Sevoflurane-Induced Dysregulation of Cation-Chloride Cotransporters NKCC1 and KCC2 in Neonatal Mouse Brain

  • O. H. CabreraEmail author
  • V. Tesic
  • Q. L. Tat
  • S. Chastain
  • N. Quillinan
  • V. Jevtovic-Todorovic


The cation-chloride cotransporters Na+–K+–2Cl–1 (NKCC1) and K+–2Cl–2 (KCC2) critically regulate neuronal responses to gamma-aminobutyric acid (GABA). NKCC1 renders GABA excitatory in immature neurons while expression of KCC2 signals GABA maturation to its inhibitory role. Imbalances in NKCC1/KCC2 alter GABA neurotransmission, which may contribute to hyperexcitability and blunted inhibition in neurocircuitry after neonatal exposure to anesthesia. Thus, we hypothesized that anesthetics may dysregulate NKCC1 and/or KCC2 in developing brain. We exposed postnatal day (PND) 7 mice to sevoflurane or carrier gases and assessed NKCC1 and KCC2 expression across three brain regions 6 h and 24 h after initial exposure. To test differences in behavior, we challenged pups receiving sevoflurane or carrier gases on PND7 with propofol on PND8 and recorded parameters of anesthesia induction and maintenance. Sevoflurane exposure increased cortical NKCC1 at 6 h (p = 0.03) and decreased cortical and hippocampal KCC2 at 24 h (p = 0.009 and p = 0.007, respectively). NKCC1/KCC2 ratio was significantly increased at both 6 h (p = 0.02) and 24 h (p = 0.03) in cortex and at 24 h (p = 0.02) in hippocampus. After propofol challenge on PND8, pups previously exposed to sevoflurane on PND7 regained righting reflex significantly faster than their non-exposed cohort (p < 0.001). Disturbing NKCC1/KCC2 balance may underlie circuit hyperexcitability and contribute to neurodevelopmental impairments we have observed in previous studies of neonatal anesthesia exposure. Human infants previously exposed to anesthesia may require higher concentrations of anesthetic drugs, potentially compounding their susceptibility for neurodevelopmental sequalae.


Developmental neurotoxicity Sevoflurane NKCC1 KCC2 Cation chloride cotransporter 


Funding Information

This study is supported in part by funds from the Department of Anesthesiology at the University of Colorado Anschutz Medical campus, R0144517, R0144517-S, R01 GM118197, R01 GM118197, R21 HD080281 and March of Dimes National Award, USA (to VJT), CU Medicine Endowments (to VJT), and R01 GM118197-11S1 (OHC).


