Perez Velazquez JL, Kokarovtseva L, Sarbaziha R, Jeyapalan Z, Leshchenko Y (2006) Role of gap junctional coupling in astrocytic networks in the determination of global ischaemia-induced oxidative stress and hippocampal damage. Eur J Neurosci 23:1–10. CrossRefPubMedGoogle Scholar
  34. Porter VR, Buxton WG, Fairbanks LA, Strickland T, O’Connor SM (2003) Frequency and characteristics of anxiety among patients with Alzheimer’s disease and related dementias. J Neuropsychiatr Clin Neurosci 15:180–186. CrossRefGoogle Scholar
  35. Psotta L, Rockahr C, Gruss M, Kirches E, Braun K (2015) Impact of an additional chronic BDNF reduction on learning performance in an Alzheimer mouse model. Front Behav Neurosci 9:58. CrossRefPubMedPubMedCentralGoogle Scholar
  36. Quesseveur G, Portal B, Basile JA, Ezan P, Mathou A (2015) Attenuated levels of hippocampal connexin 43 and its phosphorylation correlate with antidepressant- and anxiolytic-like activities in mice. Front Cell Neurosci 9:490. CrossRefPubMedPubMedCentralGoogle Scholar
  37. Roberts JC, Francetic DJ (1993) The importance of sample preparation and storage in glutathione analysis. Anal Biochem 211:183–187. CrossRefPubMedGoogle Scholar
  38. Sedlák J, L’Hanus (1982) Changes of glutathione and protein bound SH-groups concentration in rat adrenals under acute and repeated stress. Endocrinol Exp 16:103–109PubMedGoogle Scholar
  39. Selkoe DJ (2008a) Biochemistry and molecular biology of amyloid β-protein and the mechanism of Alzheimer’s disease. Handb Clin Neurol:245–260Google Scholar
  40. Selkoe DJ (2008b) Soluble oligomers of the amyloid β-protein impair synaptic plasticity and behavior. Behav Brain Res 192:106–113. CrossRefPubMedPubMedCentralGoogle Scholar
  41. Selkoe DJ (2008c) Soluble oligomers of the amyloid beta-protein impair synaptic plasticity and behavior. Behav Brain Res 192:106–113. CrossRefPubMedPubMedCentralGoogle Scholar
  42. Sharma S, Verma S, Kapoor M, Saini A, Nehru B (2016) Alzheimer’s disease like pathology induced six weeks after aggregated amyloid-beta injection in rats: increased oxidative stress and impaired long-term memory with anxiety-like behavior. Neurol Res 38:838–850. CrossRefPubMedGoogle Scholar
  43. Sharma S, Nehru B, Saini A (2017) Inhibition of Alzheimer’s amyloid-beta aggregation in-vitro by carbenoxolone: insight into mechanism of action. Neurochem Int 108:481–493. CrossRefPubMedGoogle Scholar
  44. Takeuchi H, Suzumura A (2014) Gap junctions and hemichannels composed of connexins: potential therapeutic targets for neurodegenerative diseases. Front Cell Neurosci 8:189. CrossRefPubMedPubMedCentralGoogle Scholar
  45. Thakur P, Nehru B (2014) Long-term heat shock proteins (HSPs) induction by carbenoxolone improves hallmark features of Parkinson’s disease in a rotenone-based model. Neuropharmacology 79:190–200. CrossRefPubMedGoogle Scholar
  46. Thakur P, Nehru B (2015) Inhibition of neuroinflammation and mitochondrial dysfunctions by carbenoxolone in the rotenone model of Parkinson’s disease. Mol Neurobiol 51:209–219. CrossRefPubMedGoogle Scholar
  47. Tönnies E, Trushina E (2017) Oxidative stress, synaptic dysfunction, and Alzheimer’s disease. J Alzheimers Dis 57:1105–1121. CrossRefPubMedPubMedCentralGoogle Scholar
  48. Tractenberg RE, Patterson M, Weiner MF, Teri L, Grundman M (2000) Prevalence of symptoms on the CERAD behavior rating scale for dementia in normal elderly subjects and Alzheimer’s disease patients. J Neuropsychiatr Clin Neurosci 12:472–479. CrossRefGoogle Scholar
  49. Uttara B, Singh AV, Zamboni P, Mahajan RT (2009) Oxidative stress and neurodegenerative diseases: a review of upstream and downstream antioxidant therapeutic options. Curr Neuropharmacol 7:65–74. CrossRefPubMedPubMedCentralGoogle Scholar
  50. Walf AA, Frye CA (2007) Estradiol decreases anxiety behavior and enhances inhibitory avoidance and gestational stress produces opposite effects. Stress 10:251–260. CrossRefPubMedGoogle Scholar
  51. Walsh DM, Klyubin I, Fadeeva JV, Cullen WK, Anwyl R (2002) Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo. Nature 416:535–539. CrossRefPubMedGoogle Scholar
  52. Wehr NB, Levine RL (2013) Quantification of protein carbonylation. Methods Mol Biol 965:265–281. CrossRefPubMedGoogle Scholar
  53. Wills ED (1966) Mechanisms of lipid peroxide formation in animal tissues. Biochem J 99:667–676CrossRefPubMedPubMedCentralGoogle Scholar
  54. Yi C, Mei X, Ezan P, Mato S, Matias I (2016) Astroglial connexin43 contributes to neuronal suffering in a mouse model of Alzheimer’s disease. Cell Death Differ 23:1691–1701. CrossRefPubMedPubMedCentralGoogle Scholar
  55. Yin X, Feng L, Ma D, Yin P, Wang X (2018) Roles of astrocytic connexin-43, hemichannels, and gap junctions in oxygen-glucose deprivation/reperfusion injury induced neuroinflammation and the possible regulatory mechanisms of salvianolic acid B and carbenoxolone. J Neuroinflammation 15:97. CrossRefPubMedPubMedCentralGoogle Scholar
  56. Zhang L, Li YM, Jing YH, Wang SY, Song YF (2013) Protective effects of carbenoxolone are associated with attenuation of oxidative stress in ischemic brain injury. Neurosci Bull 29:311–320. CrossRefPubMedPubMedCentralGoogle Scholar
  57. Zussy C, Brureau A, Keller E, Marchal S, Blayo C (2013a) Alzheimer’s disease related markers, cellular toxicity and behavioral deficits induced six weeks after oligomeric amyloid-beta peptide injection in rats. PLoS One 8:e53117. CrossRefPubMedPubMedCentralGoogle Scholar
  58. Zussy C, Brureau A, Keller E, Marchal S, Blayo C (2013b) Alzheimer’s disease related markers, cellular toxicity and behavioral deficits induced six weeks after oligomeric amyloid-beta peptide injection in. PLoS One 8:e53117. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Sheetal Sharma
    • 1
  • Neha Sharma
    • 1
  • Avneet Saini
    • 1
  • Bimla Nehru
    • 1
    Email author
  1. 1.Department of Biophysics, Basic Medical Sciences Block IIPanjab UniversityChandigarhIndia

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