Relative Resilience of Cerebellar Purkinje Cells in a Cardiac Arrest/Resuscitation Rat Model



In studies on cardiac arrest (CA)/resuscitation (R) injury, Purkinje cell degeneration was described, however, with inconsistent data concerning severity and time point of manifestation. Moreover, CA/R studies paid only limited attention to inhibitory stellate interneurons. To this aim, the hypothesis that cerebellar could be relatively resilient toward CA/R because of diverse cellular defense mechanisms including interaction with stellate cells was tested.


We examined rats with survival times of 6, 24, and 48 h, and 7 and 21 days in comparison with sham- and nonoperated animals. Thereby, we focused on the immunohistochemical expression of cfos, MnSOD, Bcl2, caspase 3, parvalbumin, calbindin D28 k, MAP2, IBA1, and GFAP, especially in the particular sensitivity to CA/R cerebellar lobule IX. Hippocampal CA1 degeneration was demonstrated by expression patterns of MAP2 and NeuN in combination with IBA1 and GFAP.


Comparative analysis of hippocampal CA1 pyramidal cells and cerebellar Purkinje cells confirmed a relative resil-ience of Purkinje cells to CA/R. We found only a notable degeneration of Purkinje cell neuronal fiber network, which, however, not necessarily led to neuronal cell death. To induce significant Purkinje cell loss, a stronger ischemic trigger seems to be needed. As possible Purkinje cell-protecting mechanisms, we would propose: (1) activation of inhibitory stellate cells, shown by cfos, MnSOD, and Bcl2 expression, balancing out ischemia-induced excitation and inhibition of Purkinje cells; (2) translocation of the calcium-buffering system, shown by parvalbumin and calbindin D28 k expression, protecting Purkinje cells from detrimental calcium overload; (3) activation of the neuron–astrocyte cross talk, protecting Purkinje cells from over-excitation by removing potassium and neurotransmitters from the extracellular space; (4) activation of the effective and long-lasting MnSOD defense system; and (5) of the anti-apoptotic protein Bcl2 in Purkinje cells itself. Moreover, the results emphasize the limited comparability of animal CA/R studies because of the heterogeneity of the used experimental regimes.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7



B cell lymphoma 2


Cardiac arrest/resuscitation




Granule cell layer


Glial fibrillary acidic protein


Hepatic nuclear receptor 4A


Ionized calcium-binding adaptor molecule 1


Integrated density


Intermittent positive pressure ventilation


Linear density


Microtubule-associated protein 2


Molecular layer


Manganese-dependent superoxide dismutase (mitochondrial SOD 2)


Neuronal nuclei antibody


Phosphate-buffered saline


Purkinje cell


Purkinje cell layer


Phosphate-buffered paraformaldehyde


Return of spontaneous circulation


Survival time


  1. 1.

    Venkatesan A, Frucht S. Movement disorders after resuscitation from cardiac arrest. Neurol Clin. 2006;24(1):123–32.

    PubMed  Google Scholar 

  2. 2.

    Hausmann R, Seidl S, Betz P. Hypoxic changes in Purkinje cells of the human cerebellum. Int J Legal Med. 2007;121(3):175–83.

    CAS  PubMed  Google Scholar 

  3. 3.

    Paine MG, Che D, Li L, Neumar RW. Cerebellar Purkinje cell neurodegeneration after cardiac arrest: effect of therapeutic hypothermia. Resuscitation. 2012;83(12):1511–6.

    PubMed  Google Scholar 

  4. 4.

    Welsh JP, Yuen G, Placantonakis DG, et al. Why do Purkinje cells die so easily after global brain ischemia? Aldolase C, EAAT4, and the cerebellar contribution to posthypoxic myoclonus. Adv Neurol. 2002;89:331–59.

    PubMed  Google Scholar 

  5. 5.