  1. 1.
    Cattano D, Young C, Straiko MMW, Olney JW (2008) Subanesthetic doses of propofol induce neuroapoptosis in the infant mouse brain. Anesth Analg 106:1712–1714. CrossRefGoogle Scholar
  2. 2.
    Jevtovic-Todorovic V, Hartman RE, Izumi Y, Benshoff ND, Dikranian K, Zorumski CF, Olney JW, Wozniak DF (2003) Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J Neurosci 23:876–882. CrossRefGoogle Scholar
  3. 3.
    Johnson SA, Young C, Olney JW (2008) Isoflurane-induced neuroapoptosis in the developing brain of nonhypoglycemic mice. J Neurosurg Anesthesiol 20:21–28. CrossRefGoogle Scholar
  4. 4.
    Creeley C, Dikranian K, Dissen G, Martin L, Olney J, Brambrink A (2013) Propofol-induced apoptosis of neurones and oligodendrocytes in fetal and neonatal rhesus macaque brain. Br J Anaesth 110:29–38. CrossRefGoogle Scholar
  5. 5.
    Schenning KJ, Noguchi KK, Martin LD, Manzella FM, Cabrera OH, Dissen GA, Brambrink AM (2017) Isoflurane exposure leads to apoptosis of neurons and oligodendrocytes in 20- and 40-day old rhesus macaques. Neurotoxicol Teratol. 60:63–68. CrossRefGoogle Scholar
  6. 6.
    Ing C, DiMaggio C, Whitehouse A, Hegarty MK, Brady J, von Ungern-Sternberg BS, Davidson A, Wood AJJ et al (2012) Long-term differences in language and cognitive function after childhood exposure to anesthesia. Pediatrics. 130:e476–e485. CrossRefGoogle Scholar
  7. 7.
    Sun LS, Li G, Miller TLK, Salorio C, Byrne MW, Bellinger DC, Ing C, Park R et al (2016) Association between a single general anesthesia exposure before age 36 months and neurocognitive outcomes in later childhood. JAMA - J Am Med Assoc. 315:2312–2320. CrossRefGoogle Scholar
  8. 8.
    Wilder RT, Flick RP, Sprung J, Katusic SK, Barbaresi WJ, Mickelson C, Warner DO (2009) Early exposure to anesthesia and learning disabilities in a population-based birth cohort. Anesthesiol.
  9. 9.
    Ben-Ari Y (2002) Excitatory actions of GABA during development: the nature of the nurture. Nat Rev Neurosci 3:728–739. CrossRefGoogle Scholar
  10. 10.
    Kaila K, Ruusuvuori E, Seja P, Voipio J, Puskarjov M (2014) GABA actions and ionic plasticity in epilepsy. Curr Opin Neurobiol 26:34–41. CrossRefGoogle Scholar
  11. 11.
    Ben-Ari Y, Khalilov I, Kahle KT, Cherubini E (2012) The GABA excitatory/inhibitory shift in brain maturation and neurological disorders. Neurosci 18:467–486. Google Scholar
  12. 12.
    Yeo M, Berglund K, Hanna M, Guo JU, Kittur J, Torres MD, Abramowitz J, Busciglio J et al (2013) Bisphenol A delays the perinatal chloride shift in cortical neurons by epigenetic effects on the Kcc2 promoter. Proc Natl Acad Sci U S A 110:4315–4320. CrossRefGoogle Scholar
  13. 13.
    DiGruccio MR, Joksimovic S, Joksovic PM et al (2015) Hyperexcitability of rat thalamocortical networks after exposure to general anesthesia during brain development. J Neurosci 35:1481–1492. CrossRefGoogle Scholar
  14. 14.
    Deng G, Orfila JE, Dietz RM, Moreno-Garcia M, Rodgers KM, Coultrap SJ, Quillinan N, Traystman RJ et al (2017) Autonomous CaMKII activity as a drug target for histological and functional neuroprotection after resuscitation from cardiac arrest. Cell Rep 18:1109–1117. CrossRefGoogle Scholar
  15. 15.
    Dalla Massara L, Osuru HP, Oklopcic A, Milanovic D, Joksimovic SM, Caputo V, DiGruccio MR, Ori C et al (2016) General anesthesia causes epigenetic histone modulation of c-Fos and brain-derived neurotrophic factor, target genes important for neuronal development in the immature rat hippocampus. Anesthesiology 124:1311–1327. CrossRefGoogle Scholar
  16. 16.