    Au AK, Chen Y, Du L, et al. Ischemia-induced autophagy contributes to neurodegeneration in cerebellar Purkinje cells in the developing rat brain and in primary cortical neurons in vitro. Biochim Biophys Acta. 2015;1852(9):1902–11.

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Drabek T, Wilson CD, Janata A, et al. Unique brain region-dependent cytokine signatures after prolonged hypothermic cardiac arrest in rats. Ther Hypothermia Temp Manag. 2015;5(1):26–39.

    PubMed  Google Scholar 

  7. 7.

    Kantor O, Schmitz C, Feiser J, et al. Moderate loss of cerebellar Purkinje cells after chronic bilateral common carotid artery occlusion in rats. Acta Neuropathol. 2007;113(5):549–58.

    PubMed  Google Scholar 

  8. 8.

    Pulsinelli WA, Levy DE, Duffy TE. Regional cerebral blood flow and glucose metabolism following transient forebrain ischemia. Ann Neurol. 1982;11(5):499–502.

    CAS  PubMed  Google Scholar 

  9. 9.

    Ebmeyer U, Keilhoff G, Wolf G, Rose W. Strain specific differences in a cardio-pulmonary resuscitation rat model. Resuscitation. 2002;53(2):189–200.

    CAS  PubMed  Google Scholar 

  10. 10.

    Quillinan N, Grewal H, Deng G, et al. Region-specific role for GluN2B-containing NMDA receptors in injury to Purkinje cells and CA1 neurons following global cerebral ischemia. Neuroscience. 2015;284:555–65.

    CAS  PubMed  Google Scholar 

  11. 11.

    Acosta SA, Mashkouri S, Nwokoye D, Lee JY, Borlongan CV. Chronic inflammation and apoptosis propagate in ischemic cerebellum and heart of non-human primates. Oncotarget. 2017;8(61):102820–34.

    PubMed  PubMed Central  Google Scholar 

  12. 12.

    Granger DN, Kvietys PR. Reperfusion injury and reactive oxygen species: the evolution of a concept. Redox Biol. 2015;6:524–51.

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Kaur C, Sivakumar V, Zou Z, Ling EA. Microglia-derived proinflammatory cytokines tumor necrosis factor-alpha and interleukin-1beta induce Purkinje neuronal apoptosis via their receptors in hypoxic neonatal rat brain. Brain Struct Funct. 2014;219(1):151–70.

    CAS  PubMed  Google Scholar 

  14. 14.

    Liao SL, Chen WY, Raung SL, Kuo JS, Chen CJ. Association of immune responses and ischemic brain infarction in rat. NeuroReport. 2001;12(9):1943–7.

    CAS  PubMed  Google Scholar 

  15. 15.

    Orzylowska O, Oderfeld-Nowak B, Zaremba M, Januszewski S, Mossakowski M. Prolonged and concomitant induction of astroglial immunoreactivity of interleukin-1beta and interleukin-6 in the rat hippocampus after transient global ischemia. Neurosci Lett. 1999;263(1):72–6.

    CAS  PubMed  Google Scholar 

  16. 16.

    Rodrigo J, Alonso D, Fernandez AP, et al. Neuronal and inducible nitric oxide synthase expression and protein nitration in rat cerebellum after oxygen and glucose deprivation. Brain Res. 2001;909(1–2):20–45.

    CAS  PubMed  Google Scholar 

  17. 17.

    Quillinan N, Deng G, Shimizu K, et al. Long-term depression in Purkinje neurons is persistently impaired following cardiac arrest and cardiopulmonary resuscitation in mice. J Cereb Blood Flow Metab. 2017;37(8):3053–64.

    PubMed  Google Scholar 

  18. 18.

    Keilhoff G, Esser T, Titze M, Ebmeyer U, Schild L. Gynostemma pentaphyllum is neuroprotective in a rat model of cardiopulmonary resuscitation. Exp Ther Med. 2017;14(6):6034–46.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Keilhoff G, Esser T, Titze M, Ebmeyer U, Schild L. High-potential defense mechanisms of neocortex in a rat model of transient asphyxia induced cardiac arrest. Brain Res. 2017;1674:42–54.

    CAS  PubMed  Google Scholar 

  20. 20.

    Katz L, Ebmeyer U, Safar P, Radovsky A, Neumar R. Outcome model of asphyxial cardiac arrest in rats. J Cereb Blood Flow Metab. 1995;15(6):1032–9.

    CAS  PubMed  Google Scholar 

  21. 21.

    Louis ED, Babij R, Lee M, Cortes E, Vonsattel JP. Quantification of cerebellar hemispheric purkinje cell linear density: 32 ET cases versus 16 controls. Mov Disord. 2013;28(13):1854–9.

    PubMed  Google Scholar 

  22. 22.

    Neukomm LJ, Freeman MR. Diverse cellular and molecular modes of axon degeneration. Trends Cell Biol. 2014;24(9):515–23.

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Wang AR, Hu MZ, Zhang ZL, et al. Fastigial nucleus electrostimulation promotes axonal regeneration after experimental stroke via cAMP/PKA pathway. Neurosci Lett. 2019;699:177–83.

    CAS  PubMed  Google Scholar 

  24. 24.

    Sotelo C. Molecular layer interneurons of the cerebellum: developmental and morphological aspects. Cerebellum. 2015;14(5):534–56.

    PubMed  Google Scholar 

  25. 25.

    Zhang F, Li C, Wang R, et al. Activation of GABA receptors attenuates neuronal apoptosis through inhibiting the tyrosine phosphorylation of NR2A by Src after cerebral ischemia and reperfusion. Neuroscience. 2007;150(4):938–49.

    CAS  PubMed  Google Scholar 

  26. 26.

    Heizmann CW, Braun K. Calcium regulation by calcium-binding proteins in neurodegenerative disorders. Neuroscience intelligence unit 1995. New York: Springer; 2013.

    Google Scholar 

  27. 27.

    Schwaller B. Cytosolic Ca2 + buffers. Cold Spring Harb Perspect Biol. 2010;2(11):a004051.

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Tortosa A, Ferrer I. Parvalbumin immunoreactivity in the hippocampus of the gerbil after transient forebrain ischaemia: a qualitative and quantitative sequential study. Neuroscience. 1993;55(1):33–43.

    CAS  PubMed  Google Scholar 

  29. 29.

    Burke RE, Baimbridge KG. Relative loss of the striatal striosome compartment, defined by calbindin-D28 k immunostaining, following developmental hypoxic-ischemic injury. Neuroscience. 1993;56(2):305–15.

    CAS  PubMed  Google Scholar 

  30. 30.

    Chen G, Racay P, Bichet S, et al. Deficiency in parvalbumin, but not in calbindin D-28 k upregulates mitochondrial volume and decreases smooth endoplasmic reticulum surface selectively in a peripheral, subplasmalemmal region in the soma of Purkinje cells. Neuroscience. 2006;142(1):97–105.

    CAS  PubMed  Google Scholar 

  31. 31.

    Timmermans JA, Van Bindels RJ, Os CH. Stimulation of plasma membrane Ca2 + pump by calbindin-D28 k and calmodulin is additive in EGTA-free solutions. J Nutr. 1995;125(7 Suppl):1981S–6S.

    CAS  PubMed  Google Scholar 

  32. 32.

    Bastianelli E. Distribution of calcium-binding proteins in the cerebellum. Cerebellum. 2003;2(4):242–62.

    CAS  PubMed  Google Scholar 

  33. 33.

    Bojarski C, Meloni BP, Moore SR, Majda BT, Knuckey NW. Na +/Ca2 + exchanger subtype (NCX1, NCX2, NCX3) protein expression in the rat hippocampus following 3 min and 8 min durations of global cerebral ischemia. Brain Res. 2008;1189:198–202.