    Sanchez V, Feinstein SD, Lunardi N, Joksovic PM, Boscolo A, Todorovic SM, Jevtovic-Todorovic V (2011) General anesthesia causes long-term impairment of mitochondrial morphogenesis and synaptic transmission in developing rat brain. Anesthesiology. 115:992–1002. CrossRefGoogle Scholar
  17. 17.
    Young SZ, Taylor MM, Wu S, Ikeda-Matsuo Y, Kubera C, Bordey A (2012) NKCC1 knockdown decreases neuron production through GABAA-regulated neural progenitor proliferation and delays dendrite development. J Neurosci 32:13630–13638. CrossRefGoogle Scholar
  18. 18.
    Dzhala VI, Talos DM, Sdrulla DA, Brumback AC, Mathews GC, Benke TA, Delpire E, Jensen FE et al (2005) NKCC1 transporter facilitates seizures in the developing brain. Nat Med 11:1205–1213. CrossRefGoogle Scholar
  19. 19.
    Dzhala VI, Kuchibhotla KV, Glykys JC, Kahle KT, Swiercz WB, Feng G, Kuner T, Augustine GJ et al (2010) Progressive NKCC1-dependent neuronal chloride accumulation during neonatal seizures. J Neurosci. 30:11745–11761. CrossRefGoogle Scholar
  20. 20.
    Cabrera OH, O’Connor SD, Swiney BS, Salinas-Contreras P, Manzella FM, Taylor GT, Noguchi KK (2017) Caffeine combined with sedative/anesthetic drugs triggers widespread neuroapoptosis in a mouse model of prematurity. J Matern Neonatal Med 30:2734–2741. CrossRefGoogle Scholar
  21. 21.
    Olney JW, Tenkova T, Dikranian K, Muglia LJ, Jermakowicz WJ, D'Sa C, Roth KA (2002) Ethanol-induced caspase-3 activation in the in vivo developing mouse brain. Neurobiol Dis 9:205–219. CrossRefGoogle Scholar
  22. 22.
    Ju LS, Yang JJ, Morey TE, Gravenstein N, Seubert CN, Resnick JL, Zhang JQ, Martynyuk AE (2018) Role of epigenetic mechanisms in transmitting the effects of neonatal sevoflurane exposure to the next generation of male, but not female, rats. Br J Anaesth 121:406–416. CrossRefGoogle Scholar
  23. 23.
    Lee HA, Hong SH, Kim JW, Jang IS (2010) Possible involvement of DNA methylation in NKCC1 gene expression during postnatal development and in response to ischemia. J Neurochem 114:520–529. CrossRefGoogle Scholar
  24. 24.
    Li X, Zhou J, Chen Z, Chen S, Zhu F, Zhou L (2008) Long-term expressional changes of Na + -K + -Cl- co-transporter 1 (NKCC1) and K + -Cl- co-transporter 2 (KCC2) in CA1 region of hippocampus following lithium-pilocarpine induced status epilepticus (PISE). Brain Res 1221:141–146. CrossRefGoogle Scholar
  25. 25.
    Karlócai MR, Wittner L, Tóth K, Maglóczky Z, Katarova Z, Rásonyi G, Erőss L, Czirják S et al (2016) Enhanced expression of potassium-chloride cotransporter KCC2 in human temporal lobe epilepsy. Brain Struct Funct 221:3601–3615. CrossRefGoogle Scholar
  26. 26.
    Ben-Ari Y, Gaiarsa J-L, Tyzio R, Khazipov R (2007) GABA: A pioneer transmitter that excites immature neurons and generates primitive oscillations. Physiol Rev 87:1215–1284. CrossRefGoogle Scholar
  27. 27.
    Hübner CA, Lorke DE, Hermans-Borgmeyer I (2001) Expression of the Na-K-2Cl-cotransporter NKCC1 during mouse development. Mech Dev 102:267–269. CrossRefGoogle Scholar
  28. 28.
    Flick RP, Katusic SK, Colligan RC, Wilder RT, Voigt RG, Olson MD, Sprung J, Weaver AL et al (2011) Cognitive and behavioral outcomes after early exposure to anesthesia and surgery. Pediatrics 128:e1053–e1061. CrossRefGoogle Scholar
  29. 29.
    Davidson AJ, Disma N, De Graaff JC et al (2016) Neurodevelopmental outcome at 2 years of age after general anaesthesia and awake-regional anaesthesia in infancy (GAS): an international multicentre, randomised controlled trial. Lancet 387:239–250. CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of AnesthesiologyUniversity of Colorado Anschutz Medical CampusAuroraUSA

Personalised recommendations