    CAS  PubMed  Google Scholar 

  34. 34.

    von Bernhardi R, Eugenin-von Bernhardi J, Flores B, Leon JE. Glial cells and integrity of the nervous system. Adv Exp Med Biol. 2016;949:1–24.

    Google Scholar 

  35. 35.

    Barreto GE, Gonzalez J, Torres Y, Morales L. Astrocytic-neuronal crosstalk: implications for neuroprotection from brain injury. Neurosci Res. 2011;71(2):107–13.

    PubMed  Google Scholar 

  36. 36.

    Helleringer R, Chever O, Daniel H, Galante M. Oxygen and glucose deprivation induces bergmann glia membrane depolarization and Ca(2 +) rises mainly mediated by K(+) and ATP increases in the extracellular space. Front Cell Neurosci. 2017;11:349.

    PubMed  PubMed Central  Google Scholar 

  37. 37.

    Cabungcal JH, Steullet P, Morishita H, et al. Perineuronal nets protect fast-spiking interneurons against oxidative stress. Proc Natl Acad Sci USA. 2013;110(22):9130–5.

    CAS  PubMed  Google Scholar 

  38. 38.

    Walson KH, Tang M, Glumac A, et al. Normoxic versus hyperoxic resuscitation in pediatric asphyxial cardiac arrest: effects on oxidative stress. Crit Care Med. 2011;39(2):335–43.

    CAS  PubMed  Google Scholar 

  39. 39.

    Kuwamura M, Yoshida T, Yamate J, Kotani T, Sakuma S. Hereditary cerebellar vermis defect in the Lewis rat. Brain Res Dev Brain Res. 1994;83(2):294–8.

    CAS  PubMed  Google Scholar 

  40. 40.

    Aravamuthan BR, Shoykhet M. Long-term increase in coherence between the basal ganglia and motor cortex after asphyxial cardiac arrest and resuscitation in developing rats. Pediatr Res. 2015;78(4):371–9.

    PubMed  PubMed Central  Google Scholar 

  41. 41.

    Manto M, Bower JM, Conforto AB, et al. Consensus paper: roles of the cerebellum in motor control–the diversity of ideas on cerebellar involvement in movement. Cerebellum. 2012;11(2):457–87.

    PubMed  PubMed Central  Google Scholar 

  42. 42.

    Putzu A, Valtorta S, Di Grigoli G, et al. Regional differences in cerebral glucose metabolism after cardiac arrest and resuscitation in rats using [(18)F]FDG positron emission tomography and autoradiography. Neurocrit Care. 2018;28(3):370–8.

    CAS  PubMed  Google Scholar 

  43. 43.

    Xu H, Lu A, Sharp FR. Regional genome transcriptional response of adult mouse brain to hypoxia. BMC Genom. 2011;12:499.

    CAS  Google Scholar 

Download references


The authors wish to thank Leona Bück for the excellent technical assistance.



Author information




All authors have made substantive contributions to the study. GK conceived the study, was responsible for data analyses, writing the manuscript and preparing the illustration of results. TMNT carried out all immunohistochemical stainings. TE carried out surgery including post resuscitation care and tissue sampling. UE established the experimental model and supervised the animal experiments and critically reviewed the manuscript.

Corresponding author

Correspondence to Gerburg Keilhoff.

Ethics declarations

Conflict of interest

The authors declare no conflict of interest.

Ethical Approval

This study was granted according to the requirements of the German Animal Welfare Act on the Use of Experimental Animals and the Animal Care and Use Committees of Saxony-Anhalt (permit number 42502-2-2-947 Uni MD).

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Keilhoff, G., Nguyen Thi, T.M., Esser, T. et al. Relative Resilience of Cerebellar Purkinje Cells in a Cardiac Arrest/Resuscitation Rat Model. Neurocrit Care 32, 775–789 (2020).

Download citation


  • Bcl2
  • Calcium-binding proteins
  • Caspase 3
  • cfos
  • MnSOD
  • Stellate cells